Proc. Natl. Acad. Sci. USAVol. 91, pp. 8935-8939, September 1994Cell Biology

Subcellular localization of the UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-mediated0-glycosylation reaction in the submaxillary gland

(Golgi apparatus/endoplasmic reticulum)

JURGEN ROTH*t, YANG WANGt, ALLEN E. ECKHARDT*, AND ROBERT L. HILLt*Division of Cell and Molecular Pathology, Department of Pathology, University of Zdrich, Schmelzbergstrasse 12, CH-8091 Zfirich, Switzerland; andtDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710

Contributed by Robert L. Hill, May 26, 1994

ABSTRACT Addition of N-acetylgalactosamine to threo-nine and serine is the first step in the synthesis of O-glycosid-ically linked oligosaccharides. A UDP-N-acetyl-D-galac-tosamine:polypeptide N-acetylgalactosaminyltrnsferase (EC2.4.1.41) from porcine submaillary glands was recently pu-rified to electrophoretic homogeneity, and polyclonal antibod-ies against the purified transferase were raised. Immunoblotsof porcine, bovine, and ovine submaxillary gland extracts withthe anti-tansferase antibodies gave a single band and theantibodies reacted equally well with the purified glycosylatedand N-glycanase-treated transferase. Immuoelectron micro-scopic localization of the trnsferase was achieved in LowicrylK4M thin sections and frozen-thawed thin sections of porcineand bovine submaxillary gland by using the protein A-goldtechnique. Specilc gold particle labeling was observed in the cisGolgi apparatus and smooth-membraned vesicular structuresin close topological relation with it. Labeling was undetectablein the rough endoplasmic reticulum, its transitional elements,and smooth-membraned structurs close to them, the transGolgi apparatus, mucin droplets, and the plasma membrane.The onset of labeling for peptide-bound GaINAc as detectedwith Vicia villosa isolectin B4 mirrored the transferase immu-nolocalization as directly shown by double labeling and ex-tended into the trans Golgi apparatus and mucous droplets.Apomucin immunolabeling was found throughout the endo-plasmic reticulum and the intermediate compartment andpartially overlapped the region of transferase labeling in theGolgi apparatus as demonstrated by double immunolabeling.Thus, the initial step of UDP-GaINAc:polypeptide N-acetylga-lactosaminyltransferase-mediated 0-glycosylation in porcineand bovine submaxillary gland cells occurs in the cis Golgiapparatus. The possible involvement of the intermediate com-partment remains to be clarified.

A major posttranslational modification of many membrane-bound and secretory proteins is the addition of oligosaccha-ride side chains. The subcellular localization of the enzymesacting in synthesis of N-glycosidically linked oligosaccha-rides is well known (1). In contrast, some disagreement existsas to the exact subcellular compartment that contains UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosami-nyltransferase (EC 2.4.1.41) (1, 2), the enzyme that catalyzesthe initial step in O-glycosylation and leads to formation ofthe GalNAc-O-threonine/serine linkages (3). Although it hasbeen proposed that this enzyme occurs in the endoplasmicreticulum (ER) (4-8), most studies indicate that 0-glycosy-lation commences and continues in the Golgi apparatus(9-18). In particular high-resolution in situ cytochemicalstudies have provided strong evidence that the addition of

GalNAc to the polypeptide takes place in the cis Golgiapparatus ofvarious mucous-producing cells (19, 20). Studieson class 2 mutant of low density lipoprotein receptors (21)and the mouse hepatitis virus A59 El glycoprotein (22)indicated that the initiation of O-glycosylation of these gly-coproteins takes place in a late ER and a post-ER/pre-Golgicompartment, respectively. To our knowledge, however,UDP-GalNAc:polypeptide N-acetylgalactosaminyltrans-ferase has not been localized in situ by immunoelectronmicroscopy.To help clarify these ambiguities, we have studied by

immunoelectron microscopy the subcellular distribution ofthis glycosyltransferase in porcine and bovine submaxillaryglands with a specific antibody. In addition, we have com-pared the distribution of the transferase immunoreactivitywith the labeling pattern of polypeptide-linked GalNAc res-idues as detected with the Vicia villosa isolectin B4 (23) andthat of apomucin, the nonglycosylated porcine submaxillarygland mucin polypeptide (24). Our results are consistent withthe view that the initial 0-glycosylation reaction in submax-illary gland acinar cells commences in the cis Golgi appara-tus.

MATERIAL AND METHODSReagents. Purified UDP-GaINAc:polypeptide N-acetylga-

lactosaminyltransferase was prepared and used to raise an-tibodies in rabbits as described (3). A rabbit polyclonalanti-apomucin antiserum was raised as described (24) andfreed from anti-blood groupA determinant antibodies (20). V.villosa isolectin B4 conjugated to horseradish peroxidase(HRP) was obtained from Sigma. This lectin exhibits anexquisite specificity for the Tn antigen, which is GaINAca-ketosidically linked to threonine or seine (23). ProteinA-gold complexes (25) were prepared using 6-nm, 8-nm, and12-nm gold particles (26). An affminty-purified polyclonalrabbit anti-HRP antibody was obtained from Jackson Immu-noResearch and complexed to 6-nm and 8-nm gold particles(27).UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltrans-

ferase Antibody. Antisera to the electrophoretically pureN-acetylgalactosaminyltransferase were prepared as de-scribed (3), and the IgG fraction was isolated from theantisera by protein A-Sepharose chromatography. Polyacryl-amide gel electrophoresis and immunoblots were performedby published methods (3).

Tissue Processing. Small pieces from porcine and bovinesubmaxillary glands were fixed by immersion in 3% (wt/vol)

Abbreviations: ER, endoplasmic reticulum; HRP, horseradish per-oxidase.tTo whom reprint requests should be addressed at: Department ofPathology, University of Zurich, Schmelzbergstrasse 12, CH-8091Zurich, Switzerland.

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paraformaldehyde and 0.1% glutaraldehyde in 10mM sodiumphosphate, pH 7.4/0.15 M NaCl (PBS) for 2 h. After a briefrinse in PBS, the tissue pieces were immersed in 50 mMammonium chloride in PBS for 60 min to quench freealdehyde groups, rinsed in PBS, and stored at 40C until usefor ultracryotomy. Further, tissue pieces were dehydrated ina graded ethanol series by progressive lowering of the tem-perature (down to -40(C), embedded in Lowicryl K4M at-400C, and polymerized by indirect diffuse UV irradiation at-400C (28, 29).Eleron McroscopicLbin. Ultrathin sections ofLowicryl

K4M-embedded tissue were cut and placed on Parlodion/carbon-coated nickel grids. Frozen-thawed thin sections wereprepared by the methods of Griffiths et aL (30) and Tokuyasu(31). Briefly, tissue pieces were infiltrated with 2.3 M sucrosecontaining 20% (wt/vol) polyvinylpyrrolidone (Mr, 25,000). Thesamples were rapidly frozen in nitrogen slush; ultrathin sectionswere cut at -120'C usinga Reichert Ultracut E ultramicrotome,mounted on Parlodion/carbon-coated nickel grids, and storedon 1% gelatin at 40C overnight.For immunolabeling, Lowicryl K4M and frozen-thawed thin

sections were conditioned for 5-10 min on droplets of PBScontaining 1% bovine serum albumin, 0.01% Triton X-100, and0.01% Tween 20 and then transferred to droplets of antibodiesappropriately diluted in the conditioning buffer or PBS contain-ing 1% bovine serum albumin and 0.05% Tween 20. After anincubation for 1 h (frozen-thawed sections) or 2 h (LowicrylK4M sections) at room temperature, the sections were washedwith PBS and transferred to droplets containing protein A-goldcomplexes for 1 h at room temperature. Protein A-gold com-plexes were diluted in PBS containing 1% bovine serum albu-min, 0.01% Triton X-100, and 0.01% Tween 20 to an OD525 Of0.06 (6-nm and 8-nm gold particles) and of 0.1-0.2 (12-nm goldparticles). Afterward, frozen-thawed sections were rinsed withPBS, postfixed in 1% glutaraldehyde in PBS for 20 min, andembedded in 2% (wt/vol) methylcellulose containing 0.2%uranyl acetate (32). Lowicryl K4M sections were rinsed withPBS and distilled water, air-dried, and contrasted with uranylacetate and lead acetate (29).For lectin labeling, Lowicryl K4M and frozen-thawed thin

sections were conditioned for 5-10 min with PBS containing0.05% Tween and incubated with HRP-conjugated V. villosaisolectin B4 (1-5 ug/ml of conditioning buffer) for 45 min atroom temperature. Sections were washed with PBS and thenincubated with gold-labeled anti-HRP antibody (8-nm goldparticles; diluted to give an OD525 of 0.1) for 45 min at roomtemperature. The following steps including the contrasting ofthe sections were as described above for immunolabeling.For double labeling, frozen-thawed sections were incu-

bated with appropriately diluted anti-transferase antibodiesfollowed by protein A-gold complexes (6 nm) and thenpostfixed by floating the grids on droplets of 2% paraformal-dehyde for 30 min. Free aldehyde groups were subsequentlyquenched with ammonium chloride. Afterward, sectionswere incubated with anti-apomucin antibodies followed byprotein A-gold complexes (12 nm), postfixed with glutaral-dehyde, and embedded as described above. In further doublelabeling experiments, frozen-thawed sections were first in-cubated with anti-transferase antibodies and protein A-goldcomplexes (12-nm gold particles) followed by HRP-conjugated V. villosa isolectin B4 and gold-labeled anti-HRPantibodies (6-nm gold particles).To control the specificity of anti-transferase and apomucin

immunolabeling, the antibodies were replaced with the respec-tive preiumune sera or omitted in the incubation sequence.Specificity oflectin labeling was controlled by incubation ofthelectin with 1-50 mM GaINAc 30 min prior to use or incubationwith anti-HRP antibody-gold complexes alone.

RESULTSThe polyclonal rabbit anti-transferase IgG used in thesestudies was raised against electrophoretically homogeneousenzyme and shown to immunoprecipitate transferase activityfrom Triton X-100 extracts of porcine submaxillary glands(3). Immunoblots of porcine, bovine, and ovine submaxillarygland homogenates with the anti-transferase IgG gave only asingle antibody-reactive protein and the proteins migratedwith an apparent molecular mass similar to those of thepurified porcine (3) and bovine (33, 34) enzymes (data notshown). Fig. 1 shows that the anti-transferase IgG reactsequally well with the electrophoretically pure glycosylatedtransferase and the enzymatically deglycosylated enzyme.The high degree of specificity indicated by these results is inaccord with the absence of immunolabeling for transferase inmucous droplets or plasma membrane of submaxillary glandcells (see below).The acini of the submaxillary gland are composed of two

secretory cell types that can be unequivocally distinguishedon morphological grounds and by immunolabeling for apo-mucin solely detectable in the mucous-producing cells (20).Despite fine differences in the subcellular structural organi-zation between the mucous and serous cells in porcine andbovine submaxillary glands, the pattern of transferase im-munolabeling and lectin staining for polypeptide-linked Gal-NAc residues was analogous as were the results obtained inLowicryl K4M and frozen-thawed sections.

Immunoreactivity for the polypeptide N-acetylgalac-tosaminyltransferase was undetectable throughout therough ER and its transitional elements in mucous andserous cells (Figs. 2-4). As reported (20), immunolabelingfor apomucin was observed in the rough ER of mucous cellsand also present in its transitional elements and the smooth-membrane vesicular structures between them and the cisside of the Golgi apparatus (Figs. 2h and 4a). Only occa-sionally were single gold particles observed in the cis sideof the Golgi apparatus and were absent in its remainingportions, in forming and mature mucous droplets, and in theplasma membrane. Immunolabeling for apomucin was un-detectable in bovine submaxillary gland cells (data notshown). Gold particle labeling indicative of transferaseimmunoreactivity was exclusively observed in the Golgiapparatus of both mucous and serous cells and principallyconfined to cis cisternae (Figs. 2 and 3). However, occa-sionally immunolabeling for transferase extended into themiddle of the cisternal stack of mucous cells (Fig. 2e). Suchvariation could be observed in different Golgi cisternalstacks present in a single section through the same cell andwas not related to morphologically recognizable differencesbetween individual cells in the same glandular acinus.Immunolabeling for transferase was undetectable in trans

up

FIG. 1. Immunoblots with anti-transferase IgG. Pure transferase was

IVhg= treated with N-glycanase as described (3)and then analyzed on an immunoblot.Lanes: 1, N-glycanase-treated transfer-ase; 2, pure transferase; 3, partial hydro-lysis ofthe transferase with N-glycanase.The high molecular mass bands in lane 3are presumably aggregated transferasethat accumulate during N-glycanase

2 ^ treatment.

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FIG. 2. Immunolabelingforpolypeptide N-acetylgalactosaminyltransferase in Lowicryl K4M (a-c) and frozen-thawed (dand e) thin sectionsof porcine submaxillary gland mucous cells is detectable in cis Golgi apparatus cisternae and smooth-surfaced vesicular structures adjacent tothem (a, arrowheads in c, and d). Occasionally, labeling extends into the middle portion of the Golgi apparatus (e). Labeling is undetectablein cisternae of the rough ER, its transitional elements (TE), smooth-surfaced vesicles adjacent to the latter (a-d), the trans Golgi apparatus, andmucous droplets (MD). Lectin labeling for polypeptide-bound GaINAc residues corresponds to that of transferase immunolabeling andadditionally is present in the trans Golgi apparatus and forming mucous droplets (fand g). Immunolabeling for apomucin is abundant in therough ER and vesicular structures in between the ER and the Golgi apparatus (h) (for more details, see ref. 18). (a and b, x54,O00; c and d,x36,000; e, x44,000; f, x50,000; g, x63,000; h, x32,000.)

cisternae and the trans Golgi network, in forming andmature secretory granules, and in the plasma membrane ofboth mucous and serous cells. A closer inspection of the

region between the rough ER including its transitionalelements and the cis side ofthe Golgi apparatus revealed thepresence of immunolabeling for transferase associated with

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&AMIVIL My

FIG. 3. Frozen-thawed thin sections ofa serous cell from porcinesubmaxillary gland with immunolabeling for transferase over the cisGolgi apparatus (a) and some associated vesicular structures facingthe rough ER cisternae (arrowheads) (a). Specific immunolabeling isundetectable in the rough ER (arrowheads) (a and b) and mucousdroplets (MD). N, nucleus. (x26,500.)

smooth-membraned vesicles (Fig. 2 a-d). Invariably, onlya fraction of these vesicular elements that was situatedtoward the cis side of the Golgi apparatus exhibited trans-ferase immunolabeling. Slightly overlapping distribution ofimmunolabeling for apomucin and transferase in thesestructures was evident by double immunolabeling (Fig. 4a).In control incubations, immunolabeling for transferase andapomucin was abolished (data not shown).To complement the results of the transferase immunola-

beling, the distribution of threonine- and seine-linked Gal-

NAc residues as specifically visualized with the V. villosaisolectin B4 (23) was studied. Lectin labeling was undetect-able throughout the rough ER and its transitional elements inmucous (Fig. 2fand g) and serous cells. Specific gold particlelabeling was present over cis Golgi cisternae and smooth-membraned vesicular structures associated with the cis sideof the Golgi apparatus (Fig. 2 f and g). Double immunola-beling for transferase and lectin labeling for GaINAc residuesproved their colocalization (Fig. 4b). Additional lectin label-ing also occurred over the trans Golgi apparatus and inmucous droplets. In the control experiments, lectin labelingwas abolished (data not shown).

DISCUSSIONThe N-acetylgalactosaminyltransferase studied here hasbeen purified to electrophoretic homogeneity from porcinesubmaxillary glands (3) and has very similar structural prop-erties to the transferase purified from bovine colostrum (34).The porcine enzyme has the same amino acid sequence as thebovine enzyme (refs. 33 and 34 and Y.W., N. Agrwal, andR.L.H., unpublished data). The enzyme incorporatesN-acetylgalactosamine into 0-glycosidic linkage with bothserine and threonine hydroxyl groups (34, 35), but the rate ofincorporation is highly dependent on the amino acid se-quences flanking these residues.

In the present study, a highly specific antibody raisedagainst pure porcine transferase was used to study by high-resolution immunoelectron microscopy the subcellular dis-tribution of immunoreactive transferase protein in mucousand serous cells of the porcine and bovine submaxillaryglands. In both secretory cell types, immunolabeling for thetransferase was detected only in the cis Golgi apparatus. Thislocation of the transferase accounts for the fact that the cisGolgi apparatus is the major compartment containing cy-

FIG. 4. Double immunolabeling for apomucin (large gold particles) and transferase (small gold particles) shows coexistence of bothimmunoreactivities in vesicular structures adjacent to the cis Golgi apparatus and portions of one or two cis Golgi cisternae (arrowheads) (a).In b codistribution of labeling for the transferase (large gold particles) and polypeptide-bound GalNAc residues (small gold particles) indicatesthe onset of 0-glycosylation in the cis Golgi apparatus. Additional single lectin labeling exists in the trans Golgi apparatus and mucous droplets(MD). TE, transitional element of the rough ER. (x102,000.)

A a--

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tochemically detectable polypeptide-linked GaINAc resi-dues. This is supported by the present studies, which showthat the transferase and GalNAc residues are colocalized inthe cis Golgi apparatus by double labeling ofultrathin frozen-thawed tissue sections. These results clearly demonstratethat in these cell types the initiation of 0-glycosylation takesplace in the cis Golgi apparatus and not in the ER or itstransitional elements. Others, using the Helix pomatia lectinand monoclonal anti-Tn antibodies, reported the presence ofGalNAc residues in limited portions of the rough ER (6, 7).It is possible that the transferase is localized differently indifferent cell types (13, 36, 37) but the primary chondrocytecultures (6) in which it appeared that GalNAc residues werein the ER may well have been structurally altered as aconsequence of the prolonged culture conditions (6). Indeed,the regions of the ER that appeared to contain GatNAcresidues were structurally different from the ER in healthycells. It is also possible that some ER resident proteins haveleaked out of the ER, and in accord with the establishedtrafficking of proteins between the ER and the cis Golgiapparatus, cycled back to the ER (38, 39). This could accountfor the observation that class 2 mutant low density lipopro-tein receptors that accumulate in the ER and its transitionalelements contain GalNAc residues (21).

It was of interest that no transferase was found in theintermediate compartment consisting of tubulovesicular ele-ments situated between the ER and the cis Golgi apparatus(40-42). This contrasts with the observation (41, 42) that p53and p58, two marker proteins for the intermediate compart-ment, are also detected in the cis Golgi apparatus. There isalso convincing evidence that 0-glycosylation of the Elglycoprotein of the mouse hepatitis virus A59 occurs in anintermediate compartment between the rough ER and theGolgi apparatus (22), but this budding compartment is in-duced in virus-infected cells and is probably not present inuninfected cells. In contrast to virus-infected cells, 0-gly-cosylation by the polypeptide N-acetylgalactosaminyltrans-ferase commences in the cis Golgi apparatus. Thus thetransferase appears to be a unique marker for the cis Golgiapparatus in the secretory cell types studied here.

We are grateful to Norbert Wey for preparing the photographs.This work was supported by the Swiss National Science FoundationGrant 31-26273.89 (to J.R.) and by Research Grant GM 25766 (toR.L.H.) from the National Institute of General Medical Sciences,National Institutes of Health.

1. Roth, J. (1987) Biochim. Biophys. Acta 906, 405-436.2. Sadler, J. A. (1984) in Biology of Carbohydrates, eds. Gins-

burg, V. & Robbins, P. W. (Wiley, New York), pp. 199-288.3. Wang, Y., Abernethy, J. L., Eckhardt, A. E. & Hill, R. L.

(1992) J. Biol. Chem. 267, 12709-12716.4. Strous, G. J. A. M. (1979) Proc. Natl. Acad. Sci. USA 76,

2695-2698.5. Patzelt, C. & Weber, B. (1986) EMBO J. 5, 2103-2108.6. Perez-Villar, J., Hidalgo, J. & Velasco, A. (1991) J. Biol. Chem.

266, 23967-23976.7. Ellinger, A. & Pavelka, M. (1992) J. Histochem. Cytochem. 40,

919-930.8. Cummings, R. D., Kornfeld, S., Schneider, W. J., Hobgood,

K. B., Tolleshaug, H., Brown, M. S. & Goldstein, J. L. (1983)J. Biol. Chem. 238, 15261-15273.

9. Elhammer, A. & Kornfeld, S. (1984) J. Cell Biol. 99, 327-331.10. Niemann, H., Boschek, B., Evans, D., Rosing, M., Tamura, T.

& Klenk, H. (1982) EMBO J. 1, 1499-1504.11. Johnson, D. & Spear, P. (1983) Cell 32, 987-997.12. Hanover, S. A., Elting, S., Mintz, G. R. & Lennarz, W. L.

(1982) J. Biol. Chem. 257, 10172-10177.13. Roth, J., Taatjes, D., Weinstein, J., Paulson, J., Greenwell, P.

& Watkins, W. (1986) J. Biol. Chem. 261, 14307-14312.14. Roth, J., Greenwell, P. & Watkins, W. (1988) Eur. J. Cell Biol.

46, 105-112.15. Abejon, C. & Hirschberg, C. B. (1987) J. Biol. Chem. 262,

4153-4159.16. Piller, V., Piller, F. & Fukuda, M. (1990) J. Biol. Chem. 265,

9264-9271.17. Pascale, M. C., Erra, M. C., Malagolini, N., Seraflini-Cessi, F.,

Leone, A. & Bonatti, S. (1992) J. Biol. Chem. 267, 25196-25202.

18. Schweizer, A., Clausen, H., van Meer, G. & Hauri, H.-P.(1994) J. Biol. Chem. 269, 4035 4041.

19. Roth, J. (1984) J. Cell Biol. 98, 399-406.20. Deschuyteneer, M., Eckhardt, A., Roth, J. & Hill, R. (1988) J.

Biol. Chem. 263, 2452-2459.21. Pathak, R. K., Merkie, R. K., Cummings, R. D., Goldstein,

J. L., Brown, M. S. & Anderson, R. G. W. (1988) J. Cell Biol.106, 1831-1841.

22. Tooze, S. A., Tooze, J. & Warren, G. (1988) J. Cell Biol. 106,1475-1487.

23. Tollefsen, S. E. & Kornfeld, R. (1983) J. Biol. Chem. 238,5172-5176.

24. Eckhardt, A. E., Timple, C. S., Abernethy, J. L., Toumadje,A., Johnson, W. C., Jr., & Hill, R. L. (1987) J. Biol. Chem. 262,11339-11344.

25. Roth, J., Bendayan, M. & Orci, L. (1978) J. Histochem.Cytochem. 26, 1074-1081.

26. Slot, J. & Geuze, H. (1985) Eur. J. Cell Biol. 38, 87-93.27. Roth, J., Saremaslami, P. & Zuber, C. (1992) Histochemistry

98, 229-236.28. Roth, J., Bendayan, E., Carlemalm, E., Villiger, W. & Gara-

vito, M. (1981) J. Histochem. Cytochem. 29, 663-671.29. Roth, J. (1989) in Vesicular Transport. Methods in CellBiology,

ed. Tartakoff, A. (Academic, San Diego), pp. 513-551.30. Griffiths, G., Simons, K., Warren, G. & Tokuyasu, K. (1983)

Methods Enzymol. 96, 446-485.31. Tokuyasu, K. (1986) J. Microsc. (Oxford, U.K.) 143, 139-149.32. Tokuyasu, K. (1989) Histochem. J. 21, 163-171.33. Homa, F. L., Hollander, T., Lehman, D. J., Thomsen, D. R.

& Elhammer, A. P. (1993) J. Biol. Chem. 268, 12609-12616.34. Hagen, F. K., VanWuyckhuyse, B. & Tabak, L. A. (1993) J.

Biol. Chem. 268, 18960-18965.35. Wang, Y., Agrwal, N., Eckhardt, A. E., Stevens, R. D. & Hill,

R. L. (1993) J. Biol. Chem. 268, 22979-22983.36. Brada, D., Keijaschki, D. & Roth, J. (1990) J. Cell Biol. 110,

309-318.37. Velasco, A., Hendricks, L., Moreman, K. W., Tulsiani,

D. R. P., Touster, 0. & Farquhar, M. G. (1993) J. Cell Biol.122, 39-51.

38. Pelham, H. R. B. (1991) Curr. Opin. Cell Biol. 3, 585-591.39. Hsu, V. W., Yuan, L. C., Nuchtern, J. G., Lippincott-

Schwartz, J., Hammerling, G. J. & Klausner, R. D. (1991)Nature (London) 352, 441-444.

40. Lippincott-Schwartz, J. (1993) Trends Cell Biol. 3, 81-88.41. Hauri, H.-P. & Schweizer, A. (1992) Curr. Opin. Cell Biol. 4,

600-608.42. Saraste, J. & Kuismanen, E. (1992) Semin. Cell Biol. 3,

343-355.

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