biosynthesis of cell walls fungi - microbiology and ...only a few (1, 98) papers dealing with the...

MICROBIOLOGICAL RzvEIws, June 1979, p. 117-1440146-0749/79/02-0117/28$02.00/0

Vol. 43, No. 2

Biosynthesis of Cell Walls of FungiV. FARJAS

Institute of Chemistry, Department ofBiochemistry ofSaccharides, Slovak Academy ofSciences, 809 33Bratislava, Czechoslovakia

INTRODUCTION ............................................................. 117FUTNGGAL C F WA S ....................................................... 117Chemical Structure ...................................... 117Wall Architecture ........................................................... 118

FORMATION OF CELL WALLS.120General.120Biosynthesis of Individual Wall Components.121

Chitin.121Glucans.124Polysaccharide-protein complexes.126

(i) Biosynthesis.126(ii) Regulation.128(iii) lization of reactions.129

Formation of Wall Fabric ............. ...................... ........... 129Metabolic Stability of theWall. 130

MORPHOGENETIC ASPECTS ............................. 131Topology of Wall Growth ................. 131Speculations About the Possible RegulatoryMe asm.................... 133

SUMMARY AND CONCLUSIONS.135LI TERATURE C ..........................................................135

ITRMODUCTIONThe surface structure of fungi, the cell wall,

with all its specialized functions in the diverselife activities of fungal cells, increasingly attractsthe attention not only of mycologists but also ofthe workers of other biological disciplines. Thefungal cell walls have been studied from differentaspects, and many excellent reviews have beenwritten in the past (e.g., 5, 9, 15-17, 23, 46, 91,205, 213).Papers dealing with the molecular mechanism

of cell wall formation in fungi started to appearwith increased frequency after the discovery ofnucleoside diphosphate-sugars and their func-tion as glycosyl donors in the biosynthesis ofcomplex saccharides (156) and the commercialavailability of these compounds labeled withradioactive nuclides. Whereas in 1965, at thetime of Aronson's review (5) on fungal cell walls,only a few (1, 98) papers dealing with the "invitro" biosynthesis of fungal wall componentshad been published, the present complete bibli-ography in this field would mount up tohundreds of papers.This by itself indicates the.increasing impor-

tance of studies on the mechanism of biosyn-thesis of cell surface structures, using the fungias models for simple eucaryotic cells. The ra-tionale for these studies is that they could notonly explain the mechanism of biosynthesis of

different cell wall components but also contrib-ute to a better understanding of various surface-related biological phenomena, such as cell-cellinteractions, immune response, morphogenesis,drug resistance, and others.Because of the great complexity of these prob-

lems, I will deal in this review mainly with themolecular aspects of cell wall formation in fungiand try to find their possible relationship toregulation of morphological development.

FUNGAL CELL WALLSDetailed discussion about the structure and

chemical composition of fungal cell walls fallsoutside the scope of this review; besides, therehave been several comprehensive articles writ-ten on this subject in the last years (e.g., 5, 9, 10,15, 16, 205). However, some general explanationsare necessary to serve as an introduction to theproblem of fungal cell walls.

Chemical StructureThe remarkable properties of fungal walls,

such as their mechanical strength, morphologi-cal features, and biological activity, are undoubt-edly based on their particular chemical compo-sition. Bartnicki-Garcia (15) pointed out that aclose correlation exists between taxonomic clas-sification and cell wall composition among fungi.

Polysaccharides, which represent about 80 to117

on April 26, 2020 by guest

http://mm

br.asm.org/

Dow

nloaded from

http://mmbr.asm.org/

118 FARKAS

90% of the dry matter of fungal cell walls, arecomposed of amino sugars, hexoses, hexuronicacids, methylpentoses, and pentoses (16). Glu-cose and N-acetyl-D-glucosamine (GlcNAc) usu-ally represent the chemical elements of skeletalwall polysaccharides, such as chitin, cellulose,noncellulosic f8-glucans, and a-glucans. Theother sugars are present mainly in the form ofvarious homo- and heteropolysaccharides, oftenin chemical complexes with proteins.

It can be anticipated that, owing to distinctivephysicochemical properties, the different poly-saccharides fulfill specific functions in the cellwalls. Whereas the crystalline polysaccharideschitin and /8-glucans are the components respon-sible for the mechanical strength of the wall, theamorphous homo- and heteropolysaccharides,often in association with proteins, play the roleof cementing substances and constitute the car-bohydrate moieties of extracellular enzymes andcell wall antigens (10, 91, 146).

Wall ArchitectureThe functional specialization of each cell wall

component is reflected also in its location withinthe wall structure. Electron microscopic and cy-tochemical evidence (5, 58, 65, 121, 161, 209, 210,266, 267), combined with results of chemical andenzymatic analyses (118, 119, 135, 146, 185, 200,255; M. Kopecka, H. J. Phaff, and G. H. Fleet,Proc. Int. Symp. Yeasts 4th, Vienna, Austria,1974, D5, p. 205), indicate that a certain degreeof stratification exists in the walls of fungi. Al-though distinct layering of building material israrely seen on ultrathin sections through the cellwalls, the general picture is that the outer sur-faces of the wall are smooth or slightly grannularin texture and composed of amorphous material(often glycoprotein in nature), whereas the skel-etal microcrystalline component is prominent inthe layer of the wall adjacent to plasmalemma.The interfibrillar spaces of the inner wall layerare filled with amorphous material, probably ofthe same chemical composition as that of theouter wall layer. The amorphous matrix materialpenetrates also into the periplasmic space (e.g.,86, 161, 242, 266, 267), where some of its constit-uents may exhibit different enzymatic activities(10, 146).The overall appearance of fungal walls varies

with age (Fig. 1). Newly sythesized portions ofthe walls are thin and smooth, with no visiblestratification. In older portions the primary wallis covered with secondary layers composed ofamorphous matrix material, and the fibrillar tex-ture of the innernost wall layer becomes morepronounced (118, 120, 121, 253, 254).There has been some discussion in the litera-

MICROBIOL. REV.

ture about the possible artifactual nature of themicrofibrillar cell wall components (37, 172).The main argument against the presence of dis-tinct layers of building material in the fungalcell walls was the possibility that the polysac-charide microfibrils might arise as a consequenceof various chemical treatments of isolated cellwalls (7, 116). These assumptions seemed to besubstantiated to a certain degree by Eddy andWoodhead (71), who observed "in vitro" reag-gregation of fibrillar elements of alkali-dissolvedyeast wall glucan.

Nevertheless, the presence of microfibrils ofskeletal wall polysaccharides can almost invari-ably be demonstrated in cell walls formed denovo on the surfaces of regenerating fungal pro-toplasts (e.g., 2, 88, 96, 197, 247, 265). Also,refined cytological and biochemical methods,such as partial enzymatic digestion of the wallsfollowed by electron microscopy, reveal the fi-brous nature of certain wall components (118,119, 121, 135; Kopecka et al., Proc. Int. Symp.Yeasts 4th, Vienna, Austria, 1974, D5, p. 205).

It should be noted, however, that observationsmade by application of the enzyme dissectiontechnique to reveal wall architecture do notallow straightforward interpretation in each caseand should be treated cautiously. Several factorsmay influence the final conclusions. (i) Thecross-linking between the individual wall com-ponents may result in the removal of the enzy-matically solubilized polymer as well as the sec-ond, linked polymer. (ii) The lytic enzymes usedmay be insufficiently purified-even traces ofcontaminating hydrolytic enzyme activities cansubstantially influence the final image; equallyimportant is knowledge on the precise modes ofaction of the enzymes used. (iii) The partialdissolution of the wall by enzymic or chemicaltreatments may lead to rearrangement and dis-placement of the remaining wall constituents,thus changing the obtained electron microscopicimage. (iv) Different accessibilities of the indi-vidual cell wall components to enzyme attackwhen working with intact cells or the isolatedwalls: in isolated walls the applied enzymes canattack the wall structure from both sides,whereas in intact cells only the components lo-cated on the outer wall surface are susceptible.The methodological difficulties described

above are perhaps the main sources of contro-versial conclusions concerning the details of wallarchitecture in fungi. This situation can be bestdemonstrated by the existing uncertainty aboutthe structure of the cell wall in Saccharomycessp., so far the best studied fungal cell envelope.Based on the available chemical, biochemical,

immunochemical, and cytological evidence,Lampen (146) suggested a structural model for


http://mm

br.asm.org/

Dow

nloaded from


BIOSYNTHESIS OF CELL WALLS OF FUNGI 119

Extension wig of primarwol WoN becomeprowgresivei extensiblewith distnce from **ll thiciis c5Onm

RWified zon, voltthices C 50#n

Secondary wall bmotionzone. Wall aream inthicnss With distencefrom tip

Wet thickns C 125 twn

Cros WON botan _W

E

R

[sJ

JM

retiulum

scret k- of protinEasily rm sble proen

lomiwnrin-Ske ghico

PoNfrotiOn of reticulmGround sptum

FIG. 1. Wall and septal structure of a hypha of Neurospora crassa. (Reproduced with permission fromreference 253.)

the cell wall of Saccharomyces cerevisiae. In hismodel the innermost microfibrillar layer, com-posed of insoluble ,8-glucan, is linked via proteinto the outer wall layer, composed of mannan-protein molecules mutually linked by phospho-diester bridges between their polysaccharidemoieties. The phosphodiester cross-linking wassupposed to form a physical barrier that holdswithin the wall structure the extracellular man-noprotein enzymes invertase, acid phosphatase,and others. The latter conclusion was arrived atfrom the effect of the so-called PR-factor, aphosphomannanase partially purified from cul-ture filtrates of Bacillus circulans (180). Treat-ment of intact cells with this preparation liber-ated the invertase and large phosphomannanmolecules and was followed by complete disso-lution of the cell wall. The PR-factor supposedlycleaved selectively the phosphodiester links be-tween the mannan molecules.The observation that invertase can be liber-

ated from intact yeast cells by mild sonication

or treatment with thiol reagents led Kidby andDavies (134) to a conclude that invertase is notchemically bound to the cell wall and that thenatural barrier against its escape from the wallstructure is the external wall layer, composed ofmannan-protein molecules linked together, assuggested by Lampen (146), by phosphodiesterbridges but also by disulfide linkages betweentheir protein moieties (Fig. 2).Apparently contradictory results were ob-

tained by Arnold (4), who demonstrated that inethyl acetate-treated cells the sulfhydryl com-pounds on their own were ineffective in releasingthe invertase entrapped within the wall; theydid, however, increase the extractability of themarker enzyme by yeast extracts containing glu-can-degrading activity. The differences as to ex-tractability of invertase by thiol reagents, whencompared with results of Kidby and Davies(134), could be explained by possible irreversiblechanges in the upper cell wall layer caused bythe ethyl acetate treatment.

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


120 FARKAS

HANNAN

ENZYME

GtUCAN

PROTEINCELL MEMBRANE

FIG. 2. Hypothetical structure of the ceU wall ofthe yeast S. cerevisiae. S-S, Disulfide linkage; P,phosphodiester bridge. (Modified from Kidby andDavies [134].)

The difficulties encountered in chemical ex-

traction of ,8-glucan from intact yeast cell wallsled Bacon et al. (7) to suggest the existence of athin chitinous membrane on the outer surface ofthe cell walls that would form a perneabilitybarrier preventing the extraction of 8-glucanwith the alkali. The problems described abovewith alkaline extraction off,-glucan did not oc-cur with isolated cell walls (7). Subsequent stud-ies from different laboratories have shown, how-ever, that the exclusive location of chitin inSaccharomyces cell walls is in the bud scars (48,229).Cytochemical staining selective for acid phos-

phatase (161) has revealed that this extracellularmannoprotein enzyme is located both in theouter wall layer and in the innermost layer,penetrating into the periplasmic space. The lo-calization of mannan-proteins in the outer andthe innermost regions ofthe yeast walls has beendemonstrated also by other cytochemical meth-ods (58, 210, 266, 267).The periplasmic space of yeasts has been also

reported to contain large amounts of glycogen(105, 106); its role, however, is still obscure.The presence of mannoprotein complexes in

the upper wall layer in yeast is well supportedby the fact that synergistic action of proteasesand ,B-glucanases is required to dissolve com-

pletely the walls of the intact cells (87, 214, 264,268, 269). The effect of proteases can be replacedby thiol reagents but not by the action ofpurifiedbacterial a-mannanase; neither is the a-mannan-ase alone effective in substantial removal of themannan located at the wall surface (268, 269).These results indicate that proteins held to-gether by disulfide bridges form a protectivebarrier against the penetration of exogenous glu-canase. On the other hand, walls isolated frommechanically disintegrated cells are readily dis-

MICROBIOL. REV.

solved by purified endo-1,3-/3-glucanase, indicat-ing that the innermost, periplasmic mannopro-tein layer is, unlike the outer one, only looselybound to the internal wall surface and can beeasily removed by washing with water (268, 269).

In the light of the present experimental evi-dence, the existence of phosphodiester links be-tween the carbohydrate moieties ofthe mannan-protein molecules in the upper wall layer, assuggested in previous models (134, 146), isdoubtful. The yeast mannans contain variableamounts of phosphate (9, 10), which, however,exists in S. cerevisiae mannan as a diester link-ing an a-(1,3)-mannobiosyl unit to position C-6of mannose, located in the terminal position ofthe short side chain (10, 250). In Kloeckerabrevis mannan the phosphorus is present in theform ofmannosyl-1-phosphate linked to positionC-6 of the mannose unit in the side chain of themannan molecule (250). However, direct evi-dence about the existence of phosphodiesterbridges linking together two macromolecularsubunits in yeast mannan is so far lacking.The penetration of certain types of large mac-

romolecules through the cell wall in both direc-tions indicates that the wall is by no means acompact structure. To allow the release of intra-cellularly manufactured enzymes and the pas-sage of certain exogenous macromolecules intothe cell (124, 201, 222, 223, 248, 276), the cell wallmust possess a certain number of pores. At pres-ent, it is difficult to decide whether the pores area permanent structure within the wall and todetermine their location. An alternate explana-tion, offered by Scherrer et al. (222), is that thephysicochemical interactions of penetratingmacromolecules with the components of the cellwall may evoke a reorganization within the wallstructure resulting in the tentative formation ofopenings large enough to accommodate the pen-etrating macromolecules.

FORMATION OF CELL WALLSGeneral

The problems connected with the process ofcell wall formation in fungi are being currentlyinvestigated from different points of view.Among the most frequently studied questionsare: (i) the molecular mechanisms of the biosyn-thesis of individual cell wall components, (ii) theinvolvement of cellular structures in these proc-esses and sites of participating enzyme activities,(iii) the participation of lytic enzymes in theprocess of cell wall formation, (iv) regulatoryaspects at different levels, and (v) cell wall for-mation in relation to morphological develop-ment of the cells.

a39...a_a"a


http://mm

br.asm.org/

Dow

nloaded from



Accordingly, different experimental systemshave been used in these studies, ranging in de-gree of complexity from isolated enzymes or

enzyme systems through subcellular fractionsand protoplasts to intact cells. The experimentalmethods vary from purely chemical and bio-chemical to cytological and physical. In the fol-lowing sections I will briefly review the presentknowledge on the process of cell wall formationin fingi, starting from the biosynthesis of theindividual principal wall polymers.

Biosynthesis of Individual WallComponents

Chitin. Chitin, a,8-(1,4) polymer of GlcNAc,is the major cell wall component in most fila-mentous fungi. Its in vitro biosynthesis in a cell-free system was first described by Glaser andBrown (98), who found that an enzyme prepa-

ration from Neurospora crassa catalyzes theincorporation of GlcNAc units from uridine 5'-diphosphate (UDP)-GlcNAc into a polymer un-

distinguishable from the authentic chitin. Thegeneral equation for this reaction is:

UDP-GlcNAc + [L-(1,4)-GlcNAcI.primer

LB-(1,4)-GIcNAc]. + I + UDPproduct

The reaction requires the presence of divalentcations and GlcNAc as activators. A single en-

zyme, UDP-2-acetamido-2-deoxy-D-glucose:chi-tin 4-fl-acetamidodeoxyglucosyltransferase (EC2.4.1.16), known under the trivial name chitinsynthase, seems to be involved in the reaction.Particulate preparations of chitin synthase havebeen prepared from a wide range of fungi (e.g.,3, 39, 55, 76, 101, 102, 123, 133, 164, 177, 181, 195,196,219,220,260), and most ofthem have similarkinetic properties (101). The results obtainedwith different enzyme preparations indicate thatchitin synthase is an allosteric enzyme, havingmore than one binding site per molecule (55, 63,181). In the absence ofGlcNAc the Hill numbersare close to 4 at low substrate concentrations(below 0.1 mM), and values close to 2 at highersubstrate concentrations are obtained (63, 101).In the presence of GlcNAc the Hill number isclose to 1 (219). The transfer of GlcNAc unitsfrom UDP-GlcNAc to a primer molecule seems

to proceed in a single step, and there is, so far,no evidence about the participation of "lipidintermediate" in this reaction (77, 183).The presence of an endogenous primer is not

absolutely necessary for the reaction to proceed.Solubilized preparations of chitin synthase are

capable offorming chitin under conditions when

evidently no macromolecular primer is present(98, 101, 102, 215).GlcNAc appears to act as a positive allosteric

effector of particulate chitin synthase (55, 181,216, 219). However, since relatively high concen-trations of a GlcNAc are needed to produce thesame activation as that produced by much lowerconcentrations ofUDP-GlcNAc, it has been sug-gested that UDP-GlcNAc is the natural effectorand that GlcNAc simply mimics its effect (216).The possibility has been considered that an ad-ditional stimulatory effect of GlcNAc on chitinbiosynthesis in vitro might reside in its functionas the primer for initiation of new chains ofchitin (55, 183); however, this has found verylittle experimental support (102, 216). Interest-ingly, N,N'-diacetylchitobiose but higher N-ace-tylchitodextrins especially stimulate the in vitrobiosynthesis of chitin both in the absence and inthe presence of GlcNAc (183, 204, 208). On theother hand, the activity of enzymes solubilizedwith butanol (98) or digitonin (63) is not affectedby GlcNAc.One of the most powerful inhibitors of fungal

chitin synthase is the pyrimidine antibiotic po-lyoxin D (e.g., 20, 77, 101, 102). The inhibitionby polyoxin D is competitive with regard to thesubstrate UDP-GlcNAc, the inhibitor constant,Ki, being two to three orders of magnitude lowerthan the Michaelis constant, Ki, for the sub-strate (20, 76, 103, 133, 164). A mechanism hasbeen suggested whereby polyoxin D, owing to itsstructural similarity with UDP-GlcNAc, com-petes for the active site of the enzyme (115).Fungal chitin synthase is normally prepared

as a particulate enzyme associated with cellularmembranes. Numerous studies with cell-free ex-tracts from various fungi have shown that thehighest specific activity of chitin synthase ispresent in a membrane fraction vaguely termed"microsomal" (3, 101, 103, 164, 188, 204). Theparticular membrane to which chitin synthase isattached was, until recently, difficult to identify.One of the factors negatively influencing theseparation of membrane fractions might be thatmechanical disintegration of the cells before en-zyme extraction as well as bursting ofprotoplastsby osmotic shock could cause fragmentation ofcellular membranes and their vesiculation (68).Using concanavalin A to preserve the integrity

of the plasmalemma, Durain et al. (69) were ableto show that yeast chitin synthase is locatedalmost exclusively in the plasmalemma. Simi-larly, isopycnic density gradient centrifugationof membrane preparations from Phycomycesblakesleeanus (123) or Candida albicans (39)points out that the plasmalemma is the principalsite of chitin biosynthesis.

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


122 FARKAS

Nevertheless, there have been repeated claims(183, 186, 204) that the bulk of chitin synthaseis located directly in the cell wall. It is highlyprobable that in these cases the wall prepara-tions were contaminated with the remnants ofcellular membranes, especially plasmalemma, asit has been demonstrated with cell walls pre-pared from S. cerevisiae by mechanical disinte-gration of the cells (245). The fact that fungalprotoplasts can be effectively used for prepara-tion of the enzyme (3, 133, 188) is also againstpredominant location of chitin synthase in thecell walls. As will be shown later, the location ofchitin synthase, and possibly also of some otherpolysaccharide synthases, in the plasmalemmacould be of essential importance for regulationand spatial organization of cell wall synthesis infungi.The membrane-bound chitin synthase can be

liberated by butanol extraction (98), by digitonintreatment (63, 70, 101), or by incubation of theparticulate enzyme with the substrate and acti-vator (215). Whereas both butanol and digitonindestroy the basic membrane structure, the sol-ubilizing effect of UDP-GlcNAc together withGlcNAc is difficult to explain.Ruiz-Herrera and Bartnicki-Garcia (215) pos-

tulated a working hypothesis according to whichthe chitin synthase is released from the mem-brane structure as soon as it starts the synthesisof chitin. If it were so, the presence of chitinsynthase in the cell wall would not be surprising.However, the mechanism by which the substratefor continuous chitin synthesis would be effec-tively supplied from the cell to the cell wall isdifficult to envisage. In later experiments of thesame authors and co-workers (215, 216, 218), itwas estabilished that the "soluble" preparationof chitin synthase consisted in fact of particlestermed "chitosomes."The molecular weight of chitin synthase from

Coprinus cinereus solubilized by digitonin treat-ment is of several millions (63); however, in thepresence of high salt concentrations the aggre-gates dissociate to smaller active subunits withmolecular weights of about 150,000 (101).

Preparations of chitin synthase solubilizedfrom membranes of Mucor rouxii by incubationwith the substrate and activator (215) or isolatedfrom the cytoplasm of mechanically disinte-grated cells (216) contain enzyme granules ofabout 35 to 100 am in diameter. On incubationwith UDP-GlcNAc, chitin microfibrils in asso-ciation with chitin synthase granules are pro-duced (38, 216). The terminal position of enzymegranules on the microfibrils formed indicatesthat the latter are elaborated by end synthesisof a large number of parallel chitin chains rather

than by spontaneous random crystallization ofde novo-synthesized chitin molecules (Fig. 3).

Ultrastructural studies revealed a rather com-plicated structure for chitin synthase particles,or, as they have been later renamed, chitosomes(38). Freshly isolated chitosomes appear on ul-trathin sections as clusters of protein granulesbound within a membranous shell. During fi-brillogenesis the chitosomes undergo a series ofultrastructural changes: the protein granules dis-appear, and, instead, a coiled microfibril of chitinappears inside the chitosome; the shell of thechitosome opens, and the chitin microfibril ex-tends from the particle (218). More recently (18),functional chitosomes have been isolated froma series of different genera of fungi, includingyeasts.

Although the chitosomes are fully capable ofsynthesizing chitin microfibrills in vitro, theirexistence in vivo has not yet been fully con-firned. With regard to their relatively large di-mensions, they cannot be considered as integralcomponents of the plasmalemma or another cel-lular membrane (average membrane thicknessis 8 to 9 nm). They could possibly representcontainers of chitin synthase conveying the en-zyme from the site of its synthesis to its desti-nation at the cell surface, or, eventually, theycould be of artifactual nature. The latter possi-bility is indicated by the presence of intracellularenzymes and, in some cases, of ribosomes in theisolated chitosomes (216). The artifactual natureof chitosomes is further indicated by the factthat molecules of chitin synthase solubilizedfrom the cell walls of M. rouxii by digitonintreatment associate with one another to formvesiculoid structures morphologically and func-tionally resembling chitosomes (S. Bartnicki-Garcia, C. E. Bracker, and J. Ruiz-Herrera,Abstr. Int. Mycol. Congr. 2nd, Tampa, Fla., p.41, 1977).Perhaps the most remarkable property of fun-

gal chitin synthase is that it exists in cells largelyin an inactive, or zymogenic, state. The inactiveenzyme can be converted to the active form bylimited proteolysis. The phenomenon of prote-olytic activation was first discovered with yeastchitin synthase (52) and later confirmed alsowith preparations from other sources (3, 50, 102,107, 122, 164, 182, 215, 219, 220, 260). The zym-ogenic character of chitin synthase was con-firmed also in solubilized preparations (70, 215).Crude preparations of yeast chitin synthase

obtained from protoplast lysates underwent aslow but significant increase in specific activityupon standing or storage at low temperatures,indicating that they might contain some intristicfactor capable of activating chitin synthase. Mild

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from


BIOSYNTHESIS OF CELL WALLS OF FUNGI

FIG. 3. Electron micrographs of isolated chitosomes from M. rouxii incubated with substrate. Chitosomegranules are marked with arrows. (A) Group of chitosomes; (B) isolated chitosome with extending chitinmicrofibril. (Reproduced with permission from reference 218.) Bar represents 0.1 ,m.

sonication of the pellets from the protoplastlystates led to solubilization of the so-called ac-

tivating factor whereby chitin synthase re-

mained insoluble (50). The chitin synthase freedfrom the activating factor had only negligibleactivity unless it was preincubated with the ac-

tivating factor or with trypsin. Subsequent stud-ies have shown that the activating factor is a

protease (54) located in some kind of intracyto-plasmic vesicle, unseparable from the vacuolarfraction (53). The properties of purified activat-ing factor were identical with those describedfor yeast proteinase B (108, 259), known to befunctional in inactivating yeast tryptophan syn-thase (109, 221). Other yeast proteases are un-able to activate chitin synthase, although manyproteases from other sources, for example, tryp-sin, chymotrypsin, papain, subtilisin, rennilase,and acid proteinase, are capable of doing so;however, the activating effects of the individualproteases are not the same in all cases (164).The supernatant fraction obtained after lysis

of yeast protoplasts has been found to contain aheat-stable protein capable of selective bindingto the activating factor, thus rendering it inef-fective (50). The inhibitor ofthe activating factorwas purified to homogeneity and found tobe a low-molecular-weight peptide (molecularweight, 8,500) lacking cysteine, methionine, ar-

ginine, and tryptophan (258). L6pez-Romero etal. (163) isolated from extracts of M. rouxiianother protein capable of inhibiting the in vitrosynthesis of chitin. In contrast to the inhibitorfrom S. cerevisiae, the isolated substance didnot interfere with the proteolytic activation ofzymogen by the activating factor, but it directlyinhibited the preactivated chitin synthase.The discovery of the activating factor and of

its specific inhibitor led to an assumption thatthey might be the components of the systemregulating yeast chitin synthase activity in vivo.The exclusive location of chitin in yeast budscars (48, 49, 229) and the observation that itssynthesis is restricted to a limited portion of thecell cycle (50, 111) strongly indicated that sucha regulatory mechanism could exist. Based onthe described properties of the individual com-ponents of the system catalyzing the formationof chitin, a working hypothesis has been pro-posed by Cabib and co-workers (47, 51) for themechanism of regulation of chitin synthase ac-tivity in yeast (Fig. 4).How this mechanism might operate in vivo

remains still unclear. The idea is that the vesiclescarrying the activating factor would coalescewith the plasmalemma at the site of bud for-mation, fuse with it, and activate the chitinsynthase zymogen at the given site (50). The

123VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


124 FARKAS

Chitin synthose

Zymogen * Active enzyme

--AF-Inhibitor

Activating factor (AF)FIG. 4. Scheme for activation of yeast chitin syn-

thase as proposed by Cabib et al. (51).

role of the soluble cytoplasmic inhibitor wouldbe to inactivate any activating factor that mightbe spilled into the cytoplasm, thus preventing itfrom uncontrolled action at other than the re-quired site.Although plausible, the proposed scheme for

regulation of chitin synthesis in yeast remainsstill in the category of working hypotheses. Thereason is that, so far, it has not been unequivo-cally shown that the proteolytic activation ofchitin synthase is a physiological phenomenon.Other mechanisms, such as phosphorylation-de-phosphorylation, group transfer, or removal ofcontact inhibition, might be involved as well inregulation of chitin synthase activity, and theproteolysis might mimic some of these effects(46). The doubts about the in vivo activation ofchitin synthase by proteinase B are strengthenedby the already mentioned fact that the yeastproteinase B, believed to play the role of acti-vating factor, functions at the same time as the"inactivating protein" of tryptophan synthase inyeast (108, 221, 259). Such bifunctionality of theproteinase B would not be surprising if we knewmore about the relations between the metabo-lism of tryptophan and the synthesis of chitin inyeast. Mutants of S. cerevisiae lacking protein-ase B but apparently having unaltered mor-phology have also been described (126). It ispossible, however, that the "real" activating fac-tor of chitin synthase is a minor protease that isdifficult to reveal in the usual protease assays.

Nevertheless, the existence of two forms offungal chitin synthase, the zymogen and theactive enzyme, seems to be well proven. Withregard to the crucial morphogenetic role of chi-tin in fungal cell walls, the interconversion be-tween the two forms of chitin synthase mightrepresent a key mechanism for regulating chitinsynthesis and, consequently, wall morphogene-s1s.Glucans. The generic name "glucan" covers

a large group of D-glucose polymers differingboth in type and in relative proportions of indi-vidual glycosidic bonds. The most abundant glu-cans of fungal cell walls are those with the f8-configuration, present usually as constituents of

the skeletal ruicrofibrillar portions of the walls.A relatively smaller group of fungi contain intheir walls glucose polymers linked by a-glyco-sidic bonds. Except for cellulose, most fungal cellwall glucans contain mixed glycosidic bonds, forexample, ,8-1,3 and ,8-1,6 in yeast ,/-glucan (169,170) (Fig. 5). For more details on occurence andstructure of fungal cell wall glucans, the readeris directed to more specialized articles (e.g., 15,16, 205, 271, 277).Contrary to the situation with chitin, surpris-

ingly little is found in the literature concerningthe molecular mechanism of glucan biosynthesisin fungi. In spite of considerable effort, the nu-merous attempts (although rarely published) todemonstrate in vitro biosynthesis of glucan fromdifferent nucleoside diphosphate-glucose pre-cursors in a cell-free system have failed (46).The lack of success in most of these experi-

ments indicates that the enzyme system catalyz-ing the synthesis of wall glucan in fungi is moredelicate and more sensitive to damage than arethe other fungal polysaccharide synthases. It canbe anticipated that the isolation of an activeglucan synthase would require special precau-tions to minimize its inactivation during theisolation procedure.

Several factors could be expected to influencenegatively the isolation and the assays of fungalglucan synthase. (i) Severe mechanical treat-ment used to disintegrate the cells for extractionof the enzyme could disturb the spatial relation-ship of the individual components of glucan-synthesizing machinery and lead to a loss of itsactivity. (ii) The proteases libertated from theirvacuolar compartments during cell breakage orprotoplast lysis could inactivate the glucan syn-thase by unspecific proteolysis (107, 272). (iii)The expression of the glucan synthase might beprevented by its combination with various spe-cific or nonspecific effectors that might comeinto contact with the enzyme during the isola-tion procedure. (iv) The endogenous glucosi-dases and glucan hydrolases present in the crude

G - 1(1 -3l - [G;I-(I -3 - Ga

6

G- 1-3) -[G; -1-3 -G-11-31-[G] -11I-3) - Gb

G.G-lt-3G-G -t3) -G...FIG. 5. Partial structure of a segment of yeast f8-

1,3-glucan. G, glucopyranose residue. a + b + c com-prise about 60 glucose residues; the exact lengths ofa, b, and c are unknown. (From Manners et al.[171].)

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



particulate enzyme preparations could hydro-lyze the product or substrate or both in thecourse of the enzyme assay (195).

Nevertheless, several cases have been re-ported in the literature demonstrating in vitrobiosynthesis ofwall glucan. Wang and Bartnicki-Garcia (270) isolated from mechanically disin-tegrated cells of Phytophthora cinnamomi aparticulate fraction catalyzing the incorporationof radioactive glucose from UDP-["4C]glucoseinto a glucose polymer containing both ,B-1,3-and ,8-1,6-glycosidic bonds. The highest specificactivity of glucan synthase was found in thefraction enriched in cell walls. The small yieldsof the product and the complexity of the enzymepreparation used precluded better characteriza-tion of the product. In more recent experimentsthe same authors (272) took precautions to min-imize the formation of proteases in the growingfungus by increasing the concentration of glu-cose in the growth medium. The mixed mem-brane fraction (devoid of cell walls) preparedfrom such cells catalyzed massive formation offl-glucan microfibrils from UDP-glucose. Inter-estingly, the treatment of the enzyme prepara-tion with typsin caused a nearly twofold in-crease of glucan synthase activity, indicatingthat at least a part of the enzyme was present inzymogenic form or was hindered from the accessof the substrate (272). Disaccharides, such ascellobiose, stimulated the synthesis of glucan inthe cell-free system. The product was character-ized on the basis of its digestibility with purifiedfungal exo-1,3-,8-glucanase and X-ray diffrac-tion. The results showed the absence of 8)-1,6-glycosidic linkages in the product, indicatingthat it consists entirely of,-1,3-glucan chains(272).Using cellular homogenates of Cochliobolus

miyabeanus, Namba and Kuroda (195, 196)demonstrated incorporation of [14C]glucose fromUDP-['4C]glucose into a polysaccharide precip-itable with ethanol. Chemical and structuralanalysis ofthe reaction product showed the pres-ence of both ,-1,3- and f-1,6-glycosidic linkagesbetween the individual glucose units (196).In whole cells of S. cerevisiae made permeable

with toluene-ethanol treatment, Sentandreu etal. (231) observed incorporation of labeled glu-cose from UDP-['4C]glucose into 8l-1,3-glucan.The radioactive glucan was recovered from thecell homogenate in a membrane fraction bymeans of differential centrifugation. However,when the membrane particles themselves wereassayed for glucan synthase activity, none wasdetected (231). Attempts to detect the partici-pation of a lipid intermediate in the describedreaction have failed.

Balint et al. (8) prepared from the yeast S.cerevisiae a particulate enzyme system com-posed of mixed membrane fraction catalyzingthe formation of fB-glucans from both UDP-[14C]glucose and guanosine 5'-diphosphate(GDP)-['4C]glucose. Practically no activity wasfound in the wall fraction. The enzyme prepa-ration appeared to contain two independent glu-cosyltransferase activities, one using UDP-glu-cose and the other one using GDP-glucose asthe respective substrates. A great portion of theradioactivity incorporated was liberated fromthe particles by mild alkali treatment, indicatingthat at least a part of the synthesized glucanswere of low molecular weight, possibly attachedto particles via bonds to protein. Digestion ofthe respective products with different, partiallypurified,8-glucanases indicated that the productfrom UDP-glucose contained a higher propor-tion of 8-1,3 bonds whereas the product fromGDP-glucose was more abundant in ,8-1,6 bonds(8).In a imilar system L6pez-Romero and Ruiz-

Herrera (165) detected the formation of mixed,B-1,3- and ,-1,6-glucan from UDP-glucose as theglucosyl donor, whereas the amount of radioac-tivity incorporated from GDP-glucose was neg-ligible. Contrary to the findings of Balint et al.(8), the highest specific activity of glucan syn-thase was found in the cell wall fraction.

Biosynthesis of /?-glucan from UDP-glucosewas described in less detail also in the Neuro-spora system (186), where glucan-synthesizingactivity was confined mostly to the cell walls.Fevre and Dumas (84) demonstrated the bio-

synthesis ofpolysaccharides ofthe cellulose typeby cell-free extracts from Saprolegnia monoica,using UDP-glucose as the substrate. The reac-tion was stimulated by the presence of Mg2" andcellobiose. High substrate concentrations in-creased the proportion of alkali-insoluble glu-cans formed in the reaction mixture.The highest specific activities of glucan-syn-

thesizing enzymes have been found in cell walland microsomal fractions, and they have beenhigher in branched hyphae than in unbranchedmycelia. Practically no glucan synthase activityhas been found in cytoplasm; however, the lattercontained a thermolabile component capable ofinactivating particulate glucan synthase.From the results described above, it is appar-

ent that also in the case of glucan biosynthesisuncertainty eists concerning the cellular loca-tion of glucan synthase. The physicochemicalproperties of,-glucan, namely, its insolubilityand high degree of crystallinity, indicate that itssynthesis occurs most probably in situ, i.e., inthe cell wall or at the outer surface of plasma-

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


126 FARKAS

lemma. The extracytoplasmic location ofglucan-synthesizing enzyme(s) is also supported to acertain degree by the observation that germi-nating cysts of Phytophthora incorporated ra-dioactivity from exogenously added UDP-['4C]-glucose into their walls (178). No proof, however,was presented to exclude the possibility thatadded UDP-[14C]glucose was split by extracel-lular hydrolases, thus enabling the permeationof radioactive glucose into the cell. Clearly, theproblem of cellular location of glucan synthase(as well as of other polysaccharide synthases)requires further careful examination.Polysaccharide-protein complexes. Poly-

saccharides covalently linked to protein repre-sent in some fungi, but especially in yeasts, aprincipal cell wall constituent (15, 205). Struc-turally and functionally, the fungal glycoproteinsshow many similarities with the protein-polysac-charide complexes of higher organisms. For thisreason, the fungal glycoproteins can be consid-ered as very appropriate models to study thegeneral aspects of glycoprotein structure, func-tion, and biosynthesis.The most systematic study on fungal glyco-

proteins has, so far, been performed with yeastmannan. Recent reviews by Ballou (9) and Cabib(46) comprehensively summarize from differentpoints of view the present knowledge on thissubject. Nevertheless, for the sake of complete-ness I will briefly review the older facts and tryto give a more updated version of the achieve-ments in this area.Yeast mannan is a polymer composed of pro-

tein and two carbohydrate moieties differing intheir structure and mode of attachment to thepeptide (Fig. 6). The polysaccharide moiety isrepresented by an a-1,6-linked polymannosebackbone to which short chains of mannosylunits linked together by a-1,2- and a-1,3-glyco-sidic bonds are attached predominantly bymeans of an a-1,2 bond. The polysaccharidemoiety is linked via a diacetylchitobiose bridgeby an N-glycosidic bond to an asparaginyl resi-due in the protein part of the molecule (191).The second carbohydrate moiety of yeast man-nan consists of short mannooligosaccharidescontaining both a-1,2- and a-1,3-glycosidic bondsattached at their reducing ends by an O-glyco-sidic linkage to serine or threonine residues orboth in the protein, from which they can beliberated by /3-elimination in weak alkali (190,234, 235). A species of mannoprotein containingonly the O-glycosidically linked carbohydratemoiety has been described in Hansenula wingei(275).The biosynthesis of mannan has been inves-

tigated from different aspects, such as the mech-

t2f -T' j2l -lt Jx-MM MMM

tl3 l3 T 3M McM

Outer chain

t2 3 f11M1M

31,

Inner coreM-_

0-glycosidicalBy linkedoligosaccheride

wThr)

FIG. 6. Structure ofyeast mannan. M, Mannopyr-anose residue; P, phosphate; Asn, asparagine; Ser,serine; Thr, threonine. (From Nakajima and Ballou[191])

anism of polymerization of mannosyl units, cel-lular location of individual reactions, mode oftransport of mannoproteins from the site of syn-thesis to the cell wall, and others.

(i) Biosynthesis. The structural complexityof the yeast mannan molecule suggests that itsformation would require participation of a mul-tienzyme system consisting of different glycosyl-transferases. It has been estimated (9) that atleast 10 mannosyltransferases, each of them cat-alyzing the formation of a specific glycosidicbond, are involved in the biosynthesis of carbo-hydrate moieties of the mannoprotein molecule.

All mannosyl units in the mannan moleculeoriginate from GDP-mannose as the mannosyldonor (1, 24). In some cases, however, the trans-fer of mannosyls to mannan does not occurdirectly but through intermediates of lipophilicnature (233, 249). The intermediates have beenisolated and characterized as belonging to thegroup of dolichol phosphates containing 16 to 18polyisoprene units in their molecules (127, 233).The participation of dolichol phosphates

seems to be undoubted, especially in transfer ofthose glycosyl units that are directly linked toprotein. For example, the attachment of the firstmannosyl unit to serine or threonine by forma-tion of an O-glycosidic bond to the peptide in-volves a two-step mechanism in which the man-nosyl unit from GDP-mannose is first trans-ferred to dolichol phosphate and, in the secondstep, transferred from the formed dolichol mono-phosphate-mannose to the protein acceptor ac-cording the following sequence (6, 236): (i) GDP-mannose + dolichol phosphate -- dolichol phos-phate-mannose + GDP; (ii) dolichol phosphate-mannose + protein (serine or threonine) -- pro-tein (serine or threonine)-mannose + dolicholphosphate. The mannosyl unit, once attached in

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



this way to protein, can be further mannosylatedwithout involvement of dolichol phosphate (6).The formation of the N-glycosidically linked

inner core involves a similar, but more compli-cated, reaction sequence. The first step in thisprocess is the formation of dolichol pyrophos-phate-GlcNAc from dolichol monophosphateand UDP-GlcNAc (152). By a sequence of fur-ther transglycosylic reactions, another unit ofGlcNAc is added, and the formed dolichol py-rophosphate-N,N'-diacetylchitobiose is furthermannosylated by one mannosyl unit attached bya ,-1,3 bond and by one or more mannosyl unitsmutually linked by a-glycosidic bonds (152, 154,194, 202, 203). There is preliminary evidence (67;W. Tanner, personal communication) to suggestthat the subsequent mannosylation of the lipid-linked N,N'-diacetylchitobiose proceeds, at leastpartly, with involvement of dolichol monophos-phate-mannose as the immediate mannosyl do-nor. In a final step the lipid-bound oligosaccha-ride is transferred to a protein acceptor withformation of an N-glycosidic bond (Fig. 7).From the experiments in vitro it is difficult to

ascertain what size the lipid-bound oligosaccha-ride can attain before it is transferred to protein.The smallest transferable unit seems to be theN,N'-diacetylchitobiose (155, 194), but the sizeof lipid-bound oligosaccharide can reach over 12hexose units (154, 202, 203). Thus, it seems verylikely that the formation of the whole inner coreof the polysaccharide moiety in the mannanmolecule, comprising about 12 to 17 mannosylunits attached to N,N'-diacetylchitobiose (192),can take place on the lipid. The reaction se-quence of biosynthesis of the N-asparaginyl-linked carbohydrate moiety in yeast mannan

FIG. 7. Scheme showing the possible sequence inbiosynthesis ofthe "inner core" ofyeast mannan andthe role of glycosylated dolichol phosphates. Dol-P,Dolichol phosphate; Dol-PP, dolichol pyrophos-phate; Pi, inorganic phosphate; Man, mannose; Asn,asparagine. x + n = 0 to 17.

strikingly resembles the situation found in var-ious mammalian (25, 59, 117, 157) and plant(149) systems.Although the syntheses of both carbohydrate

moieties in the molecule of yeast mannan pro-ceed independently (82), it is not known whetherthe same dolichol monophosphate is used fortransfer of mannosyl units to both the polyman-nose portion and the O-glycosidically linked ol-igosaccharides. That this could be is indicatedby the observation that in liver microsomes (262)the same dolichol phosphate serves for transfersof mannose, glucose, and N-acetylglycosaminefrom their respective nucleoside diphosphates toendogenous acceptors.

Once linkage of the inner core oligosaccharideto the peptide acceptor has been established, thefurther elongation and branching of the poly-mannose region of the glycopeptide occur ap-parently by a direct transfer of mannosyl unitsfrom GDP-mannose (155). The order of the for-mation of individual types of glycosidic bondsbetween the mannosyl units and the sizes of theside chains are most probably determined by thepresence of individual mannosyl transferasesand their substrate specificities.The latter assumption comes from the results

of experiments with yeast mannan-synthesizingenzyme systems where mannose and short man-nooligosaccharides with defined structures wereused as exogenous acceptors for transfer of man-nosyl units from GDP-mannose (81, 151, 193,243). For example, free mannose can serve asthe acceptor for the a-1,2-mannosyltransferase(81, 151, 193) while the a-1,6-mannooligosac-charides serve as substrated for both the a-1,6-mannosyltransferase (81, 193) and the a-1,2-mannosyltransferase (193). The mannotetraosecontaining an a-1,3-linked mannosyl unit at thenonreducing end accepts a new mannosyl unitat position C-6 of the reducing terminal mannose(193). A mannosylphosphate transferase isolatedfrom S. cerevisiae catalyzes the transfer of man-nosyl-l-phosphate from GDP-mannose to posi-tion C-6 of the penultimate mannosyl unit inreduced a-1,2-linked mannotetraose (129). Noevidence was obtained that a lipid-bound man-nosylphosphate derivative was involved in thereaction.The molecular mechanisms of mannoprotein

synthesis in other yeast species and differentfungal genera have not been studied to such anextent as with S. cerevisiae. In the biosynthesisof phosphomannan of Hansenula yeast species,GDP-mannose serves as the donor of both man-nosyl units and the phosphate (41, 138, 179), thelatter being transferred from the sugar nucleo-tide in the form of mannosyl-l-phosphate (41).

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


128 FARKAS

The enzyme system isolated from Hansenulaspecies catalyzes the formation of-both N-gly-cosidically and O-glycosidically linked carbohy-drate moieties (40) with the participation ofdolichol monophosphate as the lipid intermedi-ate (42, 43).A particulate enzyme system isolated from

Cryptococcus laurentii catalyzes the transfer ofmannose from GDP-mannose to endogenous aswell as to exogenous low-molecular-weight ac-ceptors with the formation of a-1,2-, a-1,3-, anda-1,6-mannosyl linkages and a mannosyl-xylosyllinkage (225, 226). In the same system UDP-xylose and UDP-galactose serve as the donorsof xylosyl (227) and galactosyl (211) units for theformation of cell wall heteropolysaccharides. InKluyveromyces lactis the UDP-GlcNAc servesas the source of terminal GlcNAc units in theside chains of the wall mannan (243).Hyphal fungi contain relatively little glycopro-

tein in their cell walls when compared withyeasts (15). Owing to this fact, studies dealingwith the mechanism of biosynthesis of the gly-coprotein components of hyphal walls are com-paratively scarce. In Aspergillus niger, GDP-mannose has been reported (160) to serve as thedonor in the transfer of mannosyl units to en-dogenous acceptors. Formation of a polyprenolphosphate-mannose serving as the intermediatein some mannosyltransferase reactions has alsobeen observed (158, 159). A similar mannosyl-1-phosphorylpolyisoprenol has been discoveredalso in N. crassa, where it apparently serves asan obligatory intermediate in the transfer ofmannosyl units to peptide acceptors (99).A particulate enzyme preparation isolated

from Penicillium charlesii catalyzed the incor-poration of mannosyl units from GDP-mannoseinto both endogenous acceptors and added pep-tidophosphogalactomannan (92). Under theseconditions, the transfer of mannose into boththe O-glycosidically linked oligosaccharide andthe phosphogalactomannan region of the glyco-peptide acceptor was observed. About 10% ofthe mannosyltransferase activity could be solu-bilized from the membranes by treatment withTriton X-100. The solubilized enzyme incorpo-rated mannosyl units from GDP-mannose pre-dominantly into the phosphogalactomannan re-gion of the acceptor, whereas the remainder ofthe activity, not solubilized by the detergent,catalyzed the formation of mannopyranosyl-(seryl/threonyl) linkages in the peptidophos-phogalactomannan (93). So far, no participationof mannosyl-linked lipid intermediates in thesereactions has been detected.

(ii) Regulation. The biosynthesis of yeastmannan in vivo appears to depend intimately on

MICROBIOL. REV.

undisturbed protein synthesis. When the for-mation of proteins is blocked by cycloheximidein intact cells (74) or protoplasts (80, 162, 256,261), the formation and extracellular appearanceof mannoproteins and mannoprotein enzymesare halted after a short delay. Cycloheximidedoes not affect the activity of the isolated man-nan-synthesizing enzyme system (80, 232); nei-ther does it influence the relatively low turnoverof the corresponding enzymes (75). Hence, it isprobable that blocking of mannan synthesis bycycloheximide reflects exhaustion of the pool ofpeptides serving as acceptors for mannosyltransfer. The inhibition ofmannoprotein synthe-sis by cycloheximide is accompanied by amarked increase in the pools of GDP-mannoseand UDP-GlcNAc (232,256), the glycosyl donorsfor biosynthesis of carbohydrate portions of themannan.Mannosylation of at least some intracellular

proteins seems to be a necessary prerequisite fortheir transport across the plasma membrane tothe cell exterior. Impediment of mannosylationby 2-deoxyglucose in the presence of glucose orfructose as respective carbon sources (80, 139,141, 162, 261) or by tunicamycin (142, 143) re-sults in inhibition of the formation and secretionof the exocellular mannoprotein enzymes inver-tase and acid phosphatase.Whereas tunicamycin interferes with the gly-

cosylation of proteins (142, 143) through inhibi-tion of the N-acetylglucosaminyl-transferasecatalyzing the formation ofthe intermediate dol-ichol pyrophosphate-GlcNAc (153), the inhibi-tion of glycosylation by 2-deoxyglucose seems tobe of a more complex nature (139). 2-Deoxyglu-cose inhibits the mannosylation of proteins byinterfering with the conversion of glucose tomannose at the level of their 6-phosphates (141,273). In addition, the GDP-2-deoxyglucoseformed in cells cultivated in the presence of 2-deoxyglucose (31) could act as a competitiveinhibitor in the mannosylation reaction (35, 150,217).There seems to operate quite an effective feed-

back mechanism controlling the synthesis ofnon-glycosylated forms of extracellular enzymesunder conditions ofinhibited glycosylation, sinceno substantial accumulation of carbohydrate-free forms of these enzymes in the cytoplasntakes place (142, 162, 261). The existence of sucha regulatory mechanism is strongly indicatedalso by the finding that a mannose-deficientmutant of Schizosaccharomyces pombe is un-able to synthesize the carbohydrate-free forn ofacid phosphatase, despite derepression condi-tions, when mannosylation is blocked by theabsence ofmannose in the growth medium (224).


http://mm

br.asm.org/

Dow

nloaded from



Somewhat contradictory resultsmere obtained less, the mechanism by which control over gly-by'Moreno'et al. (189), who observed'an'accu- coprotemi secretionby'glyc'syl'ation is executedmulation of the light and intermediate, partially remains still a subject of speculation. One of theglycosylated forms of yeast invertase at the ex- possibilities is that the glycosylation of nascent,pense of heavy, fully glycosylated enzyme when membrane-associated proteins facilitates, bycells were incubated in the presence of 2-deox- mutual interaction of hydrophilic carbohydrateyglucose. groups, the pinching-off and vesiculation of por-

(iii) Localization of reactions. The experi- tions of the membranes of the endoplasmic re-mental evidence indicates that the biosynthesis ticulum whereby the glycosylated parts of theof mannan starts at an early stage in the for- glycoproteins are facing the vesicle interior andmation of its protein moiety. Short pulses of the protein moieties remain temporarily associ-["C]mannose to growing yeast protoplasts fol- ated with the enclosing membrane.lowed by their lysis and subcellular fractionation Another open question is the mechanism ofrevealed that the nascent polypeptide is glyco- glycoprotein secretion. Two models for glycopro-sylated by small amounts of mannose, glucosa- tein transport have been proposed: (i) reversemine, and, surprisingly, glucose (217). This ini- pinocytosis of carrier vesicles which after fusiontial glycosylation apparently represents the for- with the plasmalemma would discharge theirmation of linkage regions between the protein contents into the cell exterior (see, for example,and carbohydrate moieties in the mannan, as Grove et al. [104]), which would be a processjudged from the participation of dolichol phos- analogous to glycoprotein secretion in higherphate-mannose in the glycosylation reaction organisms (175), (ii) free "diffusion" of glycopro-(147). tein macromolecules across the plasmalemmaThe cellular location of mannosyltransferases without involvement of secretory vesicles (184).

was studied by using different techniques, in- The participation of vesicles in glycoproteincluding autoradiography (136), cytochemical transport in fungi seems to be well established.staining (267), and subcellular fractionation on Numerous examples can be found in the litera-urografin gradients (61, 136, 148). The latter ture showing an accumulation of vesicles nearexperiments have shown that the highest spe- the extending zones of the cell walls (28, 57, 114,cific activity of mannosyltransferases resides in 121, 176, 187). Chemical and histochemical anal-the membranes of the endoplasmic reticulum; yses of the contents of the secretory vesicleshowever, a significant portion of activity has shows that they contain a large proportion ofbeen found also in other membrane fractions, cell wall matrix material (28, 62, 104). Anotherincluding the plasmalemma. function of vesicles could be that they might

Analysis of products synthesized by different supply new material for the extension of themembrane fractions confirmed the observation plasmalemma (17, 253).(147) that the membranes of the endoplasmic What forces direct the secretory vesicles to-reticulum possess the highest specific activity of wards the sites of active cell wall growth remainsthose mannosyltransferases requiring dolichol to be established. Theoretically, the movementphosphates as intermediates for mannosyl trans- of organelles in the cytoplasm could be organizedfer (148). On the other hand, the cytoplasmic either by cytoplasmic streaming, by a system ofvesicles and plasmalemma exhibited increased microtubules, or by electric forces. The existenceactivity of enzymes participating in the elonga- of an electrical potential between the apex andtion of oligo- and polymannose chains, not re- subapical regions in fungal hyphae (241, 253)quiring the participation of dolichol phosphates would speak in the favor of the latter possibility.(148,174). However, the participation of microtubules inThese data imply that the fornation of man- directing the secretory vesicles cannot be ex-

noproteins is a vectorial process involving the cluded, especially in view of the finding of Byerstransfer of nascent polypeptides along the mem- - and Goetsch (45), who observed bundles of mi-branes of the rough and smooth endoplasmic crotubules extending from the nuclear plaquereticula and the Golgi cistemae and finally trans- into early buds in S. cerevisiae.port across the plasmalemma, accompanied onthis route by stepwise addition ofmannosyl units Formaton of Wall Fabrcto the growing carbohydrate chains. With regard to the structural complexity andThe role of glycosylation in this translocation mechanical integrity of the cell wall, it is un-

is far from clear. The results obtained with doubted that the final steps in the formation ofyeasts as models seem to support Eylar's (78) the cell wall take place extracellularly, i.e., inproposal that the glycosylation of proteins ena- situ. The formation of the cell wall fabric frombles their passage to the cell exterior. Neverthe- its macromolecular constituents must involve

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


130 FARKAS

association of subunits, their cross-linking, andtheir aggregation into a supramolecular struc-ture with defined chemical composition andmorphology.

In contrast to the well-established presence ofpolysaccharidehydrolases in the cell walls andperiplasmic space (e.g., 14, 83, 85, 168, 206), thereis, so far, no evidence about the existence ofligase-type enzymes catalyzing the formation ofchemical linkages between the individual mac-romolecular components of the wall. Besides thenoncatalytic formation of disulfide bridges be-tween the protein moieties of wall glycoproteins,a great role undoubtedly is played by the self-assembly of subunits in the formation of wallfabric with participation of physicochemical in-teractions. As such can be considered, for ex-ample, the formation of fibrils of insoluble skel-etal polysaccharides by rapid crystallization offormed subunits (71) or polysaccharide frag-ments produced by the attack of polysaccharidehydrolases in the process of cell wall extension.Some information about the process of cell

wall assembly in fungi could be obtained byexperiments in vitro involving dissociation andreassociation of wall components. Similar exper-iments have been successful, so far, with rela-tively simple biological structures, such as ribo-somes (144), virus particles (72), bacterial flag-ella, and the like (145). Hills (113) was able todemonstrate in vitro dissociation and reassem-bly of glycoprotein-composed cell walls ofChlamydomonas reinhardi after their dissolu-tion in 8 M lithium chloride and subsequentdialysis ofthe solution against water. A "nucleat-ing agent," insoluble in 8 M lithium chloride,was required to initiate cell wall reassembly.However elegant they may be, such experimentsdo not necessarily indicate the real sequence ofevents in the assembly process when proceedingmn vivo.Although at present no means exist to disso-

ciate the complex fungal walls without denatur-ation of their basic components, certain possi-bilities for overcoming this obstacle are repre-sented by the use of fungal protoplasts. Theprotoplasts under suitable conditions produceand secrete practically all the cell wall constitu-ents (146, 198, 265). The insoluble wall compo-nents remain during this process in the form ofa microfibrillar network in close proximity to theprotoplast surface while the soluble componentsof glycoprotein nature diffuse away into thesurrounding medium. When the fibrillar poly-saccharide net formed at the protoplast surfaceis dense enough or when the diffusion of solublematerial is prevented by an artificial permeabil-ity barrier, such as embedding the protoplasts ina gel, regeneration of the cell wall takes place

MICROBIOL. REV.

(198). The increased concentration of cell wallbuilding elements at the surface of the proto-plasts apparently facilitates their mutual contactand cross-linking by chemical and physico-chemical interactions and leads to the formationof the new compact wall (for more comprehen-sive descriptions of the process of cell wall re-generation, see, for example, 198, 205, 263, 265).The formation of new portions of the cell wall

in vegetative fungal cells may involve a similarmechanism. The difference is that here the nas-cent cell wall constituents are being locally in-corporated into the preexisting cell wall struc-ture playing the role of template by ordering theproper position and assembly of the newly ar-rived macromolecules.The number of chemical and physicochemical

links in the cell walls apparently increases withthe cell age, as is indicated by the observationthat the older portions of the walls are moreresistant to attacks by endogenous (207) as wellas exogenous (44, 198, 263) polysaccharide hy-drolases.

Metabolic Stability of the WallSome bacteria exhibit extensive turnover of

cell wall components during the active growth(e.g. 36, 274). Although the exact mechanism isnot known, it is assumed that the autolytic en-zymes (autolysins) present in the periplasmicspace or in the wall cause the dissolution of wallmaterial, its release into the medium, and itseventual reutilization by the cells.The isolated cell walls of many fungi exhibit,

due to the presence of different polysaccharidehydrolases, appreciable autolytic activity (e.g.14, 83, 85, 206). Although these enzymes aresupposed to be involved primarily in cell wallweakening during growth and other morphoge-netic processes, a question arises as to whethertheir action on the cell wall could cause appre-ciable turnover of the individual cell wall com-ponents and their exchange with the monomersin the cytoplasm during vegetative growth.

Pulse-chase experiments with cells of Asper-gillus clavatus labeled with radioactive glucoserevealed that glucose and GlcNAc became met-abolically inert once they had been incorporatedinto the wall polymers (60). A similar conclusionhas been drawn from experiments with cells ofS. cerevisiae whose cell wall mannan was selec-tively labeled with 2-deoxy-D-[3H]glucose, D-[I4C]mannose, or when the cell wall was univer-sally labeled with D-[14C]glucose (140). Aftertransferring the yeast cells from radioactive intonon-radioactive growth media, the radioactivityin the cell walls persisted for at least threegenerations.

Nevertheless, there are indications that under


http://mm

br.asm.org/

Dow

nloaded from



limiting conditions, or in certain periods of thelife cycle, the cell wall polysaccharides may serveas a metabolic reserve. In a quantitative studyon the changes of a-1,3-glucan in the cell wall ofAspergillus nidulans, Zonneveld (278) has ob-served a marked decrease of a-1,3-glucan con-tent in the cell walls, accompanied by an increaseof a-1,3-glucanase activity during cleistotheciumdevelopment induced by glucose depletion fromthe medium.

Stationary-phase yeast cells when transferredto a fresh growth medium exhibit a sudden fallin the mannan content of their walls accompa-nied by an increase in a-mannanase activity(257). A similar reutilization of the mannan frac-tion of yeast cell walls is observed in the courseof starvation of the cells (167). The contents ofglucose, xylose, and other saccharides in the cellwall gradually decrease also during prolongedspontaneous autolysis of the mycelium ofAsper-gillus terreus (145a). Capsular polysaccharidesproduced and released into the medium by some'yeasts exhibit in some cases striking structuralsimilarities with the cell wall components (205).Capsular material could either originate fromthe upper glycoprotein layer of the walls orrepresent surplus production of cell wall com-ponents.These examples point out that the cell wall of

fungi is by no means a stable, metabolically inertstructure serving merely as a protective shell forthe metabolically active protoplast. It is a "liv-ing" organelle whose functions may vary withenvironmental conditions and in the course ofcell and life cycles.

MORPHOGENETIC ASPECTSTopology of Wall Growth

From the accumulated knowledge on the proc-ess of cell wall construction, it becomes increas-ingly obvious that the wall's morphology de-pends not only on its chemical structure but alsolargely on the mode of its construction, i.e., onthe topology of wall growth.The idea that fungal hyphae grow by incor-

porating new cell wall material at the wall apexhas become one of the basic dogmas of fungalcytology (17, 212, 242, 253). The data supportingthis view originate from numerous studies basedon microscopy (97, 212), autoradiography (19,22, 89, 90, 100, 131), and fluorescent labeling (90,121, 173, 253), among other methods. On theother hand, a spherical shape is considered to bethe result of uniform layering of new cell wallmaterial over the entire cell surface (19, 22).Autoradiographic observations indicate that theellipsoidal shape of yeast cells is acquired byparticipation of both polarized growth and

spherical extension (33, 79, 251).Supposing that in principle the molecular

mechanisms involved in the process of cell wallformation are essentially the same or very simi-lar in various morphological types, a basic ques-tion arises about how these processes are regu-lated so that the characteristic shape is gener-ated.

Several hypotheses have been put forward inthe past to explain the mechanism of cell wallformation and morphogenesis in fungi. Johnson(125) suggested that the cell wall is elaboratedwith participation of both synthetic and lyticprocesses. According to his hypothesis, thepreexisting rigid cell wall structure is first at-tacked by specific cell wall-bound polysaccha-ride hydrolases, and the gaps formed are thenfilled with newly synthesized cell wall material.The continual repetition of this process wouldensure the growth of the cell wall without dis-turbing its overall integrity.The results from different laboratories have

shown that, indeed there exists a delicate bal-ance between the synthetic and lytic processesin the cell wall (21). Whenever this balance isdisturbed, morphological aberrations or lysis orboth occur. For example, inhibition of chitinsynthesis by polyoxin D causes bulging and lysisof hyphal cells of M. rouxii (20). Similarly, in-hibition of wall glucan synthesis in yeast by 2-deoxyglucose or 2-deoxy-2-fluoroglucose leads tomorphological changes and often to lysis of cells(32, 34, 35, 125, 239). Inhibition of chitin synthe-sis and stimulation of cell wall hydrolases byincreased temperature causes distortions andlysis of cell walls in hyphae ofA. nidulans (132).On the other hand, inhibition of wall-plasticizingglucanases by 8-gluconolactone causes cessationof wall growth in auxin-induced yeast cells (237).The morphogenic effects of polyoxin D and thedeoxyanalogs of glucose in fungal cells resemblethe action of penicillin on the cell wall formationand morphogenesis in bacteria (e.g., 228).To explain the morphological development of

fungal cell walls, Bartnicki-Garcia (17) postu-lated the so-called unitary model of wall growth.In his model cell wall growth is considered as aresult of cumulative action of minute hypothet-ical units of wall growth at the cell surface. Theunits of wall growth are supposed to containboth lytic and synthetic enzymes involved in theformation of wall fabric. Accordingly, cell mor-phology is determined by the distribution pat-tern of the hypothetical growth units. Thus,apical growth would be the result of concentra-tion of growth units at the hyphal tip, whereasspherical morphology would result from uniformdistribution of growth units over the whole cellsurface. Although plausible, the above hypoth-

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


132 FARKASM

esis lacks an explanation of how the distributionpattern of postulated growth units is determinedand maintained and how their activity is regu-lated.From the observation that the yeast form of

Paracoccidioides brasiliensis contains a-1,3-glucan as the main cell wall component, whereasthe mycelial wall of the same species is com-posed predominantly of ,B-1,3-glucan (56), Ka-netsuna et al. (128) proposed a hypothesis ac-cording to which the temperature-inducedyeast-to-mycelial reversion is due to local acti-vation of f,-glucan synthesis at the expense ofa-glucan formation.

If we are to find a more realistic answer to theproblem of regulation of morphological devel-opment, we must consider the characteristicsand regulatory properties of the enzymes in-volved in cell wall construction, as well as allfactors that could influence this process in livingcells. A good starting point in this directionmight be the already described hypothesis ofCabib and co-workers (46, 50, 51) explaining theformation of yeast primary septum by tempo-rally and spatially localized activation of plas-malemma-bound chitin synthase. Although orig-inally intended to explain only the special caseof septum formation, the concept of regulationof morphogenetic events through changes in theactivities of preexisting polysaccharide syn-thases could find a more general application.The latter possibility seems to be supported bynumerous, often unaimed observations madewith fungi and fungal protoplasts, as can be seenfrom the following examples.Removal of the fungal cell wall by enzymic or

other suitable treatment liberates protoplaststhat under certain conditions are capable toregenerate their cell walls. The polysaccharidesynthases are set into operation, and usuallyafter a short period of time a network of poly-saccharide microfibrils is formed on the proto-plast surface (e.g., 198, 265). In all cases, how-ever, the formation ofnew a cell wall around theportoplast is isodiametric, i.e., the shape of theregenerated cell is independent from the mor-phology of the parent cell type (e.g. 88, 94, 198,265); A. Svoboda and 0. Necas, Proc. Int. Symp.Yeasts 4th, Vienna, Austria, 1974, D13, p. 215).The shape of the regenerated protoplast isrounded or irregular, depending on the proper-ties of the surrounding medium, and the thick-ness of the newly formed wall is uniform overthe whole protoplast surface. It is usually onlyafter several generations that regenerated cellsregain their original characteristic morphology(Fig. 8).Experimental evidence shows that the for-

mation of skeletal, microfibrillar components ofthe wall on the protoplast surface is to a greatdegree independent from protein synthesis (64,198, 199). In some cases it was observed thatblocking protein synthesis in regenerating pro-toplasts (64, 256) or intact cells (74) even stim-ulates to some extent the formation of skeletalwall polysaccharides. The diminished presenceof glycoprotein wall components at the surfaceof the protoplast of Candida utilis regeneratingin liquid medium is accompanied by an about 20times higher accumulation of chitin in the regen-erated walls in comparison with the chitin con-tent ofnormal walls (95). An increased formationof chitin wall component was observed also inprotoplasts of S. cerevisiae regenerating in aliquid medium containing [14C]glucose as thesole carbon source (V. Farkas and A. Svoboda,unpublished data).Another example of the independence of skel-

etal wall polysaccharide formation from proteinsynthesis is provided by the encystment of zoo-spores of Blastocladiella emersonii, Phyto-phthora palmivora, and others. In these casesalso, blocking of protein synthesis by cyclohexi-mide does not prevent the formation of a chitin-ous sheath during the encystment of zoospores(166, 230, 244).A conclusion may be drawn from these obser-

vations that the enzymes responsible for skeletalwall polysaccharide synthesis are synthesizedconstitutively and that they are relatively stableagainst turnover. The absence of protein synthe-sis does not inhibit their function; on the con-trary, in some cases the absence of protein syn-thesis even stimulates their function. At thispoint it is tempting to speculate that this prop-erty of fungal cell wall-synthesizing enzymesmay be of vital importance for survival in manyfungi; it enables them to complete the life cycleand undergo sporulation even under unfavorableconditions, such as the limitation of cell metab-olism by lack of nutrients or other negativecircumstances.

Furthermore, the above results seem to indi-cate that it is essentially the presence of the cellwall or contact with some extracytoplasmic com-ponents that hinders the expression of crypticpolysaccharide synthases in the plasmalemma.

Conceivably, loosening the contact betweenthe cell wall and plasmalemma could bring aboutthe activation of polysaccharide synthesis. Dis-tortion ofthe contact between the plasmalemmaand cell wall can be achieved not only by com-plete removal of the wall, as is the case withprotoplasts, but also in the intact cells by purelyphysical means, such as osmotic shock or me-chanical vibration. For example, rapid transfer

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from


BIOSYNTHESIS OF CELL WALLS OF FUNGI

FIG. 8. Regeneration and reversion ofprotoplasts oftheyeast Trigonopsis variabilis after 40 h ofincubationin osmotically stabilized growth medium containing 30% gelatin. (Courtesy ofA. Svoboda.) Bar represents 10

of cells from diluted buffer into medium withhigh osmolarity causes plasmolysis in A. nigerwhereby the normal apical pattern of wallgrowth is disturbed and formation of abnormalnumbers ofbranchings and septa is induced (131,246). Repeated changes in the osmolarity of thegrowth medium induces the formation of multi-ple layers of wall material on the surface ofregenerating yeast protoplasts (A. Svoboda, un-published data).

Autoradiography of hyphal cell walls isolatedby mechanical disintegration of M. rouxii cellsincubated with UDP-[3H]GlcNAc revealed thatin a great number of cases the apical pattern oflabeling was lost, possibly as a consequence ofthe mechanical treatment of cells before auto-radiography (unpublished data of J. Mc-Murrough, A. Flores-Carre6n, and S. Bartnicki-Garcia, mentioned in reference 17).To a special category belong the various mor-

phogenetic effects induced in fungi by certainantibiotics. Such changes as curling, bulging,abnormal spherical growth, frequent branching,and increase in wall thickness have been ob-served when different antibiotics have been ap-plied to growing fungi (i.e. 11-13, 20, 26, 29, 130,131, 252). In most cases the mechanisms of ac-tion of different morphogenetic antibiotics onthe processes of cell wall formation in fungi havenot been studied in detail, if at all. Nevertheless,from comparison of the data it becomes clearthat inhibition of protein (27, 73, 74, 131, 246,256) or glycoprotein (130, 142) synthesis usually

has as a consequence increased synthesis of skel-etal wall polysaccharides, very often accompa-nied by a change in wall morphology.

In these cases it is possible to obtain delocal-ized synthesis of structural wall polysaccharides,apparently without disturbing the contact be-tween the cell wall and the plasmalemma. Hereit can be hypothesized that direct or indirectinhibition of protein synthesis may lead to de-pletion of some metabolically unstable protein-aceous extracytoplasmic components inhibitingthe activity of cryptic polysaccharide synthasesin the plasmalemma. A similar explanation maybe offered for the delocalized synthesis of chitinobserved at nonpermissive temperature in atemperature-sensitive mutant of S. cerevisiae(240) as well as for the observation that yeastcells in which deoxyribonucleic acid and proteinsyntheses were inhibited by inositol deficiencycontinued the synthesis of wall glucan, resultingin the formation of abnormally thick cell walls(66).

Speculations About the PossibleRegulatory Mechanism

If we state that the cell wall determines theshape of the cell, we must bear in mind that thereverse is even more true: the cell determinesthe shape of the wall. The mechanism by whichthis control of the cell over the cellular mor-phology is executed represents one of the mostintriguing problems of modern biology.

133VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


134 FARKAS

The experimental data summarized in the pre-ceding pages strongly indicate that the key rolein the regulation of fungal morphogenesis isplayed by the activation or inactivation or bothof polysaccharide synthases located at the plas-malemma. Whereas the activation proceeds inthe intact cells apparently through enzymaticdisturbance of the contact between the plasma-lemma and the constituents of the cell wall orperiplasm, the inactivation must involve the re-verse process, i.e., the combination of plasma-lemma-bound polysaccharide synthases withspecific proteinaceous inhibitors present in theperiplasm or the cell wall. The proteinaceousnature of these postulated inhibitors is judgedmainly from the fact that blocking protein (orglycoprotein) synthesis leads to activation ofcryptic polysaccharide synthases and delocalizedsynthesis of wall. Although there is, so far, noexperimental evidence for the existence of suchinhibitors, their presence is clearly missing inthe present schemes of regulation of cell wallpolysaccharide synthesis (viz., Fig. 4). An inhib-itory protein acting directly on active chitin syn-thase was isolated from the cytoplasm of myce-Hal (182) as well as yeast (164) cells ofM. rouxii.How the activation-inactivation processes couldactually participate in the regulation of cell wallgrowth is the subject of the following specula-tions.The morphological development of the fungal

cell begins by the activation of cell wall synthesisat some site on the preexisting cell wall. It canbe assumed that the outgrowth of the new cellwall is preceded by the concentrated action ofcell wall polysaccharide hydrolases at the givensite. The localized dissolution of the wall poly-mers causes a loosening of the contact betweenthe cell wall and plasmalemma and, in turn,activates the cryptic polysaccharide synthaseslocated in plasmalemma at the given site. Thissequence of events would create on the cellsurface a region ofactive well growth, henceforthcalled the "primary growth region" (Fig. 9). Asa result of this activity, a tiny bud is formed onthe surface of the preexisting wall.Cytoplasmic vesicles containing enzymes, in-

hibitors, plasmalemma precursors, and wall ma-trix material move to the primary growth region,fuse with the plasmalemma, and discharge theircontents into the periplasmic space. The poly-saccharide synthases might be incorporated intoplasmalemma in the fully active state, or theymay be stabilized and become active only inassociation with lipophilic components of theplasmalemma. Such a mode ofactivation is com-mon with some plasmalemma-bound enzymes(70, 137).

MICROBIOL. REV.

Cell wall

ZZZZZZ _,i-~,'.i4z[Z1KL.1f} PlosmalemmaVesicles

Amorphous component //, Microfibrils

, Wall-lysing enzymes

oo Activeo Polysacchoride synthoses:- InactiveFIG. 9. Diagram illustrating formation of a

primary growth region in a preexisting cell wall.

The highest frequency of fusion of carriervesicles with the plasmalemma would be at thebud apex (e.g., 17, 253). If the polysaccharidesynthases coming to the newly formed apex getinto contact with the postulated extracyto-plasmic inhibitors and become rapidly inacti-vated, a gradient of polysaccharide synthase ac-tivity is formed on the surface of the bud. Thehighest synthesizing activity would be at theapex, where new active molecules of polysaccha-ride synthases are supplied. Consequently, thehemispherical bud would turn into a hypha (Fig.10A).

If, on the other hand, the concentration ofinhibitors or the rate of inactivation of polysac-charide synthases at the apex is low, all polysac-charide synthases incorporated into plasma-lemma remain active, resulting in sphericalgrowth of the bud (Fig. 10B). It can be imaginedthat any intermediate rates of inactivation ofnascent polysaccharide synthases would resultin the formation of an ellipsoid.

It is highly probable that the distribution pat-tern of active polysaccharide synthases at theplasmalemma is reflected also in the rate of cellwall growth. The latter term is meant here asthe number of precursor molecules incorporatedinto the whole cell wall in a unit of time. Inhyphal walls, where rapid inactivation of poly-saccharide synthases causes restriction of thegrowth zone to the wall apex, the constant rateof cell wall growth is maintained practically overthe whole growth period (253).On the other hand, in spherical or nearly

spherical cells, where the number of active pol-ysaccharide synthases in the plasmalemma con-stantly increases, the rate of cell wall growth canbe expected to increase proportionally to theincrease in plasmalemma area for most of thecell cycle. Data supporting this assumption canbe deduced from results obtained with synchro-nously growing yeasts (30, 110, 112, 238).The regulatory mechanism suggested here


http://mm

br.asm.org/

Dow

nloaded from



I

I

A

h1ALXFIG. 10. Hypothetical distribution of active (0)

and inactive (0) molecules of polysaccharide syn-thases in theplasmalemma ofhyphae (A) and spher-ical cells (B). v, Cytoplasmatic vesicles carrying en-

zymes and building material to theplasmalemma; p,plasmalemma; cw, cell wall.

represents an attempt to explain morphologicaldevelopment in fungi in terms of molecular in-teractions. It is limited in many respects andlacks direct experimental support. The restric-tion of the problem of fungal morphogenesis tothe level of the cell wall necessarily brings manysimplifications into our speculations and the ex-perimental approach. It should be kept in mindalways that formation of the cell wall is not anisolated process but is the result of metabolicactivity of the whole cell.

SUMMARY AND CONCLUSIONS

The fungal cell wall is a complex structurecomposed mainly of polysaccharides and theirchemical complexes with proteins. The crystal-line polysaccharides, chitin and fl-glucans, con-stitute the skeletal portion of the wall, whereasthe amorphous polysaccharides and protein-polysaccharides are components of the wall ma-trixThe biosynthesis of each of these components

proceeds in a different way. The protein-poly-saccharide complexes are polymerized fromtheir activated precursors by enzymes located inthe rough and smooth endoplasmic reticula. Thepolymerized products are packed into vesiclesderived from membranes of the endoplasmicreticulum and conveyed to sites on the plasma-lemma adjacent to the growing regions of thewall.

Biosynthesis ofskeletal polysaccharides is cat-alyzed by constitutively formed polysaccharidesynthases uniformly distributed in the plasma-lemma. The unique property of these enzymesis that they can exist in an active or in tempo-rarily inactive, zymogen, state. Only the portionof polysaccharide synthases located at thegrowth zone is active during wall growth. Inhyphal cells the growth of the wall is restrictedto the apical region of the wall, whereas in

spherical cells new cell wall material is beingincorporated over the whole surface of the wall.The activation-inactivation process with poly-saccharide synthases seems reversible, and it isassumed that it represents the principal mech-anism by which the fungal morphogenesis isregulated.

In this review I have tried to summarizebriefly our present knowledge on the extremelycomplicated process of cell wall formation infungi. In spite of the great progress that hasbeen made in recent years in understanding themechanism of cell wall formation in fungi, nu-merous detailed questions have to be answeredbefore we can compose a more precise picture ofthe whole process. Future research will undoubt-edly bring, among other solutions, the answersto such questions as: (i) the mechanisms of bio-synthesis of various cell wall polymers and theparticipation of glycosylated phospholipids asintermediates in individual transglycosylic re-actions, (ii) the participation of cellular organ-elles in the biosynthesis of individual cell wallpolymers, (iii) the mechanism of transport ofprefabricated wall polymers from the site offormation to the cell exterior, (iv) the polymer-ization of subunits in the wall and formation ofwall fabric, and (v) the regulation of the bio-syntheses of individual cell wall componentswith relation to morphological development andcell and life cycles.To achieve these goals would necessarily re-

quire the exploitation of all the existing meth-odological potential of the modem biologicalsciences. Not only is the effort expended in thisresearch likely to be rewarding in this specialfield of microbiology, but the knowledge ob-tained can contribute to a better understandingof related processes in types of cells other thanfungi.

ACKNOWLEDGMENTSI thank A. Svoboda for many useful discussions, W.

Tanner for the communication of previously unpub-lished results, S. Bartnicki-Garcia and A. Svoboda fororiginal photographic material, and A. P. J. Trinci forpermission to reproduce one figure from his paper. Apart of this work was written during my stay at theDepartment of Microbiology, Attila J6zsef University,Szeged (Hungary), and I thank L. Ferenczy, head ofthat Department, for providing ideal conditions formy work and giving me access to his private collectionof reprints and scientific literature. Last but not least,I thank my wife for her help and encouragementthroughout the work on this review.

LITERATURE CITED1. Algranati, I. D., H. Carminatti, and E. Cabib.

1963. The enzymic synthesis of yeast mannan.

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


136 FARKAS

Blochem. Biophys. Res. Commun. 12:504-509.2. Ann6, J., H. Eyssen, and P. De Somer. 1974.

Formation and regeneration of Penicilliumchrysogenum protoplasts. Arch. Microbiol. 98:159-166.

3. Archer, D. B. 1977. Chitin biosynthesis in pro-toplasts and subcellular fractions of Aspergil-lus fumigatus. Biochem. J. 164:653-658.

4. Arnold, W. N. 1972. The structure of the yeastcell wall. Solubilization of a marker enzyme ,B-fructofuranosidase, by the autolytic enzymesystem. J. Biol. Chem. 247:1161-1169.

5. Aronson, J. M. 1965. The cell wall, p. 49-76. InG. C. Ainsworth and A. D. Sussman (ed.), Thefungi, vol. 1. Academic Press Inc., New Yorkand London.

6. Babezinski, P., and W. Tanner. 1973. Involve-ment of dolichol monophosphate in the for-mation of specific mannosyl linkages in yeastglycoproteins. Biochem. Biophys. Res. Com-mun. 54:1119-1124.

7. Bacon, J. S. D., V. C. Farmer, D. Jones, andL. F. Taylor. 1969. The glucan components ofthe cell wall of baker's yeast (Saccharomycescerevisiae) considered in relation to its ultra-structure. Biochem. J. 114:557-567.

8. Baint, S., V. Farkas, and S. Bauer. 1976.Biosynthesis of ,B-glucans catalyzed by a par-ticulate enzyme preparation from yeast. FEBSLett. 64:44-47.

9. Ballou, C. E. 1976. Structure and biosynthesis ofthe mannan component of the yeast cell enve-lope. Adv. Microb. Physiol. 14:93-158.

10. Ballou, C. E., and W. C. Raschke. 1974. Poly-morphism of the somatic antigen of yeast. Sci-ence 184:127-134.

11. Barathova, H., and V. Betina. 1976. Antibiotic-induced changes of mycelial growth of Botrytiscinerea. Folia Microbiol. (Prague) 21:355-361.

12. Barathova, H., V. Betina, Z. Barath, and P.Nemec. 1975. Morphogenic effects of ramihy-phin A in filamentous fungi. Folia Microbiol.(Prague) 20:97-102.

13. Barathova, H., V. Betina, and P. Nemec. 1969.Morphological changes induced in fungi by an-tibiotics. Folia Microbiol. (Prague) 14:475-483.

14. Barras, D. R. 1972. A,-glucan endohydrolasefrom Schizosaccharomyces pombe and its rolein cell wall growth. Antonie van LeeuwenhoekJ. Microbiol. Serol. 38:65-80.

15. Bartnicki-Garcia, S. 1968. Cell wall chemistry,morphogenesis and tsxonomy of fungi. Annu.Rev. Microbiol. 22:87-108.

16. Bartnicki-Garcia, S. 1970. Cell wall composi-tion and other biochemical markers in fungalphylogeny, p. 81-103 In J. B. Hasborne (ed.),Phytochemical phylogeny. Academic PressInc., New York and London.

17. Bartnicki-Garcia, S. 1973. Fundamental as-pects of hyphal morphogenesis. Symp. Soc.Gen. Microbiol. 23:245-267.

18. Bartnicki-Garcia, S., C. E. Bracker, E. Reyes,and J. Ruiz-Herrera. 1978. Isolation of chi-tosomes from taxonomically diverse fungi and

synthesis of chitin microfibrils in vitro. Exp.Mycol. 2:173-192.

19. Bartnicki-Garcia, S., and E. Lippman. 1969.Fungal morphogenesis: cell wall constructionin Mucor rouxii. Science 165:302-304.

20. Bartnicki-Garcia, S., and E. Lippman. 1972.Inhibition of Mucor rouxii by polyoxin D: ef-fects on chitin synthetase and morphologicaldevelopment. J. Gen. Microbiol. 71:301-309.

21. Bartnicki-Garcia, S., and E. Lippman. 1972.The bursting tendency of hyphal tips of fungi:presumptive evidence for a delicate balancebetween wall synthesis and wall lysis in apicalgrowth. J. Gen. Microbiol. 73:487-500.

22. Bartnicki-Garcia, S., and E. Lippman. 1977.Polarization of cell wall synthesis during sporegermination of Mucor rouxii. Exp. Mycol. 1:230-240.

23. Bartnicki-Garcia, S., and I. McMurrough.1971. Biochemistry of morphogenesis in yeast,p. 441491. In A. H. Rose and J. S. Harrison(ed.) The yeasts, vol. 2. Physiology and bio-chemistry of yeasts. Academic Press Inc., NewYork and London.

24. Behrens, N. H., and E. Cabib. 1968. The bio-synthesis of mannan in Saccharomyces cere-visiae. J. Biol. Chem. 243:502-509.

25. Behrens, N. H., H. Carminatti, R. J. Stane-loni, L. F. Leloir, and A. I. Cantareila. 1973.Formation of lipid-bound oligosaccharides con-taining mannose. Their role in glycoproteinsynthesis. Proc. Natl. Acad. Sci. U.S.A. 70:3390-3394.

26. Bent, K. J., and R. H. Moore. 1966. The modeof action of griseofulvin. Symp. Soc. Gen. Mi-crobiol. 16:82-110.

27. Berthe, M. C., N. Ansary, and R. Bonaly.1977. Effect of cycloheximide on the chemicalcomposition of the cell walls of two strains ofRhodotorula. Mycopathologia 62:167-172.

28. Beteta, P., and S. Gasc6n. 1971. Localization ofinvertase in yeast vacuoles. FEBS Lett. 13:297-300.

29. Betina, V., and D. Micekova. 1973. Morpho-genetic activity of cytochalasins, cyanein andmonorden in Botrytis cinerea. Z. Allg. Mikro-biol. 13:287-298.

30. Biely, P. 1978. Changes in the rate of synthesisof wall polysaccharides during the cell cycle ofyeast. Arch. Microbiol. 119:213-214.

31. Biely, P., and S. Bauer. 1968. The fornation ofguanosine diphosphate 2-deoxy-D-glucose inyeast. Biochim. Biophys. Acta 156:432-434.

32. Biely, P., V. Farkas, and Z. Kratky. 1974.Some aspects of wall formation in Schizosac-charomyces pombe. A study with 2-deoxyglu-cose. Biologia (Bratislava) 29:919-925.

33. Biely, P., J. Kovahik, and S. Bauer. 1973. Cellwall formation in yeast. An electron micro-scopic autoradiographic study. Arch. Mikro-biol. 94:365-371.

34. Biely, P., J. Kovaiik, and S. Bauer. 1973. Lysisof Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



glucan synthesis. J. Bacteriol. 115:1108-1120.35. Biely, P., Z. Kritky, J. Kovahik, and S.

Bauer. 1971. Effect of 2-deoxyglucose on cellwall formation in Saccharomyces cerevisiaeand its relation to cell growth inhibition. J.Bacteriol. 107:121-129.

36. Boothbay, D., L Daneo-Moore, M. L. Hig-gins, J. Coyette, and G. D. Shockman. 1973.Turnover of bacterial peptidoglycans. J. Biol.Chem. 248:2161-2169.

37. Bowden, J. K., and B. Hodgson. 1970. Evi-dence against the presence of fibres or chemi-cally distinct layers in the cell wall of Saccha-romyces. Antonie van Leeuwenhoek J. Micro-biol. Serol. 36:81-108.

38. Bracker, C. E., J. Ruiz-Hererra, and S. Bart-nicki-Garcia. 1976. Structure and transfor-mation of chitin synthetase particles (chito-somes) during microfibril synthesis in vitro.Proc. Natl. Acad. Sci. U.S.A. 73:4570-4574.

39. Braun, P. C., and R. A. Calderone. 1978. Chitinsynthesis in Candida albicans. comparison ofyeast and hyphal forms. J. Bacteriol. 133:1472-1477.

40. Bretthauer, R. K., and G. C. Tsay. 1974. En-zymatic transfer of mannose from guanosinediphosphate mannose to yeast mannan-proteincomplexes. Arch. Biochem. Biophys. 164:118-126.

41. Bretthauer, R. K., C. P. Kozak, and W. E.Irwin. 1969. Phosphate and mannose transferfrom guanosine diphosphate mannose to yeastmannan acceptors. Biochem. Biophys. Res.Commun. 37:820-827.

42. Bretthauer, R. K., and S. Wu. 1975. Synthesisof the mannosyl-O-serine (threonine) linkageof glycoproteins from polyisoprenyl phosphatemannose in yeast (Hansenula holstii). Arch.Biochem. Biophys. 167:151-160.

43. Bretthauer, R. K., S. Wu, and W. E. Irwin.1973. Enzymatic transfer ofmannose from gua-nosine diphosphate mannose to dolichol phos-phate in yeast (Hansenula holstii). A possiblestep in mannan biosynthesis. Biochim. Bio-phys. Acta 304:736-747.

44. Brown, J. P. 1971. Susceptibility of the cell wallsof some yeasts to lysis by enzymes of Helixpomatia. Can. J. Microbiol. 17:205-208.

45. Byers, B., and L Goetach. 1975. Behavior ofspindles and spindle plaques in the cell cycleand conjugation of Saccharomyces cerevisiae.J. Bacteriol. 124:511-523.

46. Cabib, E. 1975. Molecular aspects of yeast mor-phogenesis. Annu. Rev. Microbiol. 29:191-214.

47. Cabib, E. 1976. The yeast primary septum: ajourney into three-dimensional biochemistry.Trends Biochem. Sci. 1:275-277.

48. Cabib, E., and B. Bowers. 1971. Chitin andyeast budding. Localization of chitin in yeastbud scars. J. Biol. Chem. 246:152-159.

49. Cabib, E., and B. Bowers. 1975. Timing andfunction of chitin synthesis in yeast. J. Bacte-riol. 124:1586-1593.

50. Cabib, E., and V. Farkas. 1971. The control of

morphogenesis: an enzymatic mechanism forthe initiation of septum formation in yeast.Proc. Natl. Acad. Sci. U.S.A. 68:2052-2056.

51. Cabib, E., V. Farkla, R. E. Ulane, and B.Bowers. 1973. Yeast septum formation as amodel system for morphogenesis, p. 105-116.In J. R. Villanueva, I. Garcia-Acha, S. Gasc6n,and F. Uruburu (ed.) Yeast, mould and plantprotoplasts. Academic Press Inc., New Yorkand London.

52. Cabib, E., and F. A. Keller. 1971. Chitin andyeast budding. Allosteric inhibition of chitinsynthetase by a heat-stable protein from yeast.J. Biol. Chem. 246:167-173.

53. Cabib, E., R. Ulane, and B. Bowers. 1973.Yeast chitin synthetase. Separation of the zy-mogen from its activating factor and recoveryof the latter in the vacuole fraction. J. Biol.Chem. 248:1451-1458.

54. Cabib, E., and R E. Ulane. 1973. Chitin syn-thetase activating factor from yeast, a protease.Biochem. Biophys. Res. Commun. 50:186-191.

55. Camargo, E. P., C. P. Dietrich, D. Sonnen-born, and J. L Strominger. 1971. Biosyn-thesis of chitin in spores and growing cells ofBlastocladiella emersonii. J. Biol. Chem. 242:3121-3128.

56. Carbonell, L. M., and J. Rodriguez. 1968. My-celial phase of Paracoccidioides brasiliensisand Blastomyces dermatitidis: an electron mi-croscope study. J. Bacteriol. 96:533-543.

57. Cartledge, T. G., and D. X. Uoyd. 1972. Sub-cellular fractionation of particles containingacid hydrolases from Saccharomyces carlsber-gensis. Biochem. J. 126:755-757.

58. Cassone, A. 1973. Improved visualization of wallultrastructure in Saccharomyces cerevisiae.Experientia 29:1303-1305.

59. Chen, W. W., and W. J. Lennarz. 1977. Metab-olism of lipid-linked N-acetylglucosamine in-termediates. J. Biol. Chem. 252:3473-3479.

60. Corina, D. L., and K. A. Munday. 1971. Themetabolic stability of carbohydrates in thewalls of hyphae of Aspergillus clavatus. J.Gen. Microbiol. 65:253-257.

61. Cortat, M., P. Matile, and F. Kopp. 1973. In-tracellular localization of mannan synthetaseactivity in budding baker's yeast. Biochem.Biophys. Res. Commun. 53:482-489.

62. Cortat, M., P. Matile, and A. Wiemken. 1972.Isolation of glucanase-containing vesicles frombudding yeast. Arch. Microbiol. 82:189-205.

63. De Rousset-Hall, A., and G. W. Gooday. 1975.A kinetic study of solubilized chitin synthetasepreparation from Coprinus cinereus. J. Gen.Microbiol. 89:146-154.

64. De Vries, 0. M. H., and J. G. H. Wessels. 1975.Chemical analysis of cel waU regeneration andreversion of protoplasts from Schizophyllumcommune. Arch. Microbiol. 102:209-218.

65. Djaczenko, W., and A. Cassone. 1972. Visual-ization of new ultrastructural components inthe cell waU of Candida albicans with fixativescontaining TAPO. J. Cell Biol. 52:186-190.

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


138 FARKAS

66. Dominguez, A., J. R. Villanueva, and R. Sen-tandreu. 1978. Innositol deficiency in Saccha-romyces cerevisiae NCYC 86. Antonie vanLeeuwenhoek J. Microbiol. Serol. 44:25-34.

67. Dominguez, A., J. R. Villanueva, and R. Sen-tandreu. 1978. Biosynthesis ofSaccharomycescerevisiae glycoproteins: nature ofsome partic-ipating glycolipids. Antonie van LeeuwenhoekJ. Microbiol. Serol. 44:183-192.

68. Dube, J., G. Setterfield, G. Kiss, and C. V.Lusena. 1973. Fate of plasma membrane ofSaccharomyces cerevisiae during cell rupture.Can. J. Microbiol. 19:285-290.

69. Duran, A., B. Bowers, and E. Cabib. 1975.Chitin synthetase zymogen is attached to yeastplasma membrane. Proc. Natl. Acad. Sci.U.S.A. 72:3952-3955.

70. Duran, A., and E. Cabib. 1978. Solubilizationand partial purification of yeast chitin syn-thase. Confirmation of the zymogenic nature ofthe enzyme. J. Biol. Chem. 253:4419-4425.

71. Eddy, A. A., and J. S. Woodhead. 1968. Analkali-soluble glucan fraction from the cellwalls of the yeast Saccharomyces carlsbergen-sis. FEBS Lett. 1:67-68.

72. Eiserling, F. A., and R. C. Dickson. 1972.Assembly of viruses. Annu. Rev. Biochem. 41:467-502.

73. Elorza, M. V., C. M. Lostau, J. R. Villaneuva,and R. Sentandreu. 1976. Cell wall synthesisregulation in Saccharomyces cerevisiae. Effectof RNA and protein inhibition. Biochim. Bio-phys. Acta. 464:263-272.

74. Elorza, M. V., and R. Sentandreu. 1969. Effectof cycloheximide on yeast cell wall synthesis.Biochem. Biophys. Res. Commun. 36:741-747.

75. Elorza, M. V., and R. Sentandreu. 1973. Theeffect of cycloheximide on mannosyl transfer-ase activity of a membrane preparation fromSaccharomyces cerevisiae, p. 205-209. In J. R.Villanueva, I. Garcia-Acha, S. Gasc6n, and F.Uruburu (ed.), Yeast, mould and plant proto-plasts. Academic Press Inc., New York andLondon.

76. Endo, A., K. Kakiki, and T. Misato. 1970.Mechanism of action of the antifungal agentpolyoxin D. J. Bacteriol. 104:189-196.

77. Endo, A., and T. Misato. 1969. Polyoxin D, acompetitive inhibitor of UDP-N-acetylgluco-samine:chitin N-acetyl-glucosaminyltransfer-ase in Neurospora crassa. Biochem. Biophys.Res. Commun. 37:718-722.

78. Eylar, E. H. 1966. On the biological role ofglycoproteins. J. Theor. Biol. 10:89-113.

79. Farkas, V., J. Kovarik, A. Kosinova, and S.Bauer. 1974. Autoradiographic study of man-nan incorporation into the growing cell walls ofSaccharomyces cerevisiae. J. Bacteriol. 117:265-269.

80. Farkas, V., A. Svoboda, and S. Bauer. 1970.Secretion of cell wall glycoproteins by yeastprotoplasts. The effect of 2-deoxy-D-glucoseand cycloheximide. Biochem. J. 118:755-758.

81. FarkaA, V., V. M. Vagabov, and S. Bauer.1976. Biosynthesis of yeast mannan. Diversity

of mannosyltransferases in the mannan-syn-thesizing enzyme system from yeast. Biochim.Biophys. Acta 428:573-582.

82. Farkas, V., V. M. Vagabov, and S. Bauer.1976. Biosynthesis of yeast mannan. Independ-ent formation of two carbohydrate moieties.Biochim. Biophys. Acta 428:583-590.

83. Fevre, M. 1977. Subcellular localization of glu-canase and cellulase in Saprolegnia monoicaPringsheim. J. Gen. Microbiol. 103:287-295.

84. Fevre, M., and C. Dumas. 1977. ,B-Glucan syn-thetases from Saprolegnia monoica. J. Gen.Microbiol. 103:297-306.

85. Fleet, G. H., and H. J. Phaff. 1974. Glucanasesin Schizosaccharomyces. Isolation and prop-erties of the cell wall associated ,B-(1-3)-glucan-ases. J. Biol. Chem. 249:1717-1928.

86. Fultz, S. A., and R. A. Wolf. 1972. Surfacestructure of Allomyces during germination andgrowth. Mycologia 64:212-218.

87. Furuya, A., and Y. Ikeda. 1966. Cell-wall lyticenzymes. II. Lytic enzyme of Streptomyces No62. J. Gen. Appl. Microbiol. 6:49-60.

88. Gabriel, M. 1968. Formation and regeneration ofprotoplasts in the mould Rhizopus nigricans.Folia Microbiol. (Prague) 13:231-235.

89. Galun, E. 1972. Morphogenesis of Trichoderna:autoradiography of intact colonies labelled by(3H)N-acetylglucosamine as a marker of newcell wall biosynthesis. Arch. Mikrobiol. 86:305-314.

90. Galun, M., A. Braun, A. Frensdorff, and E.Galun. 1976. Hyphal walls of isolated lichenfungi. Autoradiographic localization of precur-sor incorporation and binding of fluorescein-conjugated lectins. Arch. Microbiol. 108:9-16.

91. Gander, J. E. 1974. Fungal cell wall glycopro-teins and peptido-polysaccharides. Annu. Rev.Microbiol. 28:103-119.

92. Gander, J. E., L. R. Drewes, F. Fang, and A.Lui. 1977. 5-O-fB-D-galactofuranosyl-contain-ing exocellular glycopeptide of Penicilliumcharlesii. Incorporation of mannose fromGDP-D-mannose into glycopeptide. J. Biol.Chem. 252:2187-2193.

93. Gander, J. E., and F. Fang. 1977. Properties ofPenicillium GDP-mannose:glycopeptide man-nosyl transferase solubilized with Triton X-100.J. Supramol. Struct. 6:579-589.

94. Garcia-Acha, I., F. L6pez-Belmonte, and J.R. Villanueva. 1966. Regeneration of mycelialprotoplasts of Fusarium culnorum. J. Gen.Microbiol. 46:515-523.

95. Garcia Mendoza, C., and M. Novaes Ledieu.1968. Chitin in the new wall of regeneratingprotoplasts of Candida utilis. Nature (London)220:1035.

96. Gibson, R. K., C. E. Buckley, and J. F. Pe-berdy. 1976. Wall ultrastructure in regenerat-ing protoplasts of Aspergillus nidulans. Pro-toplasma 89:381-387.

97. Girbardt, M. 1969. Die Ultrastraktur der Api-kalregion von Pilzhyphen. Protoplasma 67:413-441.

98. Glaser, L., and D. H. Brown. 1957. The syn-

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



thesis of chitin in cell free extracts of Neuro-spora crassa. J. Biol. Chem. 228:729-742.

99. Gold, M. H., and H. J. Hahn. 1976. Role of amannosyl lipid intermediate in the synthesis ofNeurospora crassa glycoproteins. Biochemis-try 15:1808-1813.

100. Gooday, G. W. 1971. An autoradiographic studyof hyphal growth of some fungi. J. Gen. Micro-biol. 67:125-133.

101. Gooday, G. W. 1977. Biosynthesis of fungal wall-mechanisxms and implications. J. Gen. Micro-biol. 99:1-11.

102. Gooday, G. W., and A. De Rousset-Hall. 1975.Properties of chitin synthetase from Coprinuscinereus. J. Gen. Microbiol. 89:137-145.

103. Gooday, G. W., A. de Rousset-Hall, and D.Hunsley. 1976. Effect of polyoxin D on chitinsynthesis in Coprinus cinereus. Trans. Br. My-col. Soc. 67:193-200.

104. Grove, S. N., C. E. Bracker, and D. J. Morre.1970. An ultrastructural basis for hyphal tipgrowth in Pythium ultimum. Am. J. Botany.67:245-266.

105. Gunja-Smith, Z., N. B. Patil, and E. E. Smith.1977. Two pools of glycogen in Saccharomyces.J. Bacteriol. 130:818-825.

106. Gunja-Smith, Z., and E. E. Smith. 1974. Evi-dence for periplasmic location of glycogen inSaccharomyces. Biochem. Biophys. Res. Com-mun. 56:588-592.

107. Hasilik, A. 1974. Inactivation of chitin synthasein Saccharomyces cerevisiae. Arch. Microbiol.101:295-301.

108. Hasilik, A., and H. Holzer. 1973. Participationof the tryptophan synthase inactivating systemfrom yeast in the activation of chitin synthase.Biochem. Biophys. Res. Commun. 53:552-559.

109. Hata, T., R. Hayashi, and E. Doi. 1967. Puri-fication of yeast proteinases. I. Fractionationand some properties of the proteinases. Agric.Biol. Chem. 31:150-159.

110. Hayashibe, M., N. Abe, and M. Matsui. 1977.Mode of increase in cell wall polysaccharidesin synchronously growing Saccharomyces cer-evisiae. Arch. Microbiol. 114:91-92.

111. Hayashibe, M., and S. Katohda. 1973. Initia-tion of budding and chitin ring. J. Gen. Appl.Microbiol. 19:23-39.

112. Hayashibe, M., N. Sando, and S. Katohda.1972. Cell wall formation in growing Saccha-romyces cerevisiae, p. 811-817. In Proceedingsof the- Fourth International FermentationSymposium. Fermentation Technology Today.

113. Hills, G. J. 1973. Cell wall assembly in vitro fromChlamydomonas reinhardi. Planta 115:17-23.

114. Holley, R. A., and D. K. Kidby. 1973. Role ofvacuoles and vesicles in extracellular enzymesecretion from yeast. Can. J. Microbiol. 19:113-117.

115. Hori, M., K. Kakiki, and T. Misato. 1974. In-teraction between polyozin and active centerof chitin synthetase. Agric. Biol. Chem. 38:699-705.

116. Houwik, A. L, and D. KL Kreger. 1953. Thecell wall ofyeasts-electron microscope and X-

ray diffraction study. Antonie van Leeuwen-hoek J. Microbiol. Serol. 19:1-24.

117. Hsu, A.-F., J. W. Baynes, and E. C. Heath.1974. The role of dolichol oligosaccharide as anintermediate in glycoprotein biosynthesis.Proc. Natl. Acad. Sci. U.S.A. 71:2391-2395.

118. Hunsley, D. 1973. Apical wall structure in hy-phae of Phytophthoraparasitica. New Phytol.72:985-990.

119. Hunsley, D., and J. H. Burnett. 1970. Theultrastructural architecture of the walls ofsome hyphal fungi. J. Gen. Microbiol. 62:203-218.

120. Hunsley, D., and G. W. Gooday. 1974. Thestructure and development of septa in Neuro-spora crassa. Protoplasma 82:125-146.

121. Hunsley, D., and D. Kay. 1976. Wall structureof Neurospora hyphal apex-immunofluores-cent localization of wall surface antigens. J.Gen. Microbiol. 95:233-248.

122. Isaac, S., N. S. Ryder, and J. F. Peberdy.1978. Distribution and activation of chitin syn-thase in protoplast fractions released duringthe lytic digestion of Aspergillus nidulans hy-phae. J. Gen. Microbiol. 105:45-50.

123. Jan, Y. N. 1974. Properties and cellular locali-zation of chitin synthetase in Phycomycesblakesleanus. J. Biol. Chem. 249:1973-1979.

124. Jaspers, H. T. A., K. Christiansen, and J.van Stevenick. 1975. An improved methodfor the preparation of yeast enzymes in situ.Biochem. Biophys. Res. Commun. 65:1434-1439.

125. Johnson, B. F. 1968. Lysis of yeast cell wallsinduced by 2-deoxyglucose at sites of glucansynthesis. J. Bacteriol. 95:1169-1172.

126. Jones, E. W. 1977. Proteinase mutants of Sac-charomyces cerevisiae. Genetics 85:23-33.

127. Jung, P., and W. Tanner. 1973. Identificationof the lipid intermediate in yeast mannan bio-synthesis. Eur. J. Biochem. 37:1-6.

128. Kanetsuna, F., L M. CarbonelL L. Azuma,and Y. Yamamura. 1972. Biochemical studieson the thermal dimorphism of Paracocci-dioides brasiliensis. J. Bacteriol. 110:208-218.

129. Karson, E., and C. E. Ballou. 1978. Biosyn-thesis of yeast mannan. Properties of a man-nosylphosphate transferase in Saccharomycescerevisiae. J. Biol. Chem. 253:6484-6492.

130. Katoh, Y., A. Kuninaka, H. Yoshino, A. Tak-atsuki, M. Yamasaki, and G. Tamura. 1976.Formation of fungal multinuclear giant cells bytunicamycin J. Gen. Appl. Microbiol. 22:247-258.

131. Katz, D., and R. F. Rosenberger. 1971. Hyphalwall synthesis in Aspergillus nidulans: effectof protein synthesis inhibition and osmoticshock on chitin insertion and morphogenesis.J. Bacteriol. 108:184-190.

132. Katz, D., and R. F. Rosenberger. 1971. Lysisof an Aspergillus nidulans mutant blocked inchitin synthesis and its relation to wall assem-bly and wall metabolism. Arch. Mikrobiol. 80:284-292.

133. Keller, F. A., and E. Cabib. 1971. Chitin and

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


140 FARKAS

yeast budding: properties of chitin synthetasefrom Saccharomyces carlsbergensis. J. Biol.Chem. 246:160-166.

134. Kidby, D. K., and R. Davies. 1970. Invertaseand disuiphide bridges in the yeast wall. J.Gen. Microbiol. 61:327-333.

135. KopeckAi, M., H. J. Phaff, and G. H. Fleet.1974. Demonstration of a fibrillar componentin the cell wall of the yeast Saccharomycescerevisiae and its chemical nature. J. Cell Biol.62:66-76.

136. Kosinova, A., V. Farkas, S. Machala, and S.Bauer. 1974. Site ofmannan synthesis in yeast.An autoradiographic study. Arch. Microbiol.99:255-263.

137. Kotyk, A., and K. Janacek. 1977. Membranetransport. An interdisciplinary approach, p.108-109. Plenum Publishing Corp., New York.

138. Kozak, L. P., and R K. Bretthauer. 1970.Studies on the biosynthesis of Hansenula hol-stii mannans from guanosine diphosphatemannose. Biochemistry 9:1115-1122.

139. KrAtky, Z., P. Biely, and S. Bauer. 1975. Mech-anism of 2-deoxy-D-glucose inhibition of cellwall polysaccharide and glycoprotein biosyn-thesis in Saccharomyces cerevisiae. Eur. J.Biochem. 54:459-467.

140. KrAtky, Z., P. Biely, and S. Bauer. 1975. Wallmannan of Saccharomyces cerevisiae. Meta-bolic stability and release into growth medium.Biochim. Biophys. Acta 404:1-6.

141. Kuo, S.-C., and J. 0. Lampen 1972. Inhibitionby 2-deoxy-D-glucose of synthesis of glycopro-tein enzymes by protoplasts ofSaccharomyces:relation to inhibition of sugar uptake and me-tabolism. J. Bacteriol. 111:419-429.

142. Kuo, S.-C., and J. 0. Lampen. 1974. Tunica-mycin-an inhibitor of yeast glycoprotein syn-thesis. Biochem. Biophys. Res. Commun. 58:287-295.

143. Kuo, S.-C., and J. 0. Lampen. 1976. Tunica-mycin inhibition of (3H)glucosamine incorpo-ration into yeast glycoproteins: binding of tun-icamycin and interaction with phospholipids.Arch. Biochem. Biophys. 172:574-581.

144. Kurland, C. G. 1972. Structure and function ofbacterial ribosome. Annu. Rev. Biochem. 41:377-408.

145. Kushner, D. J. 1969. Self-assembly of biologicalstructures. Bacteriol. Rev. 33:302-345.

145a.Lahoz, R. 1967. Quantitative changes in the con-tent of nonnitrogenous compounds during au-tolysis of Aspergillus terreus. J. Gen. Micro-biol. 46:451-456.

146. Lampen, J. 0. 1968. External enzymes of yeast:their nature and formation. Antonie van Leeu-wenhoek J. Microbiol. Serol. 34:1-18.

147. Larriba, G., M. V. Elorza, J. R. Villanueva,and R. Sentendreu. 1976. Participation ofdolichol phosphomannose in the glycosylationof yeast wall mannoproteins at the polysomallevel. FEBS Lett. 71:316-320.

148. Lehle, L., F. Bauer, and W. Tanner. 1977. Theformation of glycosidic bonds in yeast glyco-proteins. Intracellular localisation of the reac-

tions. Arch. Microbiol. 114:77-81.149. Lehle, L., F. Fartaczek, W. Tanner, and H.

Kauss. 1976. Formation of polyprenol-linkedmono- and oligosaccharides in Phasoleus au-reus. Arch. Biochem. Biophys. 175:419-426.

150. Lehle, L., and R T. Schwarz. 1976. Formationof dolichol monophosphate 2-deoxy-D-glucoseand its interference with the glycosylation ofmannoproteins in yeast. Eur. J. Biochem. 67:239-245.

151. Lehle, L, and W. Tanner. 1974. Membrane-bound mannosyl transferase in yeast glycopro-tein biosynthesis. Biochim. Biophys. Acta 350:225-235.

152. Lehle, L., and W. Tanner. 1975. Formation oflipid-bound oligosaccharides in yeast. Biochim.Biophys. Acta 399:364-374.

153. Lehle, L., and W. Tanner. 1976. The specificsite of tunicamycin inhibition in the formationof dolichol-bound N-acetylglucosanine deriv-atives. FEBS Lett. 71:167-170.

154. Lehle, L., and W. Tanner. 1978. Biosynthesisand characterization of large dolichylphos-phate-linked oligosaccharides in Saccharomy-ces cerevisiae. Biochim. Biophys. Acta 539:218-229.

155. Lehle, L., and W. Tanner. 1978. Glycosyl trans-fer from dolichyl-phosphate sugars to endoge-nous and exogenous glycoprotein acceptors inyeast. Eur. J. Biochem. 83:563-570.

156. Leloir, L. F. 1971. Two decades of research onthe biosynthesis of saccharides. Science 172:1299-1303.

157. Lennarz, W. J. 1975. Lipid linked sugars inglycoprotein synthesis. Science 188:986-991.

158. Letoublon, R. C. P., J. Combe, and R. Got.1973. Transfert du mannose, a partir de GDP-mannose a des accepteurs lipidiques chez As-pergiUus niger. Eur. J. Biochem. 40:95-101.

159. Letoublon, R., and R. Got. 1974. R6le d'unintermediaite dans le transfert du mannose ades accepteurs glycoproteiques endogenes chezAspergillu8 niger. FEBS Lett. 46:214-217.

160. Letoublon, R., M. Richard, P. Louisot, and R.Got. 1971. gtude du transfert enzymatique deD-('4C)mannose a partir de GDP-(4C) man-nose, sur un accepteur endogene dans les mi-crosomes d'Aspergillus niger. Eur. J. Biochem.18:194-200.

161. Linnemans, W. A. M., P. Boer, and P. F.Elbers. 1977. Localization of acid phosphatasein Saccharomyces cerevisiae. a clue to cell wallformation. J. Bacteriol. 131:638-644.

162. Liras, P., and S. Gascon. 1971. Biosynthesisand secretion of yeast invertase. Effect of cy-cloheximide and 2-deoxy-D-glucose. Eur. J.Biochem. 23:160-165.

163. LMpez-Romero, E., S. Bartnicki-Garcia, andJ. Ruiz-Herrera. 1978. Purification of inhibi-tory protein of chitin synthetase from Mucorrouxii. Biochim. Biophys. Acta 525:338-345.

164. Lopez-Romero, E., and J. Ruiz-Herrera.1976. Synthesis of chitin by particulate prepa-rations from Aspergillus flavus. Antonie vanLeeuwenhoek J. Microbiol. Serol. 42:261-276.

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from



165. Lopez-Romero, E., and J. Ruiz-Herrera.1977. Biosynthesis of 8-glucans by cell freeextracts from Saccharomyces cerevisiae.Biochim. Biophys. Acta. 500:372-384.

166. Lovett, J. S. 1968. Reactivation of ribonucleicacid and protein synthesis during germinationof Blastocladiella zoospores and the role ofthe ribosomal nuclear cap. J. Bacteriol. 96:962-969.

167. Lyons, T. P., and J. S. Hough. 1970. Role ofyeast cell walls in brewing. Brew. Dig. 45:52-60.

168. Mahadevan, P. R., and U. R. Mahadkar. 1970.Role of enzymes in growth and morphology ofNeurospora crassa: cell-wall-bound enzymesand their possible role in branching. J. Bacte-riol. 101:941-947.

169. Manners, D. J., and A. J. Masson. 1969. Thestructure of two glucans from yeast cell walls.FEBS Lett. 4:122-124.

170. Manners, D. J., A. J. Masson, and J. C. Pat-terson. 1973. The structure of a ,8-(1,3)-D-glu-can from yeast cell walls. Biochem. J. 135:19-30.

171. Manners, D. J., A. J. Masson, J. C. Patterson,H. Bjorndal, and B. Lindberg. 1973. Thestructure of a j8-(1,6)-D-glucan from yeast cellwalls. Biochem. J. 135:31-36.

172. Marchant, R. 1966. Wall structure and sporegermination in Fusarium culmorum. Ann. Bot.(London) 30:821-830.

173. Marchant, R., and D. G. Smith. 1968. A sero-logical investigation of hyphal growth in Fu-sarium cubnorum. Arch. Mikrobiol. 63:85-94.

174. Mariott, M. S. 1977. Mannan-protein locationand biosynthesis in plasma membranes fromthe yeast form of Candida albicans. J. Gen.Microbiol. 103:51-59.

175. MarshalL, R. D. 1972. Glycoproteins. Annu. Rev.Biochem. 41:673-702.

176. Matile, P., M. Cortat, A. Wiemken, and A.Frey-Wysling. 1971. Isolation of glucanase-containing particles from budding Saccharo-myces cerevisiae. Proc. Natl. Acad. Sci. U.S.A.68:636-40.

177. Matsumae, A., and E. C. Cantino. 1971. Sen-sitivity of the spores of Blastocladiella emer-sonii and related fungi to antibiotics and someother drugs. J. Antibiot. 24:77-84.

178. Mayer, R., R. W. Parish, and H. R. Hohl. 1976.Hyphal tip growth in Phytophthora. Gradientdistribution and ultrahistochemistry of en-

zymes. Arch. Microbiol. 110:215-224.179. Mayer, R. M. 1971. The enzymatic synthesis of

the phosphomannan of Hansenula capsulata.Biochim. Biophys. Acta 252:39-47.

180. McLellan, W. L., Jr., and J. 0. Lampen. 1968.Phosphomannanase (PR-factor), an enzyme

required for the formation of yeast protoplasts.J. Bacteriol. 95:967-974.

181. McMurrough, I., and S. Bartnicki-Garcia.1971. Properties of a particulate chitin synthe-tase from Mucor rouxii. J. Biol. Chem. 246:4008-4016.

182. McMurrough, I., and Bartnicki-Garcia. 1973.

Inhibition and activation of chitin synthesis byMucor rouxii cell extracts. Arch. Biochem.Biophys. 158:812-816. '

183. McMurrough, L, A. Flores-Carreon, and S.Bartnicki-Garcia. 1971. Pathway of chitinsynthesis and cellular localization of chitin syn-thetase in Mucor rouxii. J. Biol. Chem. 246:3999-4007.

184. Meyer, J., and P. Matile. 1975. Subcellulardistribution of yeast invertase isoenzymes.Arch. Microbiol. 103:51-55.

185. Michalenko, G. O., H. L. Hohl, and D. Rast.1976. Chemistry and architecture of the my-celial wall ofAgaricus bisporus. J. Gen. Micro-biol. 92:251-262.

186. Mishra, N. C., and E. L Tatum. 1972. Effect ofL-sorbose on polysaccharide synthetases ofNeurospora crassa. Proc. Natl. Acad. Sci.U.S.A. 69:313-317.

187. Moor, H. 1967. Endoplasmic reticulum as theinitiator of bud formation in yeast. Arch. Mik-robiol. 57:135-146.

188. Moore, P. M., and J. F. Peberdy. 1976. Aparticulate chitin synthetase from Aspergillusflavus Link: the properties, location and thelevels of activity in mycelium and regeneratingprotoplast preparations. Can. J. Microbiol. 22:915-921.

189. Moreno, F., A. G. Ochoa, S. Gase6n, and J.R. Villanueva. 1975. Molecular forms of yeastinvertase. Eur. J. Biochem. 50:571-579.

190. Nakajima, T., and C. E. Ballou. 1974. Charac-terization of the carbohydrate fragments ob-tained from Saccharomyces cerevisiae man-nan by alkaline degradation. J. Biol. Chem.249:7679-7684.

191. Nakajima, T., and C. E. Ballou. 1974. Structureof the linkage region between the polysaccha-ride and protein parts of Saccharomyces cere-visiae mannan. J. Biol. Chem. 249:7685-7694.

192. Nakajima, T., and C. E. Ballou. 1975. Micro-heterogeneity of the inner core region of yeastmannoprotein. Biochem. Biophys. Res. Com-mun. 66:870-879.

193. Nakajima, T., and C. E. Ballou. 1975. Yeastmanno-protein biosynthesis: solubilization andselective assay of four mannosyltransferases.Proc. Natl. Acad. Sci. U.S.A. 72:3912-3916.

194. Nakayama, K., Y. Araki, and E. Ito. 1976. Theformation of a mannose-containing trisacchar-ide on a lipid and its transfer to proteins inyeast. FEBS Lett. 72:287-290.

195. Namba, H., and H. Kuroda. 1974. Studies onfungicides. X. Biosyntheses of f)-glucan andchitin-like substance of cell wall from Coch-liobolus miyabeamus. (1). Chem. Pharm. Bull.(Japan) 22:610-616.

196. Namba, H., and H. Kuroda. 1974. Studies onfungicides. XII. Biosyntheses of f8-glucan andchitin-like substance of cell wall from Coch-liobolus miyabeanus. (2). Chem. Pharm. Bull.(Japan) 22:1895-1901.

197. Ne(as, 0. 1965. Mechanism of regeneration ofyeast protoplasts. III. Electron microscopy ofgrowing protoplasts. Folia Biol. (Prague) 11:

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


142 FARKAS

371-377.

198. Necas, 0. 1971. Cell wall synthesis in yeast pro-

toplasts. Bacteriol. Rev. 35:149-170.199. Necas, O., A. Svoboda, and M. Kopecka. 1968.

The effect of cycloheximide (actidione) on cell

wall synthesis in yeast protoplasts. Exp. CellRes. 53:291-293.

200. Northcote, D. H., and R. W. Horne. 1952. Thechemical composition and structure of theyeast cell wall. Biochem. J. 51:232-236.

201. Ottolenghi, P. 1967. The uptake of bovine se-

rum-albumin by a strain ofSaccharomyces andits physico-pathological consequences. C. R.Trav. Lab. Carsberg 36:95-111.

202. Parodi, A. J. 1977. Synthesis of glucosyl-dolicholderivatives in yeast and their role in proteinglycosylation. Eur. J. Biochem. 75:171-180.

203. Parodi, A. J. 1978. Lipid intermediates in thesynthesis of inner core of yeast mannan. Eur.J. Biochem. 83:253-259.

204. Peberdy, J. F., and P. M. Moore. 1975. Chitinsynthetase in Mortiella vinacea, properties,cellular location and synthesis in growing cul-tures. J. Gen. Microbiol. 90:228-236.

205. Phaff, H. J. 1971. Structure and biosynthesis ofthe yeast cell envelope, p. 135-210. In A. H.Rose and J. S. Harrisson (ed.) The yeasts, vol.2. Academic Press Inc., New York and London.

206. Phaff, H. J. 1977. Enzymatic yeast cell walldegradation. Adv. Chem. Ser. 160:244-282.

207. Polacheck, Y., and R. F. Rosenberger. 1975.Autolytic enzymes in hyphae of Aspergillusnidulans: their action on old and newly formedwalls. J. Bacteriol. 121:332-337.

208. Porter, C. A., and E. G. Jaworski. 1966. Thesynthesis of chitin by particulate preparationsof Allomyces macrogynus. Biochemistry 5:1149-1154.

209. Potgieter, H. J., and M. Alexander. 1965. Pol-ysaccharide components of Neurospora crassahyphal walls. Can. J. Microbiol. 11:122-125.

210. Poulain, D., G. Tronchin, J. F. Dubremetz,and J. Biguet. 1978. Ultrastructure of the cellwall of Candida albicans blastospores: studyof its constitutive layers by the use of a cyto-chemical technique revealing polysaccharides.Ann. Microbiol. (Paris) 129:141-153.

211. Raizada, M. K., H. G. Kloepfer, J. S. Schutz-bach, and H. Ankel. 1974. Biosynthesis ofoligogalactosyl side chains of the cell envelopeglycoprotein of Cryptococcus laurentii. J. Biol.Chem. 249:6080-6086.

212. Robertson, N. F. 1965. The fungal hypha. Trans.Br. Mycol. Soc. 48:1-8.

213. Romano, A. H. 1966. Dimorphism, p. 181-209.In G. C. Ainsworth and A. S. Sussman (ed.)The fungi, vol. 2. Academic Press Inc., NewYork and London.

214. Roussel, I., I. Harrison, and G. Stewart. 1973.Studies on the formation of spheroplasts fromstationary phase Saccharomyces cerevisiae. J.Inst. Brew. London 73:48-54.

215. Ruiz-Herrera, J., and S. Bartnicki-Garcia.1974. Synthesis of cell wall microfibrils in vitro

MICROBIOL. REV.

by a "soluble" chitin synthetase from Mucorrouxii. Science 186:357-359.

216. Ruiz-Herrera, J., E. L6pez-Romero, and S.Bartnicki-Garcia. 1977. Properties of chitinsynthetase in isolated chitosomes from yeastcells ofMucor rouxii. J. Biol. Chem. 252:3338-3343.

217. Ruiz-Herrera, J., and R. Sentandreu. 1975.Site of initial glycosylation of mannoproteinsfrom Saccharomyces cerevisiae. J. Bacteriol.124:127-133.

218. Ruiz-Herrera, J., V. 0. Sing, V. J. van derVoude, and S. Bartnicki-Garcia. 1975. Mi-crofibril assembly by granules of chitin synthe-tase. Proc. Natl. Acad. Sci. U.S.A. 72:2706-2710.

219. Ryder, N. S., and J. F. Peberdy. 1977. Chitinsynthetase in Aspergillus nidulans: propertiesand proteolytic activation. J. Gen. Microbiol.99:69-76.

220. Ryder, N. S., and J. F. Peberdy. 1977. Chitinsynthase activity in dormant conidia of Asper-gillus nidulans. FEMS Microbiol. Lett. 2:199-201.

221. Saheki, T., and H. Holzer. 1974. Comparisonsof the tryptophan synthase inactivating en-zymes with proteinases from yeast. Eur. J.Biochem. 42:621-626.

222. Scherrer, R., L. Louden, and P. Gerhardt.1974. Porosity of the yeast cell wall and mem-brane. J. Bacteriol. 118:534-540.

223. Schlenk, F., and J. L Dainko. 1965. Action ofribonuclease preparations on viable yeast cellsand spheroplasts. J. Bacteriol. 89:428-436.

224. Schmidt, R., and S. Jannsen. 1974. The effectof mannose on the synthesis of the glycoproteinacid phosphatase in a mannose-deficient mu-tant of Schizosaccharomycespombe. Biochim.Biophys. Acta 362:13-16.

225. Schutzbach, J. S., and H. Ankel. 1969. Biosyn-thesis of oligosaccharide components of Cryp-tococcus laurentii cell wall. FEBS Lett. 5:145-148.

226. Schutzbach, J. S., and H. Ankel. 1971. Multi-ple mannosyl transferases in Cryptococcus lau-rentii. J. Biol. Chem. 246:2187-2194.

227. Schutzbach, J. S., M. K. Raizada, and H.Ankel. 1974. Heteroglycan synthesis in Cryp-tococcus laurentii. J. Biol. Chem. 249:2953-2958.

228. Schwarz, U., A. Asmus, and H. Frank. 1969.Autolytic enzymes and cell division in Esche-richia coli. J. Mol. Biol. 41:419-429.

229. Seichertova, O., K. Beran, Z. Holan, and V.Pokorny. 1973. The chitin-glucan complex ofSaccharomyces cerevisiae. II. Location of thecomplex in the encircling region of the budscar. Folia Microbiol. (Prague) 18:207-211.

230. Selitrenikoff, C. P., D. Allin, and D. R. Son-neborn. 1976. Chitin biosynthesis during Blas-tocladiella zoospore germination: evidencethat hexosamine biosynthetic pathway is pos-translationally activated during cell differentia-tion. Proc. Natl. Acad. Sci. U.S.A. 73:534-538.


http://mm

br.asm.org/

Dow

nloaded from



231. Sentandreu, R., M. V. Elorza, and J. R. Vil-lanueva. 1975. Synthesis of yeast wall glucan.J. Gen. Microbiol. 90:13-20.

232. Sentandreu, R., and J. 0. Lampen. 1970. Bio-synthesis of yeast mannan: inhibition of syn-thesis of mannose acceptor by cycloheximide.FEBS Lett. 11:95-99.

233. Sentandreu, R., and J. 0. Lampen. 1972. Bio-synthesis of mannan in Saccharomyces cere-

visiae. Isolation of a lipid intermediate and itsidentification as a mannosyl-l-phosphoryl po-lypenol. FEBS Lett. 27:331-334.

234. Sentandreu, R., and D. H. Northeote. 1968.The structure of a glycopeptide isolated fromthe yeast cell wall. Biochem. J. 109:419432.

235. Sentandreu, R., and D. H. Northeote. 1969.The characterization of oligosaccharides at-tached to threonine and serine in mannan gly-copeptides obtained from the cell wall of yeast.Carbohydr. Res. 10:584-585.

236. Sharma, C. B., P. Babcinski, L Lehle, andW. Tanner. 1974. The role of dolichol mono-phosphate in glycoprotein biosynthesis in Sac-charomyces cerevisiae. Eur. J. Biochem. 46:35-41.

237. Shimoda, C., and N. Yanagishima. 1971. Roleof cell wall-degrading enzymes in auxin-in-duced cell expansion in yeast. Physiol. Plant.24:46-50.

238. Sierra, J. M., R. Sentandreu, and J. R. Vil-lanueva. 1973. Regulation of wall synthesisduring S(ccharomyces cerevisiae cell cycle.FEBS Lett. 34:285-290.

239. Sipiczki, M., and V. Farka. 1979. Morpho-genic effects of 2-deoxy-D-glucose on Rhodos-poridium toruloides. Folia Microbiol. (Pra-gue), in press.

240. Sloat, B. F., and J. R Pringle. 1978. A mutantof yeast defective in cellular morphogenesis.Science 200:1171-1173.

241. Slyman, C. L*, and C. W. Slyman. 1962. Mea-surements of membrane potentials in Neuro-spora. Science 136:876-877.

242. Smith, J. E., and D. R. Berry. 1974. An intro-duction to the biochemistry of fungal develop-ment, p. 106-155. Academic Press Inc., NewYork and London.

243. Smith, W. L, T. Nakajima, and C. E. Ballou.1975. Biosynthesis of yeast mannan. Isolationof Kluyveromyces lactis mannan mutants anda study of the incorporation of N-acetyl-D-glucosamine into the polysaccharide sidechains. J. Biol. Chem. 250:3426-3435.

244. Sol, D. R., R. Bromberg, and D. R. Sonne-born. 1971. Zoospore germination in Blasto-cladiella emersonii: cell differentiation with-out protein synthesis. Proc. Natl. Acad. Sci.U.S.A. 68:459-463.

245. Soumalainen, H., and T. Nurminen. 1973.Structure and function of the yeast cell enve-

lope, p. 167-186. In J. R. Villanueva, I. Garcia-Acha, S. Gasc6n, and F. Uruburu (ed.), Yeast,mould and plant protoplasts. Academic PressInc., New York and London.

246. Sternlicht, E., D. Katz, and R. F. Rosenber-ger. 1973. Subapical wall synthesis and wallthickening induced by cycloheximide in hy-phae of Aspergillus nidulans. J. Bacteriol.114:819-823.

247. Strunk, C. 1969. Licht und elektronenmikros-kopische Untersuchungen an regenerierendenProtoplasten von Polystictus versicolor. Z.Allg. Mikrobiol. 9:205-216.

248. Svihla, G., J. L. Dainko, and F. Schlenk. 1969.Ultraviolet micrography of penetration of ex-ogenous cytochrome c into the yeast cell. J.Bacteriol. 100:498-504.

249. Tanner, W. 1969. A lipid intermediate in man-nan biosynthesis in yeast. Biochem. Biophys.Res. Commun. 35:144-150.

250. Thieme, T. R., and C. E. Ballou. 1971. Natureof the phosphodiester linkage of the phos-phomannan from the yeast Kloeckera brevis.Biochemistry 10:4121-4129.

251. Tkacz, J. S., and J. 0. Lampen. 1972. Wallreplication in Saccharomyces species: use offluorescein-conjugated concanavalin A to re-veal the site of mannan insertion. J. Gen. Mi-crobiol. 72:243-247.

252. Touimi-Benjelloun, A., and R. Bonaly. 1976.etude des parois de Rhodotorula rubra. VII.Influence du chloramphenicol sur la morphol-ogie et les phosphatases des paroi de Rh. rubra.Chem. Biol. Interact. 13:403-414.

253. Trinci, A. P. J. 1978. Wall and hyphal growth.Sci. Prog. London 65:75-99.

254. Trinci, A. P. J., and A. J. Coolinge. 1975.Hyphal wall growth in Neurospora crassa andGeotrichium candidum. J. Gen. Microbiol. 91:355-361.

255. Troy, F. A., and H. Koffler. 1969. The chem-istry and molecular architecture of Penicilliumchrysogenum. J. Biol. Chem. 244:5563-5576.

256. Tsiomenko, A. B., Yu. A. Shabalin, V. V.Dmitriev, V. M. Vagabov, B. A. Fikhte,and I. S. Kulaev. 1975. Biosynthesis of cellwall components by yeast protoplasts in thepresence of cycloheximide and 2-deoxy-D-glu-cose (in Russian). Izv. Akad. Nauk. SSSR 225:1446-1449.

257. Tsiomenko, A. B., V. M. Vagabov, J. Augus-tin, and L. S. Kulaev. 1974. About relationshipof turnover of inorganic polyphosphates andmannan in yeast (in Russian). Dokl. Akad.Nauk SSSR 215:478-480.

258. Ulane, R. E., and E. Cabib. 1974. The activatingsystem of chitin synthetase from Saccharo-myces cerevisiae. Purification and propertiesof an inhibitor of the activating factor. J. Biol.Chem. 249:3418-3422.

259. Ulane, R. E., and E. Cabib. 1976. The activatingsystem of chitin synthetase from Saccharo-myces cerevisiae. Purification and propertiesof the activating factor. J. Biol. Chem. 251:3367-3374.

260. Van Laere, A. J., and A. R. Carlier. 1978.Synthesis and proteolytic activation of chitinsynthetase in Phycomyces blakesleanus Bur-

VOL. 43, 1979


http://mm

br.asm.org/

Dow

nloaded from


144 FARKAS

geff. Arch. Microbiol. 116:181-184.261. Van Rijn, H. J. M., P. Boer, and E. P. Steyn-

ParvC. 1972. Biosynthesis of acid phosphataseof baker's yeast. Factors influencing its produc-tion by protoplasts and characterization of thesecreted enzyme. Biochim. Biophys. Acta 268:431-441.

262. Vessey, D. A., N. Lysenko, and D. Zakim.1976. Evidence for multiple enzymes in thedolichol utilizing pathway of glycoprotein bio-synthesis. Biochim. Biophys. Acta 428:138-145.

263. Villanueva, J. R. 1966. Protoplasts of fungi, p.

3-62. In G. C. Ainsworth and A. E. Sussman(ed.) The fungi, vol. 2. Academic Press Inc.,New York and London.

264. Villanueva, J. R., M. Gacto, and J. Sierra.1973. Enzymic composition of a lytic systemfrom Micromonospora chalcea AS, p. 3-24. InJ. R. Villamueva, I. Garcia-Acha, S. Gasc6n,and F. Uruburu (ed.), Yeast, mould and plantprotoplasts. Academic Press Inc., New Yorkand London.

265. Villanueva, J. R., and L. Garcia-Acha. 1972.The phenomenon of regeneration in fungalprotoplasts. Rev. Latinoam. Microbiol. 14:49-64.

266. Vorisek, J., J. Ludvik, and Z. ftehacek. 1974.Cytochemical localization ofpolysaccharides inClaviceps paspali ultrastructure during sub-merged formation of alkaloids. J. Bacteriol.118:285-294.

267. Vorisek, J., and V. Pokorny. 1975. Cytochem-ical detection of polysaccharides on the surfaceof the cell membrane complex in fungi. Arch.Microbiol. 102:293-298.

268. Vrsanski, M., P. Biely, and Z. Kratky. 1977.Enzymes of the yeast lytic system produced byArthrobacter GJM-1 bacterium and their rolein the lysis of yeast cell walls. Z. Allg. Mikro-biol. 17:465-480.

269. Vrjanski, M., Z. Kreitky and P. Biely. 1977.Lysis of intact yeast cells and isolated cell wallsby an inducible enzyme system ofArthrobacterGJM-1. Z. Allg. Mikrobiol. 17:391-402.

270. Wang, M. C., and S. Bartnicki-Garcia. 1966.Biosynthesis of ,B-1,3 and P-1,6 linked glucanby Phytophthora cinnamomi hyphal walls.Biochem. Biophys. Res. Commun. 24:832-837.

271. Wang, M. C., and S. Bartnicki-Garcia. 1970.Structure and composition of walls of the yeastform of Verticillium albo-altrum. J. Gen. Mi-crobiol. 64:41-54.

272. Wang, M. C., and S. Bartnicki-Garcia. 1976.Synthesis of /?-1,3-glucan microfibrils by a cell-free extract from Phytophthora cinnamomi.Arch. Biochem. Biophys. 175:351-354.

273. Wick, A. N., D. R. Drury, H I. Nakada, andJ. B. Wolfe. 1957. Localization of the primarymetabolic block produced by 2-deoxy-glucose.J. Biol. Chem. 224:963-969.

274. Wong, W., F. E. Young, and A. N. Chatterjee.1975. Regulation of bacterial cell walls: turn-over of cell wall in Staphylococcus aureus. J.Bacteriol. 120:837-843.

275. Yen, P. H., and C. E. Ballou. 1973. Compositionof a specific intercellular agglutination factor.J. Biol. Chem. 248:8316-318.

276. Yphantis, D. A., J. L. Dainko, and F.Schlenk. 1967. Effect of some proteins on theyeast cell membrane. J. Bacteriol. 94:1509-1515.

277. Zevenhuizen, L P. T. M., and S. Barnicki-Garcia. 1969. Chemical structure of the insol-uble hyphal wall glucan of Phytophtora cin-namomi. Biochemistry 8:1496-150.

278. Zonneveld, B. J. M. 1972. Morphogenesis inAspergillus nidulans. The significance of thecell wall and a-1,3-glucanase for cleistotheciumdevelopment. Biochim. Biophys. Acta 273:174-187.

MICROBIOL. REV.


http://mm

br.asm.org/

Dow

nloaded from


biosynthesis of cell walls fungi - microbiology and ...only a few (1, 98) papers dealing with the...

Documents