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Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 7227-7231, August 1992 Biochemistry Distinct processes mediate and glycopeptide export from the in Saccharomyces cerevisiae KARIN RoMISCH AND RANDY SCHEKMAN Department of Molecular and and Howard Hughes Research Institute, University of California, 401 Barker Hall, Berkeley, CA 94720 Communicated by David D. Sabatini, April 17, 1992 (received for review March 6, 1992)

ABSTRACT and export from the Saccha- degraded in a chloroquine-sensitive fashion suggesting their romyces cerevisiae endoplasmic reticulum was examined in vitro transport to the . using the pro-a-factor and a synthetic tri- The results described above suggest that the transport peptide containing the acceptor site for N-linked routes of the tripeptides are dependent on the biological as substrates. The release of both glycosylated pro-a-factor system in which they are used. Our aim was to determine and glycotripeptide from the endoplasmic reticulum was de- whether a glycosylation acceptor tripeptide, Ac-Asn-Tyr- pendent on , temperature, and ATP. Antibodies against Thr-NH2 (Ac-NYT-NH2), is a suitable marker for fluid-phase two essential for the formation of transport vesicles, transport in the yeast secretory pathway. In Saccharomyces Sec23p and p105, inhibited glyco-pro-a-factor exit from the cerevisiae, protein transport from the ER to the Golgi com- endoplasmic reticulum but did not affect the release of the plex has been reconstituted in vitro and used to characterize glycosylated tripeptide. Furthermore, in contrast to pro-a- the components involved in this process (10-15). Vesicular factor, the exported glycopeptide was not associated with a intermediates of ER-to-Golgi transport can be isolated from membrane fraction and did not acquire Golgi-specific a(1-6)- reactions in which or permeabilized yeast linked residues. We conclude that the glycosylated spheroplasts are the donor membranes (11, 12, 15). We have tripeptide leaves the yeast endoplasmic reticulum by a route examined the transport of the tripeptide in the yeast in vitro different from the secretory pathway, possibly through an systems in direct comparison with the yeast secretory protein ATP-driven pump. pro-a-factor. We have found that the glycosylated acceptor tripeptide does not exit from the yeast ER in membrane- The secretory pathway ofeukaryotic cells consists ofa series bounded vesicles, that peptide export is not inhibited by of distinct membrane-bounded between which antibodies that block the formation oftransport vesicles from proteins are moved in a vectorial fashion by consecutive the ER, and that the core glycosylated tripeptide does not vesicle budding and fusion events (1). A set of resident acquire Golgi-specific modifications of its oligosaccharyl soluble and membrane proteins and a specific compo- moiety. Thus the glycosylation acceptor tripeptide cannot be sition oftheir membranes define the structure and function of used as a bulk flow marker for the secretory pathway in S. each of these organelles (1). Proteins destined for the secre- cerevisiae but may serve as a tool in the characterization of tory pathway carry a that targets them from the an as yet unknown export mechanism from the yeast ER. cytosol to the endoplasmic reticulum (ER), the first of the secretory pathway (2). Many ER-resident proteins contain sequences that are responsible for their retention in MATERIALS AND METHODS this organelle (3-5). Therefore, current views favor the idea Strains, Growth Conditions, and Preparation of Cell Frac- that export from the ER does not require a specific signal and tions. The S. cerevisiae strains used were RSY255 (MATa, that, with the exception of large complexes, all proteins ura3-52, leu2-3,112) and RSY607 (MATa, ura3-52, leu2- translocated into the of the ER are packaged into 3,112, PEP4:: URA3). Yeast cells were grown to early loga- transport vesicles (6). Wieland et al. (7) tested this hypothesis rithmic phase at 30°C in YP (2% Bacto-peptone and 1% yeast directly by using iodinated acyltripeptides containing the extract, both from Difco) containing 2% or 5% . acceptor site for N-linked glycosylation (Asn-Xaa-Thr/Ser) Anti-a(1-6)-mannose serum has been described (10). Antisera as markers for the fluid phase of the ER to measure the bulk were raised against p105 and Sec23p (24). IgG fractions from flow rate from the ER to the surface of mammalian cells. The these sera were purified (16) and were a gift from Tohru tripeptides were glycosylated in the ER and secreted with a Yoshihisa (this laboratory). 35S-labeled prepro-a-factor was halftime of 10 min, which was taken as an indication that fast synthesized in vitro as described (10, 17) except that the S-100 and efficient export from the cell does not require a specific fraction used for the of prepro-a-factor mRNA signal in the molecule that is secreted (7). In studies from the was prepared by liquid lysis (18). Cytosol for the same laboratory (8) the temperature and cytosol dependence semi-intact transport assay was prepared from RSY255 by of the exit of a glycotripeptide from semi-intact mammalian the bead beating method (12) in the presence or absence of cells were found to be similar to that of secretory protein ATP as indicated. The final protein concentration was 10 export. mg/ml. Semi-intact cells were prepared from logarithmically Geetha-Habib et al. (9) investigated the fate of three growing RSY255 as described (10). Ac-NYT-NH2 was syn- different acceptor tripeptides after their injection into the thesized by David King (University of California, Berkeley) of Xenopus oocytes. The were core- and iodinated as described (7). Specific radioactivity of the glycosylated in this system; however, their glycosyl side 1251-Ac-NYT-NH2 ranged from 1.2 to 2.2 x 107 cpm/nmol. chains were not processed to complex , In Vitro Transport in Semi-Intact Cells. In vitro transport suggesting a failure to progress to the , and reactions were performed essentially as described (12). Stage none of the glycopeptides was secreted. Glycopeptides were I (translocation) reaction mixtures contained either S-100 lysate with 35S-labeled prepro-a-factor or 107 cpm of 125I-Ac- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ER, endoplasmic reticulum; MSP, medium-speed in accordance with 18 U.S.C. §1734 solely to indicate this fact. pellet; MSS, medium-speed supernatant.

7227 Downloaded by guest on September 28, 2021 7228 Biochemistry: Romisch and Schekman Proc. Natl. Acad. Sci. USA 89 (1992) NYT-NH2 in a final volume of 160 ul. For stage II (transport) reactions, permeabilized yeast spheroplasts containing either glycosylated pro-a-factor or glycosylated acceptor tripeptide were incubated in B88 (20 mM Hepes-KOH, pH 6.8/150 mM potassium acetate/5 mM magnesium acetate/250 mM sorbi- 10 6C10lo + tol) at 20'C or at the temperature indicated in the figure 8-20 0C, + legend. Both stage I and stage II reaction mixtures contained 50 ,uM GDP-mannose. Reaction mixtures without ATP 6 lacked ATP and the ATP-regenerating system, and cytosol o a- for these assays was prepared in the absence of ATP. i-u Samples were then centrifuged to obtain a medium-speed supernatant (MSS) fraction and a medium-speed pellet (MSP) 2 fraction and digested with trypsin as described (12). Glyco- sylated material in both fractions was analyzed by precipi- 0 tation with Con A-Sepharose. The ratio of glycosylated 0 20 40 60 80 100 120 140 [35S]pro-a-factor or 1251-labeled acceptor tripeptide in the minutes MSS to total glycosylated [35S]pro-a-factor or 125I-labeled acceptor tripeptide (MSS plus MSP) is expressed as percent is,/; ,P 4 - 8 -?1-~~~~~~~~~~~~~"C, release. Time courses, Con A precipitations, and precipita- -0- 10o C, - 7 tions with anti-a(1-6)-mannose antibodies were performed as ~~~~~~~~~20"C, - described (12). Inhibition of budding from the ER by anti- Sec23p and anti-plO5 antibodies was achieved by incubating 6 10 ,ul of washed stage I membranes with 1 dul of each of the intac yeat5 A20tC, + IgG fractions of anti-Sec23p immune (5.7 mg/ml) and pre- immune (4.7 mg/ml) and anti-plO5 immune (16.1 mg/ml) and o preimmune (15.3 mg/ml) sera for 5 min on ice prior to the >.oE. 3- stage II reactions. 2- Flotation in Sucrose Gradients. Samples (100 ,ul) of MSP FIG 1."C+yon eprtr eurmnsfrterlaeo fractions of stage II reactions were mixed with sucrose and 1.3 ml and a final B88 without sorbitol to a final volume of 0 sucrose concentration of 50% (wt/vol). Gradients were gen- 0 20 40 60 80 100 1 20 140 erated by layering 1.3 ml of each 60%o, 50% (containing the minutes sample), 40%, and 30%o sucrose solution in B88 without sorbitol in 5.3-ml Ultraclear thin-wall tubes (Beckman). Gra- FIG. 1. Cytosol and temperature requirements for the release of dients were centrifuged for 34 hr at 40,000 rpm and 4°C in an glycosylated pro-a-factor and glycosylated Ac-NYT-NH2 from semi- SW50.1 rotor (Beckman). Gradient fractions (200 ,u) were intact yeast cells. At the indicated times, stage II reactions were collected by hand from the top. The density of each fraction stopped by cooling the aliquots to 4"C and fractionation into a MSP was measured with a Zeiss refractometer and expressed as (containing the semi-intact cells) and a MSS. Glycosylated pro-a- factor (Upper) or glycosylated Ac-NYT-NH2 (Lower) at each time percent (wt/wt) sucrose. The content of [35S]pro-a-factor point was quantified by Con A precipitation. Closed symbols indicate was determined by liquid scintillation counting, and the the presence of90 pg ofcytosol per 25-,ul transport reaction mixture; content of 125I-labeled tripeptide by y counting. Glycosylated open symbols represent reactions that lacked cytosol. Incubation [35S]pro-a-factor and 125I-labeled tripeptide were quantified temperatures for the transport reactions are indicated. All samples by Con A precipitation. were done in duplicate. The zero time point of each set of samples was subtracted as background from the corresponding values. Max- imal release of Con A-precipitable material into the MSS for both RESULTS AND DISCUSSION pro-a-factor and Ac-NYT-NH2 was -50%o efficient. Exit of Glycosylated Acceptor Tripeptide from the Yeast ER in Vitro Is Cytosol- and Temperature-Dependent. Protein sylated pro-a-factor in the MSS at the last two time points transport from the S. cerevisiae ER to the Golgi complex has may have been due to partial cell lysis after these prolonged been reconstituted in vitro in well-characterized semi-intact incubations, which may have resulted in release of ER cell systems (10, 12, 14, 15). The exit ofthe secretory protein membranes into the MSS. Glycosylated pro-a-factor not pro-a-factor from the ER in this system is dependent on the enclosed within membranes was not measured under the presence of cytosol and a physiological temperature (10, 12, assay conditions because it is susceptible to digestion with 14, 15). In a mammalian semi-intact cell system the trypsin, in contrast to the glycotripeptide. of a glycosylated acceptor peptide into the culture medium is The export of glycosylated acceptor tripeptide from the S. stimulated by raising the temperature from 15°C to 37°C and cerevisiae ER in vitro displayed similar features. At 20TC, the addition of cytosol (8). Fig. 1 shows a direct comparison half-maximal release in the presence ofcytosol (Fig. 1 Lower, ofthe kinetics ofexport from the ER in semi-intact yeast cells 10 Release of glycosyl- of core-glycosylated pro-a-factor and core-glycosylated ac- filled circles) was reached after min. ceptor tripeptide. Pro-a-factor did not exit from the yeast ER ated Ac-NYT-NH2 into the MSS was maximal after about 40 in the absence of cytosol (Fig. 1 Upper, open symbols) or at min. As for glycosylated pro-a-factor, glycotripeptide exit at 4°C (triangles). At 20°C, half-maximal budding of pro-a- 10"C in the presence of cytosol (filled squares) reached its factor in the presence of cytosol (filled circles) was reached half-maximal value at 30 min and glycotripeptide release into after 15 min, and incubation times longer than 40 min resulted the MSS was maximal after 60 min. In contrast to pro-a- in no further significant release of glycosylated pro-a-factor factor, however, some glycosylated tripeptide exited from into the MSS. When the assay temperature was lowered to the ER at 40C (triangles) and in the absence of cytosol (open 10°C (filled squares) the time required to reach half-maximal symbols). At 10TC and 20WC, glycotripeptide release in the pro-a-factor exit in the presence of cytosol was increased to absence ofcytosol reached about 30% ofthat in the presence 30 min and maximum release ofpro-a-factor from the ER was of cytosol (compare open and filled circles, open and filled achieved after 60 min. The slightly elevated levels of glyco- squares). Downloaded by guest on September 28, 2021 Biochemistry: R6misch and Schekman Proc. Natl. Acad. Sci. USA 89 (1992) 7229 Release of Glycosylated Acceptor Tripeptide from the Yeast ER Is ATP-Dependent. Baker et al. (10) and Rexach and 0 Schekman (12) have shown that export of the secretory t 20- protein pro-a-factor from the ER of permeabilized yeast Cu 8 b 45 i spheroplasts is dependent upon energy in the form of ATP. ° 15- Helms et al. (8) demonstrated a requirement for ATP in the 9- x 40w release of a glycosylated octanoyltripeptide into the medium E 0 ° CL 10- from permeabilized CHO 15B cells. We directly compared _ -o 35 w the energy dependence ofpro-a-factor budding and Ac-NYT- .O- NH2 release from the ER in the semi-intact yeast cell system. 30 of Export glycosylated pro-a-factor from the yeast ER in 0 25 vitro was entirely ATP-dependent. The presence of 1 mM 5 ATP and the ATP-regenerating system stimulated the exit of tom glycosylated acceptor tripeptide about 5-fold as compared with a reaction performed in the absence of ATP. In contrast 1 0 55 to glycosylated pro-a-factor, however, some glycosylated Ac-NYT-NH2 was released into the MSS even in the absence z m 8- 50 of ATP and the ATP-regenerating system. Glycosylated Acceptor Tripeptide in the MSS Is Not Mem- 45.j brane-Associated. In MSS fractions derived from semi-intact 0 40 ' 0 spheroplasts or a yeast -based budding assay, 0 E 4- glycosylated pro-a-factor is protected from digestion with o 35 ' trypsin (10, 12, 13) and sediments with a membrane fraction 7- 2 -.O in sucrose gradients (12). These results suggest that glyco- sylated pro-a-factor is exported from the S. cerevisiae ER in --,2 5 membrane-bounded vesicles. In studies that investigated the 05 10 15 2~~~20 25 export of glycosylated octanoyl acceptor tripeptide from top fraction # bottom intact mammalian cells, the glycotripeptide was found to float in sucrose gradients at positions indicative of its location in FIG. 2. Flotation properties of pro-a-factor (Upper) and Ac- the ER and Golgi compartments (7). The position of ER to NYT-NH2 (Lower) from MSS samples in sucrose density gradients. Golgi transport vesicles in these gradients is not known. In Fractions were collected from the top. Glycosylated pro-a-factor, addition, the glycosylated octanoyltripeptide was unable to Ac-NYT-NH2 content and the density of each fraction were quan- cross the membranes when added to the Golgi fraction (7). tified as described in Materials and Methods. Filled circles represent fractions derived from a transport reaction performed in the presence Glycosylated octanoyltripeptide secreted from permeabi- of ATP. Open squares represent fractions derived from a reaction lized CHO cells in vitro is not membrane-enclosed, as shown performed in the absence of ATP. Small open circles indicate the by precipitation with Con A-Sepharose performed in the density of each fraction in percent (wt/wt) sucrose. presence or absence of detergent (8). Because the glycopeptide is not -sensitive, we gradients irrespective of whether the transport reaction was could not use a protease protection assay to determine performed in the presence or absence of ATP and an ATP- whether it was released from the yeast ER in membrane- regenerating system (Fig. 2 Lower). We conclude that in bounded vesicles. Therefore we investigated the sedimenta- contrast to glycosylated [35S]pro-a-factor, glycosylated 1251- tion properties of glycosylated Ac-NYT-NH2 in the MSS labeled Ac-NYT-NH2 is released from the yeast ER in a free fraction. We found that during a 1-hr at 300,000 form and not in membrane-bounded vesicles. x g, 90%0 of the glycosylated 1251-labeled acceptor peptide The data presented thus far do not exclude the possibility from the MSS remained in the supernatant whereas about that some ofthe budded vesicles are "leaky" or that yeast ER 80o of the glycosylated [35S]pro-a-factor released into the membranes are permeable to glycopeptides. However, the MSS sedimented into the pellet fraction. The MSS fractions requirement for ATP and incubation at physiological tem- containing either glycosylated [35S]pro-a-factor or glyco- perature and the stimulation of glycopeptide export by a sylated Ac-NYT-NH2 were further analyzed by density sed- cytosol fraction are consistent with an active transport pro- imentation in 20-70%o (wt/vol) sucrose step gradients. Gly- cess. Glycopeptide formation in semi-intact yeast cells is cosylated pro-a-factor sedimented at 30o (wt/wt) sucrose, ATP-independent (unpublished data) but takes place in the consistent with the results from Rexach and Schekman (12), lumen of the ER as the acceptor tripeptide competes for the who found that ER-to-Golgi transport vesicles from semi- posttranslational glycosylation of pro-a-factor (unpublished intact yeast cells sedimented at 32% (wt/wt) sucrose. In data). ATP is required for a-factor precursor translocation contrast to [35S]pro-a-factor, all 1251-labeled acceptor tripep- across the yeast ER membrane in vitro. When ATP is tide in the MSS remained on top ofthe velocity sedimentation omitted, no glycosylation of urea-denatured prepro-a-factor gradient. is observed (unpublished data), suggesting that oligosaccha- In addition we investigated the flotation properties of ryltransferase is not accessible to a nontranslocated sub- glycosylated tripeptide and glycosylated pro-a-factor after strate. Further, under conditions that do not allow export their release from the yeast ER. The MSS fractions were from the ER in semi-intact cells, glycosylated pro-a-factor adjusted to 50%o (wt/vol) sucrose and layered into 30-60o and glycotripeptide are protected from digestion with en- (wt/vol) sucrose step gradients. Glycosylated [35S]pro-a- doglycosidase H in the absence but not in the presence of 1% factor from a MSS derived from a transport reaction per- Triton X-100 (unpublished data), which demonstrates that the formed in the presence of ATP and an ATP-regenerating ER membranes are intact. system floated up to about 32% (wt/wt) sucrose (Fig. 2 Antibodies Against Sec23p and p105 Inhibit Release of Upper, filled circles, fractions 4-6). Glycosylated pro-a- Glycosylated Pro-a-Factor, But Not of Glycosylated Acceptor factor from a budding reaction performed in the absence of Tripeptide, from the Yeast ER. To address the possibility that ATP remained in the load fractions (Fig. 2 Upper, open budded vesicles are selectively glycopeptide-porous, we squares, fractions 14-18). Glycosylated 125I-labeled Ac- sought conditions that specifically inhibit vesicle budding. NYT-NH2 remained in the load fractions of the flotation The formation of ER transport vesicles requires the function Downloaded by guest on September 28, 2021 7230 Biochemistry: Romisch and Schekman Proc. NatL Acad. Sci. USA 89 (1992) of several proteins that have been identified by genetic the peptide is exported from these cells via the Golgi com- screening procedures (19) and partially characterized bio- plex. We investigated whether glycosylated 1251-Ac-NYT- chemically and morphologically (10-13, 20-22). One ofthese NH2 contained a(1-6)-linked mannose after in vitro transport proteins, Sec23p, is peripherally associated with the yeast ER in semi-intact cells by immunoprecipitation with anti-a(1-6)- membrane and purifies as a complex with a 105-kDa poly- mannose antibodies. Glycosylated pro-a-factor served as a peptide, p105 (20). Antibodies against both proteins have control. Within 40 min at 200C, about 40%o of the Con been shown to specifically inhibit the budding reaction in A-precipitable glycosylated [35S]pro-a-factor acquired a(1- vitro (20, 24) and thereby block the exit of secretory proteins 6)-linked mannose residues (Fig. 4 Upper, compare light gray such as pro-a-factor from the yeast ER. We compared the with dark gray bars). In contrast, even after 100 min at 200C influence of anti-Sec23p and anti-plO5 antibodies on the no significant fraction of the glycosylated 1251-labeled accep- export of glycosylated 125I-Ac-NYT-NH2 and [35S]pro-a- tor peptide became precipitable with the anti a(1-6)-mannose factor. Semi-intact cells containing either [35S]pro-a-factor or antibodies (Fig. 4 Lower, compare light gray with dark gray 1251-Ac-NYT-NH2 were incubated in the absence or presence bars). Therefore, the majority of glycosylated Ac-NYT-NH2 of the indicated amounts of preimmune or immune IgG released from the ER of semi-intact cells in vitro does not directed against p105 or Sec23p (see legend to Fig. 3). seem to enter the Golgi apparatus. However, we cannot Antibodies against p105 and Sec23p inhibited the release of the glycosylated pro-a-factor into the MSS (Fig. 3, gpaf, com- exclude that the core-glycosylated peptide enters Golgi pare dark gray bars with light gray bars) but had no effect on complex but is a poor substrate for a(1-6)-mannosyltrans- the export of glycosylated 1251-Ac-NYT-NH2 (g-Ac-NYT- ferase. Our data are consistent with the results of Geetha- NH2, compare dark gray bars with light gray bars). Control Habib et al. (9), who demonstrated that glycosylation accep- experiments showed that antibody inhibition of pro-a-factor tor,tripeptides do not acquire resistance budding was prevented by titration of the antibody with pure in Xenopus oocytes. Sec23p or p105 (24). We have compared the behavior of the glycosyl acceptor Core-Glycosylated Acceptor Tripeptide Does Not Become tripeptide Ac-NYT-NH2 with that of the yeast secretory Modiied in Golgi Membranes. In S. cerevisiae, secretory protein pro-a-factor in a S. cerevisiae cell-free reaction proteins that contain the appropriate glycosylation acceptor site in their protein sequence are core-glycosylated in the ER C. A and further modified by addition of a(1-6)-, a(1-2)-, and n a(1-3)-linked mannose residues in the Golgi complex (23). Antibodies raised against a(1-6)-linked mannose have been used to demonstrate the transport of [35S]pro-a-factor to Golgi membranes in semi-intact yeast cells (10, 12). In mammalian cells, addition of N-acetylglucosamine residues to the glycosyl side chains in the Golgi complex results in resistance to cleavage by endoglycosidase ,.E {, 1 Fo ~ ~ ~~in H, whereas Golgi-modified yeast remain en- doglycosidase H-sensitive (23). Wieland et al. (7) have re- ported that a fraction of the glycosylated octanoyl acceptor tripeptide secreted from intact mammalian cells is resistant to digestion with endoglycosidase H and have concluded that ...Co6.. o g puf g-Ac-NYT -NH

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FIG. 3. Antibodies against p105 and Sec23p inhibit the release of glycosylated pro-a-factor (gpat), but not of glycosylated (g) Ac- FIG. 4. Pro-a-factor, but not Ac-NYT-NH2, acquires a(1-6)- NYT-NH2, into the MSS. Washed semi-intact cells (10 Al) containing mannose residues in semi-intact cells. Semi-intact celis containing either glycosylated pro-a-factor or glycosylated Ac-NYT-NH2 were glycosylated pro-a-factor (Upper) or glycosylated Ac-NYT-NH2 incubated with anti-p105 IgG (aplO5) (16.1 ,ug) or the corresponding (Lower) were incubated at 20"C in the presence of cytosol, 1 mm preimmune IgG (pre) (15.3 ,ug) with anti-Sec23p IgG (5.7 jig) or the ATP, and an ATP-regenerating system for the indicated times and corresponding preimmune IgG (4.7 Ag), or in the absence of IgG for subsequently fr-actionated into MSS and MSP. Half of each MSS 5 min on ice prior to the stage II reaction. Glycosylated material fr-action was analyzed by precipitation with Con A-Sepharose, the released into the MSS before the stage II incubation was subtracted other half by precipitation with antibodies direted against a(1-6)- as background. All reactions were performed in duplicate and the linked mannose residues and protein A-Sepharose. Samples at all maximal variation between duplicate samples was 3%. time points were taken in duplicate. Downloaded by guest on September 28, 2021 Biochemistry: R6misch and Schekman Proc. Natl. Acad. Sci. USA 89 (1992) 7231

designed to examine protein export from the ER and trans- supported by grants from the National Institutes of Health and the port to the Golgi apparatus. The temperature, cytosol, and Howard Hughes Research Institute. ATP requirements for the release of glycosylated pro-a- factor and glycotripeptide from the yeast ER are similar. 1. Pelham, H. R. B. (1989) Annu. Rev. Cell Biol. 5, 1-23. in contrast to pro-a-factor, the exported glyco- 2. Walter, P. & Lingappa, V. R. (1986) Annu. Rev. Cell Biol. 2, However, 499-516. tripeptide does not reside in membrane-bounded vesicles, its 3. Munro, S. & Pelham, H. R. B. (1987) Cell 48, 899-907. export is not inhibited by antibodies against proteins that 4. Nilsson, T., Jackson, M. & Peterson, P. A. (1989) Cell 58, have been shown to be involved in the formation of transport 707-718. vesicles from the ER, and the glycosyl side chain of the 5. Jackson, M., Nilsson, T. & Peterson, P. A. (1990) EMBO J. 9, tripeptide does not acquire Golgi-specific modifications. We 3153-3162. achieved similar results when we used the yeast microsome- 6. Rothman, J. E. (1987) Cell 50, 521-522. A. & Rothman, assay (13) instead of semi-intact cells. We 7. Wieland, F. T., Gleason, M. L., Serafini, T. based transport J. E. (1987) Cell 50, 289-300. conclude that the exit route of the glycosylated acceptor 8. Helms, J. B., Karrenbauer, A., Wirtz, K. W. A., Rothman, tripeptide from the yeast ER is different from the secretory J. E. & Wieland, F. T. (1990) J. Biol. Chem. 265, 20027-20032. pathway followed by proteins such as pro-a-factor and may 9. Geetha-Habib, M., Park, H. R. & Lennarz, W. J. (1990) J. Biol. instead be mediated by an ATP-driven pump in the mem- Chem. 265, 13655-13660. brane of the yeast ER that requires a cytosolic component. 10. Baker, D., Hicke, L., Rexach, M., Schleyer, M. & Schekman, The potential physiological role of a glycopeptide pump in the R. (1988) Cell 54, 335-344. D. & yeast ER membrane may be the recycling to the cytosol of 11. Baker, D., Wuestehube, L., Schekman, R., Botstein, Segev, N. (1990) Proc. Natl. Acad. Sci. USA 87, 355-359. short peptides that result from protein degradation in the ER. 12. Rexach, M. F. & Schekman, R. (1991) J. Cell Biol. 114, Although we cannot formally exclude that S. cerevisiae is 219-229. fundamentally different from mammalian cells with respect to 13. d'Enfert, C., Wuestehube, L., Lila, T. & Schekman, R. (1991) glycopeptide export, our data do cast some doubt on the J. Cell Biol. 114, 663-670. interpretation of the experiments performed with glycosyla- 14. Ruohola, H., Kabcenell, A. K. & Ferro-Novick, S. (1988) J. tion acceptor tripeptides as bulk flow markers in mammalian Cell Biol. 107, 1465-1476. cells (7, 8). Wieland et al. (7) and Helms et al. (8) have shown 15. Groesch, M. E., Ruohola, H., Bacon, R., Rossi, G. & Ferro- Novick, S. (1990) J. Cell Biol. 111, 45-53. no evidence that the endoglycosidase H-resistant oligosac- 16. Harlow, E. & Lane, D. (1988) Antibodies: A Laboratory charide corresponds to authentic "complex" structures or Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), that the glycopeptide resides inside membrane-bounded p. 310. transport vesicles; thus it is conceivable that glycotripeptide 17. Hansen, W., Garcia, P. D. & Walter, P. (1986) Cell 45, 397-406. export from mammalian cells does not occur via the secretory 18. Dunn, B. & Wobbe, C. R. (1989) in Current Protocols in pathway but rather by ATP-driven pumps in the ER and the Molecular Biology, eds. Ausubel, F. M., Brent, R., Kingston, plasma membrane, respectively. As the experiments by R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene/Wiley Interscience, New York), Vol. 2, pp. 13.13.5- Wieland et al. (7) provided the key data on which the bulk 13.13.6. flow model for secretory protein export was based, our 19. Novick, P., Field, C. & Schekman, R. (1980) Cell 21, 205-215. results reopen the question of selectivity in the packaging of 20. Hicke, L. & Schekman, R. (1989) EMBO J. 8, 1677-1684. secretory proteins for export from eukaryotic cells. 21. Kaiser, C. A. & Schekman, R. (1990) Cell 61, 723-733. 22. Orci, L., Ravazzola, M., Meda, P., Holcomb, C., Moore, We thank David King for the synthesis of Ac-NYT-NH2, Arlene H.-P., Hicke, L. & Schekman, R. (1991) Proc. Natl. Acad. Sci. Eun-Sapsis for the preparation of lyticase, and Tohru Yoshihisa for USA 88, 8611-8615. the antibodies against Sec23p and p105. We are especially grateful to 23. Kornfeld, R. & Kornfeld, S. (1985) Annu. Rev. Biochem. 54, Michael Rexach and Tom Yeung for their assistance and helpful 631-664. discussions throughout the course of this work. K.R. was supported 24. Hicke, L., Yoshihisa, T. & Schekman, R. (1992) Mol. Biol. Cell by a fellowship ofthe Boehringer Ingelheim Funds. The research was 3, 667-676. Downloaded by guest on September 28, 2021