proteases and proteolytic cleavage of storage proteins in

Journal ofExperimentalBotany

Journal of Experimental Botany, Vol. 47, No. 298, pp. 605-622, May 1996

REVIEW ARTICLE

Proteases and proteolytic cleavage of storage proteins indeveloping and germinating dicotyledonous seeds

K. Miintz1

Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany

Received 25 July, 1995; Accepted 16 January 1996

Abstract

Proteolytic cleavage plays an important role instorage protein deposition and reactivation in seeds.Precursor polypeptides are processed by limitedproteolysis to mature subunits of reserve proteins instorage tissue cells of developing seeds. Steps of pro-teolytic processing are closely related to steps inintracellular protein transfer through the endomem-brane system and to the deposition in the storagevacuole. In germinating seeds special endopeptidasestrigger storage protein breakdown by limited proteo-lysis. The induced conformation changes of storageproteins open them to attack by additional endo- andexopeptidases which degrade the protein reservescompletely. Proteases that catalyse limited cleavageor complete degradation are synthesized as pre-cursors which also undergo stepwise limited proteol-ysis when they are formed in cotyledons of developingor germinating seeds. In general, this processingtransforms enzymatically inactive proenzymes intoactive proteases. Different compartments participatein the processing steps. Many of the proteases areencoded by small multigene families. Different mem-bers of the corresponding protease families seemto act during seed development and germination.Proteolytic processes that contribute to the molecularmaturation and to the reactivation of storage proteinsin dicotyledonous seeds seem to be controlled by (1)differential expression of members of the protease-encoding gene families; (2) stepwise processing andactivation of protease precursor polypeptides; (3) tran-sient differential compartmentation of precursors andmature polypeptides of proteases and storage pro-teins, respectively; and (4) interacting changes instorage protein structure and protease action. Thepresent knowledge on these processes is reviewed.

Key words: Dicotyledons, seeds, storage proteins,proteolytic cleavage, proteases.

Introduction

Developmental^ regulated changes in cellular proteinpatterns result from the degradation of proteins whichare no longer needed, as well as from the biosynthesis ofnew proteins. The latter depends on the supply of aminoacids which, at least in part, are recycled from proteinbreakdown. Similarly, cellular protein patterns change inresponse to environmental influences, like temperaturestress (heat shock proteins), water supply (desiccationproteins), or pathogen attack (pathogenesis related (PR)proteins). Developmentally and environmentally inducedchanges in the cellular proteins take place at qualitative(pattern changes) as well as quantitative levels, forexample, increase or decrease in enzyme activity by chan-ging the amount of the corresponding protein. Proteinbiosynthesis and degradation represent important factorsin the regulation of nitrogen sink/source relationshipswhich are controlled by developmental as well as environ-mental factors, such as the storage and reactivation ofvegetative storage proteins in soybean (Staswick, 1990,1994) or storage proteins in seeds (Mflntz, 1994).Misfolded and damaged proteins have to be continuouslyeliminated by protein degradation and to be replaced bynewly formed proteins. In addition, limited proteolysiscontributes to precursor protein maturation and modi-fication which often are related to intracellular proteinsorting and targeting processes, like the detachment ofsignal peptides from precursor proteins that aresynthesized at membrane-bound polysomes and co-trans-lationally sequestered into the lumen of the endoplasmicreticulum (ER). Limited proteolysis and complete poly-peptide degradation are closely interacting processes.

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606 Muntz

Detached protein fragments like signal and additionaltargeting peptides of secretory and vacuolar proteins, ofnuclear-encoded plastid and mitochondria! proteins orthe propeptides which result from the subsequent pro-cessing of various precursor polypeptides, for example,from the activation of zymogens, undergo rapid break-down. The major part of these protein precursors, how-ever, remains functionally intact and might be furthertransported into other cellular compartments.

Proteases, subclassified into endo- and exopeptidases,catalyse limited and complete proteolysis (for reviews seeBarrett, 1994; Rawlings and Barrett, 1993). Different setsof proteases and protein degradation systems have beenassociated with different cellular compartments (Vierstra,1993) like the ubiqutin-dependent protein breakdownsystem and the proteasomes in the cytosol or the plastidprotein degradation system. A system should exist todegrade glyoxysomal proteins when these microbodiesare transformed into peroxisomes in lipid storing cotyle-dons of germinating seeds. Vacuoles in storage tissues ofseeds, tubers or stems of trees harbour compartment-specific sets of proteolytic enzymes that play a criticalrole in processing and breakdown of storage proteindepositions (Mtlntz et al. 1985; Shutov and Vaintraub,1987; Wilson, 1986).

Seed storage proteins in the restricted sense are globu-lins which are characteristic of dicotyledonous seeds anda few cereals, like oats and rice, and the prolamins andglutelins from cereal grains acccording to the classificationof Osborne (1924). They are only formed in specificstorage tissues (e.g. endosperm or cotyledon mesophyll)during seed development. At the time of storage proteinaccumulation the respective organs and storage tissuesrepresent nitrogen and carbon sinks. They become nitro-gen and carbon sources when the protein reserves arereactivated. Within the frame of changing sink/sourcerelations storage protein metabolism represents a specialexample for the developmentally and environmentallycontrolled formation and degradation of proteins. In thestorage tissue cells the storage vacuole harbours themajority of participating compartment-specific proteolyticenzymes.

Most storage proteins are multimeric. The subunits arepolymorphic and encoded by multigene families. Theprimary translation products of storage protein mRNArepresent precursors of the subunits formed at membrane-bound polysomes (rough endoplasmic reticulum, rER).Most of the precursor polypeptides (e.g. storage globulins)are transported into the storage vacuole. On their waythey pass the Golgi apparatus, where at the trans-Golgithey are entrapped into transfer vesicles which are thentargeted to the vacuole (for reviews see Chrispeels, 1991;Chrispeels and Raikhel, 1992; Vitale et al, 1993). Asprotein deposition proceeds the storage vacuole differen-tiates into protein bodies. In the endosperm of developing

maize kernels the protein bodies are directly formed fromthe prolamin (zein)-accumulating ER (Larkins andHurkman, 1978). It has also been postulated that theprolamin (gliadin) in wheat is directly transferred tostorage vacuoles without passing the Golgi apparatus(Galili et al, 1995). Several of the globulin polypeptidesare modified by glycosylation in the ER and Golgiapparatus and/or undergo additional processing by lim-ited proteolysis in the vacuole. This latter step of molecu-lar maturation then transforms the molecular specieswhich fits to intracellular transport into a species whichcan be deposited in the vacuole. Clearly, the processingof precursors of storage globulin subunits by limitedproteolysis is closely related to changing structure func-tion relations which evolutionarily have been adapted onthe one side to the constraints acting during intracellularprotein transfer and targeting as well as on the other sideto the constraints governing deposition and accumulationinside the storage vacuoles. This includes controlled lim-ited proteolysis as well as the protection against prematurestorage protein degradation which might be mediated byspecial structural characters of the storage proteins,by maintaining proteases in an inactive state and/or byappropriate compartmentation.

A third group of constraints which have most certainlyplayed a role in the evolution of these proteins, as wellas the proteases that degrade them, are those imposed bystorage protein reactivation. The storage proteins mustbe made susceptible to proteolytic attack by appropriatestructural changes, protease activation and changes in thelocalization of various components among various com-partmentations. Limited proteolysis is essential for theinitiation of storage protein breakdown. The triggeringendopeptidases may be present, in an inactive state withtheir substrates inside the protein bodies or they may benewly synthesized during seed germination. The structuralchanges induced by the initial cleavages of the storageproteins result in conformation changes that open themto further degradation. In storage tissue cells this isaccompanied by the reformation of the vacuole fromprotein bodies.

The processes of storage protein deposition and react-ivation have been most extensively investigated usingglobulins of dicotyledonous plants. Close analogues tothe globulins exist in the major storage proteins of oatsand rice, and in minor storage proteins of other monoco-tyledonous seeds. A knowledge of the structure of thestorage globulins is essential for understanding theirinteractions with the proteases that degrade them.

Globulin structure

According to their sedimentation characteristics in sucrosegradients dicotyledonous storage proteins are classifiedinto 1 IS, 7S and 2S proteins. Whereas 1 IS and 7S proteins


belong to the globulins, except rice where the 1 IS proteinwas classified as a glutelin, the 2S class comprises globu-lins as well as albumins. The 11S and 7S globulins arenamed legumin-like or legumins and vicilin-like or vicilins,respectively, in accordance to the predominating storageglobulins in pea (Pisum sativum L.) and faba beans (Viciafaba L.). Trivial names, which frequently have beenderived from the botanical name of the correspondingplant, are in use for many of these storage proteins.

Legumins (11S globulins)

Legumin holoproteins, which have a molecular weight of300—400 kDa, are composed of six nearly identical sub-units with molecular weights of 50-60 kDa. Each subunitis composed of two differently sized polypeptide chains(Fig. 1). The larger more hydrophilic one (mol. wt.30-40 kDa) has a weak acidic pi and is named a-chain,whereas the smaller more hydrophobic one (mol. wt.20 kDa) has a strongly basic pi and is named /-chain.Both chains are linked by a disulphide bridge betweencysteine residues at highly conserved positions in the a-(amino acid residue 87) and /-chain (amino acid residue7). The amino acid sequences of /-chains are morehomogeneous than those of the a-chains which varyconsiderably in length because of different numbers ofrepeats in its C-terminal part (Heim et al, 1989, 1994;Horstmann et al, 1993). In addition, much larger a-chains are present in a minor group of large leguminsubunits which, for example, have been found in seeds ofVicia faba L. The sequence of one of these large subunits(mol. wt. 64 502 kDa) supports the hypothesis that thelarger subunits have evolved through amplification andmutation of repeats in the C-terminal domain of the a-chain (Heim et ai, 1994). There is no indication that thelegumin holoprotein molecules represent homo-oligomers.In the quaternary structure of the holoprotein, two trimersare layered upon and twisted against each other at anangle of 60°. The hydrophobic /-chains are mostly buriedinside the holoprotein. The more hydrophilic a-chain ismainly located at the surface and forms loops whichextend out of the protein. The major loop is formed bythe variable and hydrophilic C-terminal region of thea-chain.

Legumin genes and the corresponding mRNAs encodesubunits. Therefore, the primary translation productcorresponds to one subunit. It has a transient N-terminalsignal peptide. a- and /-chains are linked by a peptidebond between the C-terminal amino acid residue of thea-chain, which always is Asn, and the N-terminal residueof the/-chain, which is almost always Gly. The disulphidebridge between both the a- and /-chain regions ofthe legumin precursor is already formed in the lumen ofthe ER into which the growing pre-prolegumin isinserted during biosynthesis. The signal peptide is

Proteolysis of seed storage proteins 607

co-translationally detached and thereby prolegumin isgenerated. Prolegumin assembles into trimers. These rep-resent the transport-competent molecular form of leguminprecursors (Fig. 3). The peptide linkage between a- and/-chains is only proteolytically cleaved after the pro-legumin trimers have reached the storage vacuole. Sinceboth chains have already previously been bridged by thedisulphide linkage, a- and /-chains which are encoded byone gene remain paired in the subunit. Prolegumins donot seem to be able to assemble directly into hexamers.Cleavage of the -Asn-Gly-linkage forms the prerequisitefor the transformation of two trimers into one hexamer.Disassembly of the subunits does not seem to occurduring the trimer to hexamer transformation which resultsfrom a very specific single proteolytic cleavage.

Vicilins (7S globulins) and their structural similarities tolegumins

Vicilin holoproteins which have molecular weightsbetween 150 and 240 kDa are trimers composed of twotypes of subunits, large convicilin-like ones with mol. wt.70-80 kDa and small subunits of about 50 kDa (Fig. 1).Homo-oligomeric as well as hetero-oligomeric trimers areknown (Thanh and Shibasaki, 1977), the latter composedof different ratios of large and small subunits (Mtintzet al, 1986). Amino acid sequences of the large and smallsubunits are very homologous. Close to their N-terminusthe large subunits contain an approximately 20 kDa large,strongly hydrophilic insertion comprising repetitive ele-ments. The major C-terminal 50 kDa fragment exhibitshigh sequence similarity to the small subunits (Doyleet al, 1986). The small subunits are polymorphic anddiffer with respect to two characters: the degree of glycos-ylation and the presence of sites for limited proteolysisby trypsin-like enzymes. Non-glycosylated subunits freeof cleavage sites have also been described, for example,the major 50 kDa subunit from Vicia faba vicilin(Bassuner et al., 1987; Weschke et al., 1988) and the70 kDa convicilin subunit from peas, Pisum sativum L.(Newbigin et al., 1990). Phaseolin, the best-known 7Sglobulin from garden bean (Phaseolus vulgaris L.) iscomposed of non-cleavable 50 kDa subunits with differentdegrees of glycosylation (Slightom et al, 1985). There isstrong evidence that vicilin and legumin have a commonevolutionary origin (Shutov et al, 1995) and that theirsubunits have principal similarity in the 3-dimensionalstructure (Lawrence et al, 1994; Shutov et al, 1995)which corresponds to the structure of phaseolin from thegarden bean (Lawrence et al, 1990, 1994) and canavalinfrom jackbean, Canavalia ensiformis (L.) DC. (Nget al, 1993).

Like legumin subunits the vicilin subunits are encodedby multigene families. Primary vicilin translation productsbear transient N-terminal signal sequences which are


608 Miintz

no.

1

2a

2b

processing scheme subunit name( processing enzyme )

N a p C

plant species

I ,subunits of tegunwvthe 12Sgfobulra

several lecteis

(signal pepWase.legumam)

zeffi, hocdeinvicttnPfl-congiyami

(signal pepddase )

pnateoinvtafin

(signal peptxjase)

Pimm satnnjm, victa laba.Glyctne max, Lupnus spec ,Avena stbva. Oryza sattva

P&utn sMtrwm,Rianus communs

Zoa mays. Homeum yulpanj

Gtyctne max

Phasa&us wtgam,Paum saUvum, Victa laDa

2c

+p Ta + p

iL

{signal popbdase,unknown enzymes )

Pisum sxtrvum, Viaa fatoa

3a

3b

N

1

N

m

m

i

i

1

N

(a

N

I

1

1

|

N

NC ^

c1

c1

1

1

c. 1

c1

1

1

1

c1c1

convicHln

a- fl-congtyanmo-p-cooglydtwi

a-gtobulln

(signal pepooase.protsaseCI)

naptn,2Sa«xjniin

Paum satiwm

Glyanemax

Gossyptum twsutum

Brasses napusBertnofctto excelsa

\ pepteJaw.legumatn,unknown enzymes?)

/A\—i

C N "0NC / \ r \ N C

concanavalmA

(signal peptidase.loguman )

Canavaia enstfotmrs

Fig. I. Processing of different storage proteins by limited proteolysis during seed maturation. N and C indicate the N- and C-terminus of thepolypeptides: <j>. glycosylation site: the transient N-terminal signal peptide is similarly hatched in all cases, different hatchings were used forpropeptides. whereas the mature chains of the subunits remained white. I, 12S globulin processing; 2a-2c, different forms of the processing of50 IcDa subunits of 7S globulins, 3a and 3b, different forms of the processing of 70 kDA subunits of 7S globulins, 4, 2S globulin processing; 5concanavalin A processing.


co-translationally detached when the growing polypeptideis sequestered from the membrane-bound polysomes intothe ER-lumen. No disulphide bridges are known to link7S globulin subunits. Core glycosylation of the respectivevicilin subunits takes place in the ER where trimers arealso assembled. Glycosyl side-chains are trimmed andmodified in the Golgi apparatus. Like prolegumin, vicilintrimers are sorted into transfer vesicles at the trans-Golginetwork and transported into the storage vacuoles.

2S storage proteins

This is a heterogeneous group of proteins where polypep-tides predominate which are structurally related to thenapin-like 2S proteins from Cruciferae (Shewry andTatham, 1990). 2S storage proteins are polymorphic andencoded by multigene families. The proteins are mono-meric and consist of disulphide-linked large and smallpolypeptide chains (Fig. 1) which are derived from acommon precursor which bears a transient N-terminalsignal peptide. After its detachment the primary transla-tion product undergoes an even more complicated proteo-lytic processing than prolegumin. In addition to the twofragments which correspond to the mature large andsmall polypetide chains, the precursor comprises a pro-peptide located between the signal peptide and theN-terminus of the small polypeptide chain. The largechain forms the C-terminal fragment in the precursor.The propeptide cleavage site as well as the cleavage sitebetween the two polypeptide chains are flanked by Asn-residues in the P-position of many 2S protein precursors.In the ER, the regions of the propolypeptide whichcorrespond to the small and large chains are alreadylinked by disulphide bridge formation. After the propep-tide is cleaved off, the peptide linkage between the smalland large chain is cleaved. In the case of the 2S albuminof Brazil nut (Bertholletia excelsa H.B.K.) this stepinvolves the elimination of a short linker peptide betweenthe small and large chains. Finally, the large chain of theBrazil nut 2S albumin is trimmed by the detachment ofshort oligopeptide fragments at the C-terminus.

Concanavalin A

The sequence of mature concanavalin A (con A) is notco-linear to the sequence of the precursor polypeptidederived from the corresponding cDNA (Carrington et al.,1984). After the N-terminal signal peptide is co-translationally cleaved off in the rER, the glycosylatedprocon A undergoes processing. An internal glycosylatedoligopeptide fragment is excised and the N- andC-terminus of the precursor are ligated in a head-to-tailposition (Bowles et al, 1986; Faye and Chrispeels, 1987).In addition, small terminal oligopeptide fragments mightbe detached (Fig. 1). Both cleavage sites of the internal


peptide fragment are flanked by Asn-residues in theP-position.

Processing of storage globulin precursors bylimited proteolysis in developing seeds

Signal peptide cleavage

Precursor polypeptides of storage globulins are seques-tered from their cytoplasmic site of formation into theER-lumen, thus entering the so-called secretory pathwaythrough the endomembrane system. This membranetransfer is mediated by the N-terminal signal sequencewhich is proteolytically detached after finishing its func-tion as a targeting and membrane transfer signal. Thesingle site cleavage is catalysed by a signal endopeptidaselocated in the ER membrane and acting at its innersurface (Dalbey and von Heijne, 1992). The enzyme hasbeen extensively investigated in eukaryotic animals andyeast (Lively et al., 1994). It is a hetero-oligomeric serineendopeptidase. The cleavage site between signal peptideand N-terminus of the polypeptide chain is characterizedby a specific arrangement of amino acid residues describedby the -l;-3-rule according to von Heijne (1986). Sincesignal peptides of plant proteins are correctly detached inheterologous cell-free systems composed of the wheatgerm in vitro translation system, completed with dogpancreas signal recognition particles and microsomalmembranes, as well as in homologous systems with plantmicrosomes, a similar signal peptidase should also bepresent in plants (Bassiiner et al., 1984). As a result ofthe signal peptide cleavage a propolypeptide is formed inmost cases. As far as presently known, this propolypeptidedoes not undergo further limited proteolysis in the ERor Golgi compartment, although there the polypeptidesfold into a conformational state and oligomerize, whichmakes them competent for intracellular transfer throughthe endomembrane system and into the storage vacuole.Several storage protein polypeptides are modified byglycosylation during their passage through the ER andGolgi compartment, but this glycosylation does not seemto be a prerequisite for their intracellular transfer andtargeting (Voelker et al., 1989).

Propolypeptide processing by limited proteolysis invacuoles

The presence of N-terminally Asn-flanked processing sitesis a common character of many US globulin (Lawrenceet al., 1994), 2S storage protein (Hara-Nishimura et al.,19936) and even protein body membrane protein pre-cursors (Inoue et al, 1995). The complete evolutionaryconservation of this Asn at the C-terminus of the largelegumin polypeptide chains indicates that it may beimportant for recognition by the corresponding pro-cessing enzyme. Mutation or deletion of this Asn renders


610 Milntz

prolegumin uncleavable in vitro as well as in vivo.Recently, cysteine endopeptidases have been describedfrom castor bean (Hara-Nishimura et al, 1991), soybean(Scott et al, 1992; Muramatsu and Fukazawa, 1993) andjack bean (Abe et al, 1993) that at least in vitro catalysethe Asn-specific limited cleavage of prolegumins, pre-cursors of 2S proteins, proconcanavalin A and someother proteins. An enzyme belonging to this class ofendopeptidases, but prepared from germinating vetchseeds, was able to catalyse in vitro the Asn-specific molecu-lar maturation of prolegumin (Becker et al, 1995a), thatnormally occurs in developing seeds. Polypeptides whichcross-react with antibodies raised against the enzymefrom castor bean were also detected in other plant organsexcept seeds (Hiraiwa et al, 1993). Therefore, this typeof enzyme may belong to a class of cysteine endopeptid-ases which specifically cleaves at the C-terminus of Asnresidues that are exposed in a way which gives theenzyme access.

Legumain in developing and mature seeds: When thecDNA corresponding to the castor bean (Ricinus com-munis L.) Asn-specific processing proteinase from seedswas sequenced and the complete amino acid sequencewas derived (Hara-Nishimura et al, 1993a, 1995), it

turned out to be a new type of cysteine endopeptidase.The sequence did not correspond with the well-knownpapain-like cysteine endopeptidases. The only similaritythat appeared on screening the databases was to thesequence of an endopeptidase from Schistosoma mansoni,a human parasite. The Ricinus mRNA encodes a 55 kDapre-propolypeptide that is presumably comprised of a 31amino acid-long N-terminal signal peptide, the mature37 kDa enzyme and an approximately 14 kDa C-terminalpropeptide sequence the exact length of which is unknownsince the C-terminus of the mature enzyme has yet to bedetermined (Fig. 2b). Antibodies have been raised againstthe purified native proteinase protein and used to localizethe enzyme to vacuoles in cells of the maturing castorbean endosperm in which US and 2S storage proteinsare also localized. This result reinforces the interpretationof previous 'grind and find' experiments in which theAsn-specific processing enzyme co-purified with the pro-tein bodies of castor bean endosperm. The same antibodycross-reacted in immunoblots with polypeptide bandsafter electrophoresis of extracts from mature and earlygerminating seeds, too. Extracts from roots, hypocotyland leaves of castor bean as well as from pumpkin andsoybean cotyledons and hypocotyls, from mung beanroots and spinach leaves were active in the enzyme-

PROTBNASE A

1 18 128 152 286 307

C C

QGQCGSCWAFST1

TDLNHGVA NSWGA i

359

/ \KDEL ER

?

?

VA?

PROTEINASE B

24 49 77 216 265 376 ( ? ) 493

ND

NYRHQ.D.CHAY EACESGS TCLGDLYSER

Fig. 2. Schematic presentation of essential characters of proteinases A and B. SP, signal peptide; PP1 and PP2, propeptides; i£, glycosylation sites;C, positions of cysteine residues; sequences of highly conserved fragments are indicated; arrows, amino acid residues of the active centre in papainwhich are conserved in proteinase A; ER, endopiasmic reticujum, and VA, vacuole, indicate the cellular compartments where processing (presumably?)occurs.


specific assay which uses a synthetic decapeptide substratecontaining an Asn-flanked internal cleavage site (Hiraiwaet al, 1993). The cDNA-derived amino acid sequence ofthe vacuolar Asn-specific processing enzyme of soybeancotyledons exhibits 77% similarity to the castor beancysteine endopeptidase and cleaves native precursor poly-peptides as well as synthetic oligopeptides like the castorbean enzyme (Shimada et al, 1994).

A similar endopeptidase was purified from maturejack bean, Canavalia ensiformis L. (Abe et al., 1993).Oligonucleotides derived from partial N-terminal aminoacid sequence of the enzyme have been used to obtainfour different cDNA clones which suggests that isoen-zymes exist which are encoded by a multigene family(Takeda et al., 1994). Since similar Asn-specific cysteineendopeptidases are present in various legumes it wasnamed legumain (Ishii, 1994; Takeda et al., 1994) accord-ing to the current edition of Enzyme Nomenclature (EC3.4.22.34). A method was published which now permitssimple and sensitive quantitation of correspondingenzyme activities even in crude extracts (Cornel andPlaxton, 1994) using benzoyl-L-asparagine-/7-nitroanilideas the substrate with subsequent diazotization of thegenerated p-nitroanilide which makes the spectrophoto-metric measurement of this reaction product moresensitive.

Polymorphism of an Asn-specific 33-33.8 kDa vacuolar11S processing enzyme has also been reported for acysteine endopeptidase preparation from mature soybean(Muramatsu and Fukazawa, 1993). The three isoformswere isolated which had isoelectric points of 4.94, 4.89,and 4.85, respectively. They specifically cleaved proglyci-nin, which was recombinantly produced in E. coli, intothe mature a- and _$-chains at the C-terminus of Asn inthe precursor. Glycinin is the legumin-like US storageglobulin from soybean and the endopeptidase was called'maturation enzyme'. Whereas the Asn-specific cysteineendopeptidases reported so far do not seem to be glycosyl-ated, Scott et al. (1992) prepared a glycosylated USglobulin maturation endopeptidase from soybean. Theenzyme which in electrophoresis under denaturing condi-tions gave 3 polypeptides with relative molecular weightsof 85, 63 and 23 kDa in vitro correctly processed pro-legumin from Vicia faba L. as indicated by N-terminalmicrosequencing of the cleavage product. Octapeptidesubstrates were synthesized with an Asn in the P-positionof the internal cleavage site, and several mutations of theAsn-residue were produced. Only octapeptides havingAsn in an internal P-position were cleaved by the enzyme.This confirms its strict Asn-specificity.

Several lines of evidence indicate that this new class ofAsn-specific cysteine endopeptidases is the prime candid-ate for the propolypeptide processing proteinase thatcatalyses the maturation of US and 2S storage proteinsin dicotyledonous seeds. (1) The enzyme has been local-


ized to the same compartment as the storage proteins,namely the vacuoles (developing protein bodies) of cellsin storage endosperm and cotyledons of developing seeds,(2) in vitro cleavage of the corresponding storage proteinprecursors occurs as expected, and (3) the time-course ofthe appearance of the corresponding enzyme polypeptideas well as the time-course of its activity correspond withthe period of biosynthesis and processing of the presump-tive in vivo subtrates in developing castor bean (Corneland Plaxton, 1994; Hiraiwa et al., 1993). Nevertheless,conclusive in vivo evidence for the suggested endopeptid-ase function is still lacking. More conclusive evidencecould be provided if it is possible to inhibit storageprotein maturation by expression of appropriate anti-sense-DNA constructs of the proteinase in the developingseeds of transgenic plants.

Legumin processing by limited proteolysis in vitro andin vivo: An in vitro processing assay for proglycinin ofsoybean has been developed by combining the in vitrotranscription/translation of US globulin propolypeptidesfollowed by oligomer reconstitution (Dickinson et al,1987, 1989) with an in vitro cleavage assay using thesoybean US globulin maturation enzyme (Scott et al,1992; Jung et al, unpublished). Prolegumin B from fababean which was produced in vitro and could be assembledinto trimers was purified by isopycnic sucrose densitycentrifugation and used as substrate in the cleavage assay(Nielsen et al., 1995). The results of these experiments,which at least in part are still unpublished, indicate thatonly correctly formed prolegumin trimers undergo thesingle site cleavage of the peptide bond linking the a- andjS-chain in the precursor and are thereby converted intothe hexamers. Prolegumin monomers are degraded prob-ably because they assume a conformation where the manyother Asn-flanked sites in the polypeptide are not pro-tected against attack by the maturation enzyme. Thisresult also suggests that no disassembly of the prolegumintrimer should take place in the process of trimer tohexamer transition in vivo.

Confirmation of these in vitro results have come fromtobacco transformation experiments. Prolegumin withoutthe C-terminal Asn of the a-chain could not be cleavedin transgenic tobacco seeds and formed only trimers butno hexamers (Jung et al, unpublished). Prolegumin chainfragments of various length were fused before the com-plete chloramphenicol acetyl transferase polypeptide(CAT). Stable a-chains were formed inside vacuoles ifthe constructs contained an intact a//8-chain cleavage site.The detached fragments comprising partial jS-chainsequences or the complete ^-chain fused to CAT weredegraded. This suggests that these fusion polypeptideswere recognized as misfolded by the vacuolar proteases.Legumin-CAT fusions with fragments shorter than thecomplete a-chain did not contain the a/$-cleavage site.


612 MOntz

They remained outside the vacuole and were not degraded(Jung et al., 1993). This processing of prolegumin-CATfusions could also be demonstrated in vitro. The enzymewhich catalyses the correct processing as well as theenzyme responsible for the degradation of the misfoldedchains should be located within the vacuole. Both activit-ies can probably be attributed to the same Asn-specificcysteine endopeptidase.

The prolegumin processing activity has been localizedto vacuoles of storage tissue cells of developing pumpkinand castor bean seeds (Hara-Nishimura et al., 1987;Harley and Lord, 1985). The Asn-specific processingcysteine endopeptidase has been purified from proteinbodies of castor bean endosperm (Hara-Nishimura et al,1991). Immunohistochemical studies of this proteasehave localized it to the vacuoles of similar cells (Hara-Nishimura et al, 1993a). Prolegumin trimers, but nohexamers, are present in the ER of developing storagetissue cells in pea (Chrispeels et al., 1982), soybean(Barton et al., 1982) and pumpkin cotyledons (Hara-Nishimura and Nishimura, 1987). Conversely, only negli-gible amounts of prolegumin have been found in vacuolesof corresponding cells in which legumin hexamers pre-dominate. All these results indicate that prolegumin trimerassembly takes place in the ER. The trimers are thentransferred into the storage vacuoles where they arecleaved by the processing enzyme. This cleavage of thea//?-chain peptide linkage transforms the prolegumintrimer directly into the mature legumin hexamer which isthen able to form deposits in the developing proteinbodies. Formation of the processing enzyme as well as ofthe prolegumin are subject of strict developmental control(Hara-Nishimura and Nishimura, 1987, 1993a). Bothconcomitantly undergo intracellular protein transferthrough similar compartments. Since no cleavage of theprolegumin seems to occur before reaching the vacuole,the protease should be inactive during its passage throughthe secretory pathway and becomes activated after itsarrival in the storage vacuole. A finely tuned interplay ofchanging structure function relations between substrateand enzyme proteins, of enzyme activation and compart-mentation is responsible for regulating this process.

Processing of a protein body membrane proteinprecursor: Recently it has been shown (Inoue et al, 1995)that protein body membrane polypeptides MP27 andMP32 arise from the translation of an mRNA into acommon precursor which must be post-translationallyprocessed into the two polypeptides. The putative cleav-age site in the P-position is flanked by an Asn-residue.The authors suggest that here also an Asn-specific cysteineendopeptidase may be the processing enzyme, since, asabove, the precursor polypeptide enters the secretorypathway and the polypeptides are associated with theinner surface of the protein body membrane.

Limited proteolysis of provicilin in vacuoles: Post-translational cleavage of vicilin precursors has beendemonstrated in developing pea (Gatehouse et al., 1981,1982, 1983) and field bean cotyledons (Scholz et al.,1983). Some of the polymorphic 50 kDa pea vicilinscontain two internal cleavage sites flanked by Arg andLys residues, whereas others are free of such sites. Inmature pea cotyledons sets of distinct polymorphic cleav-age products of 33, 19, 16, and 13.5 kDa have been foundwhich are generated by cleavage at both or only one ofthe two preformed sites. The 16 kDa fragment was glycos-ylated. In field bean 50 kDA vicilin subunits predominatethat are free of such cleavage sites. The proportion of thevicilin subunits that were cleaved to sizes comparable tothose found in pea was strongly dependent upon thepreparation method. Since in the same legume plantcleavable as well as non-cleavable forms of 50 kDa vicilinsubunits co-exist and in certain legumes, like gardenbeans, no cleavage of the vicilin-homologous phaseolinsubunits takes place, it is difficult to assign a function tothis processing step.

This type of limited proteolysis does not appear tooccur in the processing of convicilin-like subunits of 7Sglobulins. Nevertheless, these proteins are subject tospecific processing. In developing cotton seeds (Gossypiumhirsutum L.) the vicilin-like storage proteins, named a-globulins, are represented by two groups of polymorp-hic subunits, the glycosylated 52 kDa and the non-glycosylated 46.5 kDa polypeptides (Dure and Chlan,1981). Both are generated by processing of a family ofpropolypeptides with molecular weights of about 67 kDa(Chlan et al., 1986, 1987). An N-terminal 20.5 kDafragment is detached by limited proteolysis from theprecursor to form the mature vicilin-like chains. Theputative cleavage site is flanked by Arg-residues, so thata processing enzyme different from the proleguminprocessing endopeptidase must be responsible. The intra-cellular site of this processing event still remains to beelucidated.

Other processing enzymes? A putative aspartate endopep-tidase has been partially purified from extracts of devel-oping and mature Brassica napus seeds and used inprocessing experiments with in vrtrosynthesized andradioactively labelled rape napin-like 2S albumin propoly-peptide from Arabidopsis thaliana as a substrate (compareFig. 1). In addition, synthethic substrates have been usedthat had been designed according to the peptide whichlinks the small and large chain in the albumin precursorand is excised during its processing (D'Hondt et al,1993). Internal peptide bonds were cleaved in thesesubstrates which suggests that the enzyme could alsoprocess the precursor. The propolypeptides were split intofragments that correspond to the size of the small andlarge chains of mature 2S albumin. Thus the authors


claim that they have found a processing protease whichat least in vitro might correctly process 2S storage albuminprecursors of cruciferous seeds. No evidence was pre-sented that the enzyme is present in a compartment where2S proalbumin processing might take place and how theN-termini of the mature chains might be generated bythis or an additional enzyme.

Limited proteolysis and storage globulindegradation in germinating seeds

In mature dry seeds storage proteins are present in theembryo axis as well as in the storage tissues proper, likethe endosperm, for example, in castor beans, or thecotyledon mesophyll, for example, in legumes, oil seedrape, sunflower or pumpkin. However, in the embryoaxis, time-course, pattern and mechanism of storage pro-tein degradation as well as its contribution to nitrogensupply for the developing embryo and its regulatoryinteraction with the major protein degradation processesin the proper storage tissues has not been investigated.Research has been focused on proteolytic enzymes andstorage protein degradation in the proper storage tissues.There, the beginning of measurable storage proteindegradation can be detected at days 2-3 after the startof imbibition (dai) depending on the species under investi-gation. Further storage protein breakdown proceedsmuch more rapidly in the cotyledons of germinatingVigna radiata (L.) Wilczek. (Baumgartner and Chrispeels,1979) or Phaseolns vulgaris L. (Nielsen and Liener, 1984;Boylan and Sussex, 1987) where 7S globulins stronglypredominate, than in seeds of Pisum sativum L. (Bashaand Beevers, 1975), Vicia faba L. (Lichtenfeld et al.,1979, 1981) or Glycine max (L.) Merr. (Wilson et al.,1986) in which nearly 50% or more of the storage proteinis made of 1 IS globulin. Nevertheless, the overall patternof storage globulin degradation is similar and, in parallelto storage globulin breakdown, protein bodies start tofuse and large vacuoles are regenerated in the storagetissue cells.

Pattern of storage protein cleavage

If globulins from field beans were analysed by electrophor-esis under non-denaturing conditions or by immuno-electrophoresis slight mobility changes were observed forvicilin and legumin during the first days of germination.Changes in mobility in immuno-electrophoresis may bedue to changes in the charge (Lichtenfeld et al., 1979).Similar observations were made with germinating vetchseeds (Shutov and Vaintraub, 1973), beans (Boylan andSussex, 1987) and buckwheat (Dunaevski and Belozerski,1989a). If samples from early germination stages wereanalysed under denaturing conditions, cleavage productswere apparent. As germination progressed, the original


bands became weaker and distinct breakdown productsappeared. Taken together these results suggest that limitedproteolysis plays an important role in initiating storageglobulin degradation. After limited proteolysis the holo-proteins nearly retain their original electrophoretic mobil-ity under non-denaturing conditions. The holoproteinsalso retain their sedimentation characters in sucrose gradi-ent centrifugation. Both results indicate that at this stagethe proteins are largely intact. Only very small fragmentsare lost at this stage. Consequently, the amount ofliberated amino acids must be small. This is reflected inthe slow initial decrease of protein nitrogen that wasobserved in germinating seeds. Whereas distinct large sizecleavage products were found the small ones have neverbeen electrophoretically demonstrated, indicating that thehalf-lives of the smaller fragments may be much smallerthan those of the large fragments. The amino acidsproduced by the rapid breakdown of the small fragmentsare transferred to the growing germling where they pro-vide the starting material for new protein biosynthesis.The breakdown of the major amount of storage globulinsoccurs from 4-8 dai depending on the plant species andit coincides with the major activity of proteolytic enzymes.

Pattern of legumin degradation: In seeds containing bothlegumins and vicilins the degradation of legumin proceedsmore slowly (Lichtenfeld et al., 1979, 1981; Wilson et al.,1986). The a-chain of legumin starts to be degraded firstwhereas the jfl-chain remains intact until 5-6 dai incotyledons of germinating field bean and soybean whenthe major storage protein degradation occurs. This mightbe attributed to the topographic position of this chain inthe holoprotein. The a-chains are thought to be exposedat the surface while the _/8-chains are located inside theholoprotein. This is supported by two observations. (1)Antibodies raised against the holoprotein predominantlyreact with a-chains, but only weakly or not at all withthe jS-chains in immunoblots; and (2) in short-timein vitro incubation experiments endopeptidases nearlyexclusively cleave a-chains, but not jff-chains. The majorcleavage intermediates exhibit relative molecular weightsof 24 000 to 30 000 which is larger than the molecularweight of j9-chains, thus indicating that these inter-mediates must have been generated from the a-chains.No detailed analysis of the cleavage products such aspartial N-terminal amino acid sequencing has so far beenperformed. Therefore, no exact localization of the cleav-age sites is known. In cleavage experiments with trypsindistinct fragments of the a-chain of glycinin, the legumin-like globulin from soybean, are generated (Shutov et al.,1993). The fragment sizes suggest that the sites that areattacked correspond to loops II and 12 in the predictedsecondary structure of legumin (Shutov et al., 1995).Similar sites should represent the targets of the first,limited, endopeptidolytic attack during legumin degrada-tion in germinating seeds.


614 MQntz

Pattern of vicilin degradation: Conglycinin is the majorvicilin-like 7S globulin in soybean. During germinationthe large a- and a'-subunits of ^-conglycinin disappearfirst (Bond and Bowles, 1983; Wilson et al, 1986).Whereas Bond and Bowles (1983) already found degrada-tion of these polypeptides in mature and imbibing seeds,Wilson et al. (1986) have reported that the breakdownof these ^-conglycinin subunits starts at the earliest 1 dafter imbibition. As the a- and a'-^-conglycinin polypep-tides disappear, a 51 200 kDa polypeptide accumulateswhich reacts with jft-conglycinin-specific antibodies and isgenerated by the detachment of an N-terminal fragmentfrom the large ^-conglycinin subunits (Qi et al., 1992).This limited proteolysis step resembles the processingreported for large vicilin subunits of cotton during seedmaturation (Chlan et al., 1986). The intermediates of a-and a'-jS-conglycinin degradation start to disappear at 6dai when new jS-conglycinin-specific intermediates withmol. wt. 25-31 kDa emerge. The 50 kDa _/?-^-conglycininchain does not seem to be degraded until 6 dai. Itsdegradation starts at the same time as that of the 51.2 kDaintermediate degradation product of the a- and a'-fi-conglycinin. Therefore, it has not been possible to deter-mine the precursors of the 25-31 kDa conglycinin cleav-age products. The degradation of j0-j?-conglycinincoincides in time with the breakdown of the jfl-chains ofglycinin, the legumin-like US globulin of soybean.

In Vigna radiata and Phaseolus vulgaris, in which themajor globulins are 7S trimers containing predominantly50 kDa subunits, globulin degradation takes place aroundthe 3rd to 4th dai (Baumgartner et al, 1979; Nielsenand Liener, 1984; Boylan and Sussex, 1987). Groups ofdegradation intermediates with Mt 20-30 kDa appear.The non-glycosylated 45.5 kDa phaseolin polypeptideseems to be degraded more easily than the glycosylated51 and 48 kDa subunits (Nielsen and Liener, 1984).

Germination proteases

By applying different protease inhibitors to germinatingcastor beans (Alpi and Beevers, 1981) and to extractsfrom germinating garden beans (Nielsen and Liener,1984) and mung beans (Chrispeels and Boulter, 1975) itwas possible to determine that sulphydryl proteinases or,as they are named today, cysteine endopeptidases, areprimarily responsible for storage globulin degradation invivo. Numerous publications in which increases in amountand activity of cysteine endopeptidases are correlatedwith the major breakdown of storage globulins, supportthis conclusion (for reviews see Wilson, 1986; Shutov andVaintraub, 1987; Vierstra, 1994). Some authors alsoreport the involvement of serine endopeptidase(Mitsuhashi et al, 1986; Qi et al, 1992) or metallo-endopeptidase (Belozerski et al, 1990; Elpidina et al,1991) in storage globulin degradation.

The pioneering work of Chrispeels's group (reviewedby Baumgartner and Chrispeels, 1979) on the proteolyticbreakdown of vicilin in germinating seeds of Vigna radiatahas demonstrated that cysteine endopeptidases synthe-sized de novo, in their case a vicilin peptidohydrolase(Baumgartner and Chrispeels, 1977), catalyse globulindegradation in the cotyledons of germinating legumeseeds. Purified vicilin peptidohydrolase has a mol. wt. of23 kDa, an IEP at pH 3.75 and exhibits maximal activityat pH5.1. The enzyme preferentially cleaves syntheticesters with asparagine and glutamine, readily degradesvicilin in vitro, and exhibits its major activity increase ingerminating seeds from 2-5 dai when most of the vicilindisappears. It is synthesized at membrane-bound poly-somes. The enzyme protein undergoes vesicular transportinto the protein storage vacuole where it initiates thedegradation of globulin (Baumgartner et al, 1978).Although a protease inhibitor with in vitro activity againstthe vicilin peptidohydrolase is present in the cotyledoncells of germinating Vigna radiata seeds, it is located in adifferent subcellular compartment so that it probablyplays no role in the regulation of vicilin degradation(Baumgartner and Chrispeels, 1976; Chrispeels et al,1976; Chrispeels and Baumgartner, 1978). Since isolatedprotein bodies from mature seeds showed only negligibleendogenous protein degradation and storage proteinbreakdown was elicited by the addition of extracts from4 dai seeds, the de novo synthesis of the vicilin peptidohyd-rolase is assumed to be the key event in vicilin degradation(Harris and Chrispeels, 1975; Baumgartner et al, 1978).This was confirmed by detailed studies on vicilin andlegumin degradation in germinating vetch, Vicia sativa L.(for review see Shutov and Vaintraub, 1987). In thissystem two proteases participate in globulin degradation:a triggering cysteine endopeptidase A with an Afr 21 kDa(cDNA-derived mol. wt. 25 244 kDa), which is activeagainst globulins prepared from mature dry seeds, and acysteine endopeptidase, termed proteinase B, with Mr

38 kDa that exhibits strict Asn cleavage specificity. Bothenzymes appear to be absent from the mature seed andto be de novo synthesized during germination. Since thesubstrate for proteinase B is globulins that have alreadybeen modified by the 'triggering' proteinase A, its activityis only apparent after proteinase A has become active. Aserine endopeptidase appears to be required for the partialhydrolysis of the large vicilin-like subunits of soybean fi-conglycinin, the a- and a'-jS-conglycinin chains (Qi et al,1992). This has confirmed an earlier study that implicateda serine endopeptidase in the limited proteolysis of a64 kDa subunit in cotyledons of germinating Vigna mungoseeds (Mitsuhashi et al, 1986). In both cases no corres-ponding enzyme activity could be measured in extractsfrom mature dry seeds.

A different model for the initiation of globulin break-down has been developed by Belozersky and his


group by studying germinating buckwheat (Fagopyrumesculentum L.). They found a Zn-dependent metallo-endopeptidase that forms an inactive complex with anendogenous protease inhibitor inside the protein bodiesof mature seeds (Belozerski et al, 1990; Elpidina et a!,1991). The enzyme was shown to degrade in vitro alegumin preparation from mature buckwheat. They pro-posed that the dissociation of the enzyme from theinhibitor which is mediated by zinc ions, triggers globulinbreakdown. In addition, a cysteine endopeptidase hasbeen isolated from germinating buckwheat whichdegrades legumin prepared from seedlings, but not thatprepared from dry seeds (Dunaevsky and Belozersky,19896). The authors proposed that limited proteolysis oflegumin by the metallo-endopeptidase, which is alreadypresent in mature seeds, forms a prerequisite for furtherdegradation of legumin by the sulphydryl endopeptidaseand that the activity of the latter protease is feedbackinhibited by degradation products.

Papain-like cysteine endopeptidases: proteinase A: Papain-like cysteine endopeptidases represent the major storageprotein degrading enzymes which appear during seedgermination, for example, in cotyledons of Phaseolusvulgaris (Boylan and Sussex, 1987), of Vigna mungo,where it is called SH-EP (Akasofu et al, 1989, 1990) aswell as of Vicia sativa (Becker et al, 19956), where theenzyme is named proteinase A. A more distantly relatedcysteine endopeptidase protein, termed P34, has beenfound in the cotyledons of developing, mature and ger-minating soybean, Glycine max (Kalinski et al, 1990,1992) although evidence is still lacking that it is enzym-atically active. A cysteine endopeptidase from Vicia fabaalso seems to belong to this papain-like class of pro-teinases (Yu and Greenwood, 1994). Only very lateduring germination it reaches maximum activity afterapproximately 50% of the stored proteins had alreadydisappeared. A member of this enzyme group has evenbeen found in pods of developing Phaseolus vulgarisfruits. It shows more than 90% sequence similarity tothe Vigna mungo SH-EP (Tanaka et al., 1991, 1993).Homologous storage protein degrading endopeptidaseshave been found in other dicotyledonous seeds as well ascereal grains, like barley and rice, during germination.Proteinase A, SH-EP, and P34 are synthesized at therough endoplasmic reticulum (rER) and eventuallyappear in the protein storage vacuole. The primary trans-lation products have mol. wts similar to or larger than43 kDa and undergo four to five limited proteolyticcleavage steps (Fig. 2). The first step is the detachmentof the N-terminal signal peptide during translation whichyields proproteinase A, a proteinase A precursor polypep-tide, that is comprised of an N-terminal propeptide ofapproximately 120 amino acid residues, and a fragmentwhich corresponds to the mature enzyme. The N-terminal


propeptide is detached by two to three consecutive cleav-age steps. Whereas the proproteinase SH-EP and its firstcleavage product are inactive, the product of the nextcleavage exhibits very low activity. The enzyme becomesfully activated by the last proteolytic processing step(s)(Mitsuhashi and Minamikawa, 1989). The tetrapeptideKDEL which is present at the C-terminus of cDNA-derived sequences of SH-EP and proteinase A precursorsis known to be a ER retention signal. It is lacking inisolated-mature SH-EP (Okamoto et al., 1994) and pro-teinase A (Becker et al., 19956) of germinating seeds.One could speculate that this step coincides with thetransfer of the proenzyme from the ER to the vacuoleand/or an intermediary compartment on the way to thevacuole. The mature enzymes have different molecularweights: SH-EP, 33 kDa (electrophoretically determined);P34, 28.6 kDa; and proteinase A 25.4 kDa (both calcu-lated from cDNA-derived sequences). Exactly where inthe cell the N-terminal propeptide fragments are detachedis still unknown. However, for several reasons discussedlater, at least the final step should take place in thevacuole. Only the propeptide regions of the precursors ofproteinase A and P34 contain glycosylation sites, whichagrees well to the finding that the mature enzymes arenot glycosylated. The P34 precursor has been shown tobe glycosylated (Kalinski et al, 1992). SH-EP also con-tains potential gJycosylation sites in the mature polypep-tide, but it is not known whether they actually bearcarbohydrate side chains. Papain-like cysteine endopep-tidases exhibit optimal activity at pH values near 5. Themature enzymes have 7 cysteine residues and containconserved elements which are known to be part of theactive centre of papain (Fig. 2a). Whereas only a singlegene appears to code for the Vigna mungo SH-EP(Yamauchi et al., 1992), small gene families with at leasttwo members have been shown to code for proteinase A(Becker et al., 19956) and P34 (Kalinski et al., 1990).

Proteinase P34 not only in its primary structure is lessrelated to SH-EP than proteinase A, but it also has noC-terminal KDEL-signal for ER-retention. In maturesoybeans P34 forms dimers which are thought to be aninactive state of the enzyme (Herman et al, 1990; Kalinskiet al., 1992). A Cys residue at position 10 seems to beresponsible for dimerization. In germinating seeds anN-terminal fragment is detached which transforms P34into P32. It was speculated that the dimer dissociates bythis step into monomers and the enzyme is activated bymonomer formation. No de novo formation has beenshown during germination.

Other papain-like cysteine endopeptidases, termedCPR1 and CPR2, have been found in the cotyledons ofgerminating Vicia sativa seeds (Becker et al., 1994). Theyare formed de novo during germination. The greatestsequence homology so far found for CPR2 is to a droughtstress-inducible pea cysteine endopeptidase that has been


616 Milntz

found in vegetative organs (Guerrero et al., 1990) and toa related enzyme from developing soybean cotyledons(Nong Van Hai et al, 1995). The vetch seed enzyme canalso be induced by drought stress (Fischer, unpublished).It is still unknown whether these vetch endopeptidasesparticipate in the storage protein degradation or not.However, they become active at the time of storageglobulin degradation, and, since the cDNA-derived aminoacid sequences contain N-terminal signal peptides, theyare probably located in the proper subcellular compart-ment. Sequence comparison with N-terminal sequencesof homologous enzymes suggest that both cysteine endo-peptidases contain propeptides that are processed to yieldthe mature enzyme. Calculated molecular weights ofmature CPR 1 and 2 are 26.1 and 24.9 kDa, respectively,and both have activity maxima in the acidic pH-range.The conservation of elements of the active centre as wellas the positions of the Cys-residues indicate that theenzymes are related to papain (see also Fig. 2a).

Proteinase B: a legumain-like cysteine endopeptidase:Proteinase B has been purified from cotyledons of germin-ating Vicia sativa and partially sequenced. The sequenceswere then used to derive oligonucleotides in order toclone and sequence the corresponding cDNA (Beckeret al., 1995a). The derived amino acid sequence of theenzyme indicates that it belongs to a class of cysteineendopeptidases, termed legumain (already referred to),that have recently been found in maturing castor beanand soybean as well as in mature jackbean. The enzymestrictly cleaves at sites with Asn or Asp in P-position andprocesses prolegumin like the legumains from developingseeds. Its pH optimum is 5.6. As is true of legumain frommaturing seeds, the enzyme is synthesized as a precursorcomposed of an N-terminal signal peptide (putatively 24amino acid residues long), followed downstream by a 24residues N-terminal propeptide, the sequence of themature enzyme and finally a 10-12 kDa C-terminal pro-peptide (Fig. 2b). To generate the mature enzyme, whichhas a mol. wt. of 37-39 kDa, the precursor has to undergoat least three proteolytic processing steps: the cotransla-tional signal peptide detachment, and the cleavage of theN-terminal and of the C-terminal propeptides, which areflanked by Asn residues at the cleavage sites. There aresome indications that the precursor undergoes autocata-lytic cleavage and activation. Two potential glycosylationsites are present in the mature enzyme. Proteinase B issynthesized de novo during vetch seed germination.

Serine endopeptidase in germinating soybean: The serineendopeptidase Cl (Qi et al., 1992, 1994) of soybean whichpartially cleaves a- and a'-jff-conglycinin chains as well asthe homologous convicilin from pea has a pH optimumat 3.5 to 4.5 and a mol. wt. of 70 kDa. Although nocorresponding activity could be measured in extracts from

mature seeds, the enzyme as well as jS-conglycinindegradation could already be detected after 1 d of imbibi-tion. Distinct cleavage products with mol. wt. 48-50 kDaare generated in a stepwise fashion. The enzyme cleavesin vitro inside specific clusters of acidic amino acid residuesindependent of whether these are present in large vicilinsubunits or in the C-terminal region of the a-chain oflegumin (Qi et al., 1994). A presumably related enzymehas been found in germinating Vigna mungo seeds(Mitsuhashi el al., 1986).

Metallo-endopeptidase in germinating buckwheat: A Zn-containing enzyme with a pH optimum between 8.0 and8.2 has been purified to homogeneity in electrophoreticanalysis. Its relative mol. wt. has been determined to be34 kDa by electrophoresis under denaturing conditionsand 39 kDa by gel chromatography. The enzyme cleavespeptide bonds in oxidized insulin 5-chains which containLeu, Tyr or Phe in the P-position (Belozerski et al., 1990)and cleaves legumin prepared from mature buckwheat aswell as from soybean seeds. The metallo-endopeptidaseappears to be reversibly inactivated by association withan endogenous proteinase inhibitor polypeptide. It hasbeen localized in protein bodies (Elpidina et al., 1991).

Protein processing by limited proteolysis and the control ofstorage protein maturation and breakdown in legume seeds:an hypothesis

Endopeptidase-specific antibodies were used along withthe appropriate cDNA probes to detect the presence ofthe enzyme and mRNA in storage tissues of developingand germinating seeds and to follow the time-course oftheir appearance. Proteinase A- (Kalinski et al., 1992;Becker et al., 19956) and proteinase B-specific polypep-tides (Hara-Nishimura et al, 1993a; Becker et al., 1995a;Okamoto et al, 1995) have so far been detected indeveloping as well as in mature and germinating seeds ofdifferent legumes, castor bean and pumpkin. Polypeptideswith the size of mature proteinase B were found in proteinbodies prepared from vetch seeds 8 h after the start ofimbibition. Precursors of proteinase A could only bedetected in fractions that did not contain protein bodiesand they corresponded in size to precursor polypeptidebands found in developing seeds. Three days after imbibi-tion, when both these polypeptide bands were no longerdetectable on Western blots, first newly formed proteinaseB mRNA appeared, closely followed by the appearanceof proteinase A mRNA. No signals for proteinase A- andB-specific mRNAs have been detected at earlier germina-tion days. Approximately 24 h later, the proenzyme bandsappeared in Western blots and these were followed bythe appearance of intermediate precursors as well as thoseof mature proteases within the next few days. Thisreinforces previous results which indicated that theseproteases are synthesized de novo during germination.


The proteinase B polypeptides that have been detected inthe cotyledons of mature, early imbibing and early ger-minating seeds must have been synthesized, processedand transported to protein bodies during seed develop-ment (Hara-Nishimura et al., 1993a). On the other hand,only precursor polypeptide bands of proteinase A havebeen found in extracts prepared from maturing vetchcotyledons and these were absent in protein body fractionsof 8 h imbibed seeds. This indicates that processing andtransport of proteinase A precursors occur either verylate during seed maturation or early after imbibition. Thiswould make sense, if as presumed by Kalinsky et al.(1992) and others (Becker et al., 19956) proteinase Bparticipates in the molecular maturation of proteinase Aprecursors which have the necessary Asp residue in theP-position at the processing site in front of the N-terminusof the mature polypeptide. Thus, proteinase A, which cancleave mature globulin holoproteins, would remain out-side the protein bodies until storage protein degradationis induced. Proteinase B, which can not attack matureglobulins, can be safely stored along with the globulinsinside the protein bodies. During protein body develop-ment its activity is necessary for the maturation of pro-legumin (Mtlntz et al., 1993), 2S protein precursors


(Hara-Nishimura et al., 1991, 1993ft) and membraneproteins (Inoue et al., 1995) at their Asn-flanked cleavagesites. In a similar way it could contribute to the finalactivation of proteinase A precursors during seed imbibi-tion when these precursors are transferred into the proteinbodies. Provided proteinase A-like precursors, which havebeen found outside the protein bodies in developing andmature seeds, have a C-terminal KDEL as found insimilar enzymes of germinating seeds, this tetrapeptidemay be required to delay its transfer into the vacuole.The detachment of this ER-retention signal would permitthe transfer of the precursor into protein bodies wherethe last N-terminal propeptide could then be cleaved byaction of proteinase B. If this hypothesis is correct thenKDEL detachment would play an important role incontrolling breakdown of globulin. In contrast to SH-EPand proteinase A the precursor of P34 has no KDEL atits C-terminus (Kalinski et al., 1990) and, as expected, ithas been localized in the protein bodies of maturingsoybean (Kalinsky et al., 1992). The metalloendo-pepidase of mature buckwheat has also been found to belocated inside the protein bodies along with the buckwheatlegumin (Elpidina et al., 1991). This enzyme and P34 aresupposed to be inactive inside protein bodies of mature

PHOTBNBOOY

PROTEIN BOOT

Fig. 3. Hypothetical scheme of compartmentation, processing of a storage protein (12S globulin taken as the example) and cysteine endopeptidaseprecursors as well as proteinase activation to integrate the data so far obtained for cotyledon cells of developing, mature and germinating vetch(Vicia saliva L.) seeds (A), for soybean (Glycine max L. (Merr.) (B), and buckwheat (Fagopvrum esculemum L.) (C). ER, endoplasmic reticulum,LEG, legumin; KDEL, C-terminal tetrapeptide, presumably acting as ER retention signal.


618 MQntz

seeds. The metallo-endopeptidase of buckwheat has beenfound to be inactivated by its binding to a proteaseinhibitor (Fig. 3c) that is also present in the proteinbodies (Elpidina et ai, 1991). The P34 dimer that hasbeen detected in protein bodies of mature soybean(Fig. 3b) is thought to represent an inactive state of theenzyme. Transformation of P34 into P32 is suggested togenerate monomers which are assumed to be the activeenzymes (Herman et ai, 1990; Kalinsky et ai, 1992).Other proteins, together with the storage globulins, thatare located inside the protein bodies have also to beprotected against an uncontrolled degradation by theproteinase B-like processing enzyme. This protectioncould either be due to an insensitive structure, as in thecase of hexameric legumin, or by subcompartmentationof the storage vacuole.

The amount of proteinase A and B protein, as visual-ized in Western blots, is much lower in cotyledons ofdeveloping, mature and early germinating seeds than it islater on during germling growth when most of the globulinis broken down. According to the model of Shutov andVaintraub (1987) proteinase A begins degradation ofglobulins by cleaving a small number of exposed surfacesites following the zipper cleavage mechanism. Thesecleavages trigger conformational changes that make theprotein accessible for proteinase B and carboxypeptidasewhich are both present in protein bodies of mature seeds.The low ratio of proteases to globulin early in germinationmay explain why only limited globulin degradation occursat that time. Through the combined action of proteinaseA, proteinase B and carboxypeptidases, the releasedglobulin fragments might be rapidly degraded down tosmall peptides and even amino acids. This would explainwhy it has not been possible to detect cysteine endopeptid-ase activity and cleavage products in extracts from earlygermination stages. It is still unclear how important thestored cysteine endopeptidases are for storage proteinbreakdown since they are eventually replaced by largequantities of de novo synthesized enzymes with similarspecificities. Two findings indicate that they are active.(1) early changes in mobility of the holoproteins whichhave been attributed to charge shifts resulting from thedetachment of fragments containing acid and/or basicamino acid residues. Clusters of such residues are locatedin the N-terminal regions of large vicilin subunits and inthe C-terminal region of a-chains from legumin. Bothregions are known to be cleaved at the initiation ofglobulin breakdown. However, these charge shifts havealso been interpreted as an effect of protein deamidationand recently a plant protein deamidase has been charac-terized in germinating wheat (Vaintraub et ai, 1992). (2)Proteinase A (called vignain) and B (called legumain)activity could be detected in extracts of mature seeds ofVigna aconitifolia by Kembhavi et al. (1993). Further-more, much higher activity was measured in extracts of

seeds during the first 3 dai, if the extracts were acidified.Without acidification the activities of both proteinasesincreased from 0 h of imbibition (hai) and peaked 36 haiwith a subsequent decrease until it reached nearly zeroafter 72 hai. Proteases of other classes have also beenreported to be present in dry seeds, among them serineendopeptidases and metallo-endopeptidases (see reviewby Wilson, 1986). As mentioned above a serine endopepti-dase has been shown to catalyse the initial cleavages oflarge vicilin subunits. In this case the de novo formationof the enzyme appears to be a prerequisite for thedegradation of these polypeptides (Qi et ai, 1992).According to Belozerski et al. (1990) the buckwheatmetallo-endopeptidase which is assumed to initiate legu-min degradation is present in an inhibitor-inactivatedstate in the mature seed and it becomes activated byinhibitor dissociation (Elpidina et al., 1991).

The histochemical analysis of germinating mung bean(Harris and Chrispeels, 1975) and soybean cotyledons(Diaz et al., 1993) has revealed that storage proteindegradation does not start synchronously in the wholestorage tissue. It is slowly initiated only in subepidermalcell layers (mung bean) or in cells adjacent to the vascularbundles (soybean). From these starting sites, which canobviously be different in different grain legumes, theincrease in endopeptidase activity as well as in proteindegradation proceeds wave-like inwards through thetissue. The low levels of proteinase and protein breakdownactivities that have been measured in extracts from seedsof these early germination stages could result from thesmall percentage of cells where protein reactivation hasalready been started in storage tissue. Therefore, initiallythe small amounts of stored endopeptidases should onlybecome active in protein degradation inside the proteinbodies of limited cell layers. It can even be speculatedthat in these cell layers enzyme de novo synthesis hasalready been started. The corresponding mRNA levelsremain below the limits of sensitivity of Northern blotswhich always have been done with extracts from a tissuewhere the majority of cells still were inactive in trans-cription of endopeptidase genes. New experimentalapproaches are needed to elucidate the mechanisms whichinitiate protein reactivation in storage tissues duringimbibition and early germination of seeds.

Protein storage vacuoles have been thought to beformed by transformation of the vegetative vacuole inthe storage tissue cells which already exists before globulindeposition starts (Chrispeels, 1991). Recently, it has beenreported that in developing pea cotyledons the proteinstorage vacuole is formed de novo (Hoh et ai, 1995;Robinson et ai, 1995). The vegetative vacuole degener-ates. In our laboratory this was also observed in develop-ing cotyledons of Vicia narbonensis L. (Hillmerunpublished). Protein bodies seem to be generatedfrom the storage vacuole by two different mechanisms:


(1) budding of the storage vacuole during early maturationstages of storage protein deposition; and (2) fragmentationduring later stages. In addition, it has been suggested thatprotein bodies could be generated directly from the ERduring late maturation (Robinson and Hinz, 1995). Thus,as suggested previously by Adler and Miintz (1993),differential protein body formation seems to occur. Itremains to be investigated whether the different proteinbodies are similarly equipped with proteinases or not.

Since small gene families encode proteinases A and B,differential gene activation in developing and germinatingseeds might lead to the formation of isozymes during thedifferent ontogenetic periods. Thus according to ourhypothesis (Fig. 3) processing as well as degradation ofstorage globulins in the storage tissues of developing andgerminating seeds might be controlled (1) by differentialexpression of genes encoding pre-propolypeptides of iso-forms of different cysteine endopeptidases, (2) by stepwiseand differential limited proteolysis of these endopeptidaseprecursors which finally leads to the generation of theactive mature enzyme in the vacuole; (3) by transientdifferential compartmentation of cysteine endopeptidaseprecursors and activation-catalysing enzyme(s) as well as(4) by transient differential compartmentation of degrada-tion-triggering cysteine endopeptidase and its storageglobulin substrate. It is predicted that other endopeptid-ases contributing to storage globulin processing ordegradation might be controlled in a similar fashion. Inaddition, specific conformational changes induced by theaction of the proteases on their substrate globulinscontribute to the control of processing and degradationof storage proteins. The rate of globulin breakdown mayalso be regulated via feedback regulation by the level ofamino acids at the site of storage protein breakdown ashas been established for the buckwheat cysteine endopep-tidase (Dunaevski and Belozerski, 1989b). Protein reserveactivation in storage tissues seems to be under the controlof the embryo axis. Cotyledons detached from the axisbefore imbibition exhibited no increase in endopeptidaseactivity and globulin breakdown. The mechanisms ofsignal transduction between axis and cotyledons or endo-sperm are still under investigation (recently reviewed byBewley and Black, 1994). Depletion of amino acids inthe growing axis, which is the major site of proteinbiosynthesis in the germinating seed (Dunaevsky andBelozersky, 1993) as well as phytohormonal signals trans-mitted from the axis to the storage tissues (Gifford el ai,1984; Nandi et ai, 1995) have been implicated in control-ling the breakdown of protein reserves.

Complete breakdown of storage proteins is the resultof massive de novo proteinase formation and the combinedaction of endo- and carboxypeptidases inside the proteinbodies and vacuoles in the storage tissue cells of mid-germination seeds. Both amino acids and short peptidesare transported from the vacuolar compartment into the


cytoplasm where amino- and dipeptidases degrade themfurther. The amino acids are then used to meet metabolicdemands in the storage tissue itself, like the biosynthesisof several reserve degrading hydrolases, and to meet thedemands imposed by the major site of protein biosynthesisin the germinating seed and growing germling, theembryo axis.

Acknowledgements

The author expresses his sincere gratitude to Dr C Becker, DrS Hillmer and above all to Dr D Waddel for critically readingthe manuscript which, in addition, has been very much improvedby the language editing done by Dr D Waddell. The skilfuldrawing of the figures by Mrs KJlian and the careful arrangementof the list of references by Mrs. E Bielig are gratefullyacknowledged.

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proteases and proteolytic cleavage of storage proteins in

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