Coat Assembly Directs v-snare Concentration into Synthetic COPII Vesicles

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1 Molecular Cell, Vol. 2, , November, 1998, Copyright 1998 by Cell Press Coat Assembly Directs v-snare Concentration into Synthetic COPII Vesicles Ken Matsuoka,* Yasujiro Morimitsu, Koji Uchida, and Randy Schekman* * Department of Molecular and Cell Biology Howard Hughes Medical Institute University of California, Berkeley Berkeley, California Laboratory of Food and Biodynamics Graduate School of Bioagricultural Sciences Nagoya University Nagoya Japan Summary (Kuehn and Schekman, 1997). COPII comprises two heterodimeric complexes (Sec23/24p, Sec13/31p) and a small GTP-binding protein Sar1p (Nakano and Muramatsu, 1989; Hicke et al., 1992; Barlowe et al., 1993; Salama et al., 1993). The GTP-bound form of Sar1p promotes the assembly of the coat and participates directly in the recruitment of cargo proteins (Barlowe et al., 1994; Kuehn et al., 1998; Springer and Schekman, 1998). Sar1p is recruited to the ER membrane to initiate the budding process by a transient interaction with Sec12p, a cytoplasmically exposed membrane protein that promotes nucleotide exchange on Sar1p (Nakano et al., 1988; d Enfert et al., 1991; Barlowe and Schekman, 1993). Sar1p-GTP binds directly to the phospholipid bilayer and recruits Sec23/24p; together those molecules comprise a sorting complex that distinguishes cargo and resident proteins (Kuehn et al., 1998). Selective transport implies the existence of sorting signals. Such signals for anterograde transport from the ER are only just beginning to be deciphered. Some mem- brane proteins contain sorting signals in the cytoplasmic domain, and others contain a positive or negative signal in the membrane spanning domain (Sato et al., 1996; COPII proteins are required to create transport vesi- cles and to select cargo molecules for transit from the ER. A reconstituted liposome budding reaction was used to detect the capture and concentration of membrane-associated v-snare molecules into synthetic COPII vesicles. A novel glutathione-phosphatidyl- ethanolamine conjugate (Glut-PE) was synthesized and incorporated into chemically defined liposomes Fielder and Rothman, 1997; Nishimura and Balch, 1997; to provide binding sites for GST hybrid proteins. Large Rayner and Pelham, 1997; Honsho et al., 1998). Soluble liposomes containing bound cytoplasmic domains of secreted proteins most likely interact with membrane the v-snares, Sec22p or Bos1p, or of the ER resident receptor proteins that contact COPII subunits directly proteins, Sec12p and Ufe1p, were exposed to COPII pro- (Schimmoller et al., 1995; Kuehn et al, 1998; Vollenteins and GMP-PNP. v-snares but not resident pro- weider et al., 1998). teins were concentrated in synthetic COPII vesicles In order to monitor directly the sorting of membrane generated from donor liposomes. We conclude that proteins into COPII vesicles, we have developed a re- COPII proteins are necessary and sufficient for cargo constituted proteoliposome budding reaction. In this reselection and vesicle morphogenesis. port, we show that the cytoplasmic domains of two SNARE proteins are sufficient to allow their selective Introduction uptake into synthetic COPII vesicles. Protein transport in the secretory pathway is mediated by vesicles that enclose cargo (membrane and soluble proteins destined for transit) and targeting (SNARE) proteins but exclude or restrict the capture of resident proteins designed to be retained in a donor compartment (Schekman and Orci, 1996). Cytoplasmic coat proteins are required for transport vesicle formation. Subunits of coat complexes such as clathrin and its assembly proteins, COPI, and COPII are known to interact directly or indirectly with cargo proteins (Cosson and Letourner, 1994; Hoflack, 1998; Kuehn et al., 1998; Springer and Schekman, 1998). In addition, coat complexes have been shown to direct the shape change that accompanies the formation of a bud on a phospholipid membrane surface (Matsuoka et al., 1998; Spang et al., 1998; Takei et al., 1998). Thus, it seems likely that coat complexes fulfill all essential aspects of transport vesicle bio- genesis. The COPII coat is responsible for all known antero- grade protein traffic from the ER to the Golgi complex To whom correspondence should be addressed ( schek man@uclink4.berkeley.edu). Results GST Hybrid Proteins Tethered to Liposomes To detect the selective capture of membrane proteins into synthetic COPII vesicles, we developed an ap- proach to tether the cytoplasmic domains of membrane proteins to pure phospholipid liposomes. We reasoned that GST hybrid proteins may be retained on the external surface of liposomes that contain a glutathione-deriva- tized phospholipid (Figure 1A). To prepare such a conju- gate, we coupled reduced glutathione and dioleoylphosphatidylethanolamine (DOPE) using a homobifunctional cross-linker, 1,4-butanediol diglycidyl ether. The prod- uct, Glut-PE, was purified by silica gel and thin layer chromatography. A single species was detected in two TLC solvent systems. A proposed structure was con- firmed by positive ion fast atom bombardment mass spectrometry (Figure 1B). Glut-PE (3.5 mol%) was included in a mixture of phospholipids, replacing a corresponding fraction of DOPE, and formulated into liposomes optimized for budding of synthetic COPII vesicles (Matsuoka et al., 1998). Liposomes supplemented with Glut-PE bound COPII proteins and formed COPII vesicles normally (data not

2 Molecular Cell 704 Figure 1. Strategy of the Tethering of GST Hybrid Proteins to Liposomes Containing Glut-PE Figure 2. Binding of GST and Sec22p-GST to Liposomes with or without Glut-PE (A) Separation of liposomes and bound GST or Sec22p-GST by Sepharose CL2B chromatography. The elution pattern of lipids (upper panel) and proteins detected by SYPRO RED staining (lower panels) are shown. (B) High affinity binding of GST hybrid proteins to liposomes containing Glut-PE. GST hybrid proteins were incubated with liposomes and separated as in (A). Proteins in the liposome fractions were separated by SDS-PAGE and detected by SYPRO RED staining. Each lane contained an equal amount of liposomes. The most promi- nent band in each Glut-PE lane corresponds to each fusion protein. The minor bands in the Ufe1p-GST lanes are degradation (A) Schematic illustration of the binding of a GST hybrid protein on a Glut-PE liposome. products. (B) Proposed structure of Glut-PE and fragmentation pattern derived by fast atom bombardment mass spectrometry. Three ion peaks [m/z 1276 (M Na), m/z 1298 (M H 2Na), and m/z 1320 GST only when Glut-PE was present in the liposome (M 2H 3Na) ] were detected. Addition of NaI in the matrix en- (Figure 2). GST hybrid proteins were similarly adsorbed onto Glut-PE liposomes (Figure 2), though in some in- stances the dependency on the modified PE was not absolute (e.g., Figure 2B, Sec12p-GST). Curiously, the GST hybrids bound more avidly than GST to Glut-PE liposomes (Figure 2A). The cytoplasmic domain may itself interact weakly with the lipid phase. In one in- stance, Bet1p-GST, the nonspecific interaction was too great to allow selective tethering. hanced the peak intensity of m/z 1320 (M 2H 3Na). Furthermore, three new ion peaks [m/z 1292 (M K), m/z 1330 (M H 2K), and m/z1368 (M 2H 3K) ] appeared in the presence of KI in the matrix. Base ion peak m/z 604 was assigned to the fragment ion from the molecular ion peak. The other fragment ions also supported the proposed structure. The units that constitute Glut-PE are written on the left, and the assignment of fragment ions is indicated in the structure. shown). Liposomes formulated with or without Glut-PE were mixed with GST or GST hybrid proteins. We chose hybrid proteins that contain the cytoplasmically oriented domains of representative v-snares (Sec22p and Bos1p) and resident ER membrane proteins (Sec12p and Ufe1p). Protein liposome combinations were separated by gel filtration, and the fractionation of lipids was monitored by the fluorescence of NBD phospholipids in the liposomes and of proteins by SDS-PAGE (Figure 2A). Liposomes in the void volume (fractions 3 and 4) contained Selective Capture of SNAREs in Synthetic COPII Vesicles Glut-PE liposomes containing bound Sec22p-GST were isolated by gel filtration and mixed with Sec13/31p, Sec23/24p, and Sar1p, and incubated with or without GMP-PNP. We showed previously that synthetic COPII vesicles bud from defined liposomes only in the presence of a nonhydrolyzable nucleotide analog (Matsuoka et al., 1998). Synthetic COPII vesicles were separated

3 Reconstitution of Membrane Protein Sorting 705 fractions. A medium density peak of phospholipid (fraction 7 9), corresponding to large coated liposomes (Matsuoka et al., 1998), contained little Sec22p-GST. Without nucleotide, most of the Sec22p-GST remained at the top of the gradient along with phospholipid. To address whether the recovery of Sec22p-GST in the high-density fraction was saturable, we prepared liposomes containing 4-fold more hybrid protein. After a COPII budding reaction, the distribution of Sec22p-GST in a density gradient was similar to that of the NBD fluorescence, and no significant enrichment of hybrid protein in the high-density fraction was observed (data not shown). These results suggest that the clustering of coat proteins necessary to bud synthetic COPII vesicles concentrates Sec22p-GST from the donor liposome in a saturable manner. COPII subunits not bound to liposomes migrated in fractions 8 13 of the sucrose density gradient; thus, it was possible that some of the Sec22p-GST dissociated from liposomes and sedimented along with unbound coat proteins. To test this possibility, we resolved unbound coat subunits from synthetic COPII vesicles by velocity sedimentation on a sucrose gradient. The phospholipids and Sec22p-GST sedimented together as a single peak coincidentally with COPII proteins (data not shown). Parallel incubations conducted with or without GMP-PNP revealed an absolute difference in the recovery of coat proteins in the velocity gradient fractions corresponding to synthetic COPII vesicles (Figure 3B, Sec13p). Budding resulted in the enrichment of Sec22p-GST within synthetic COPII vesicles. Starting liposome fractions were compared to gradient-purified COPII vesicles by quantitative immunoblot normalized to arbitrary units of NBD phospholipid fluorescence (Figures 3B and 4C). A range of 3- to 4-fold enrichment of Sec22p-GST was obtained in five repetitions of this experiment. We considered the possibility that some or all of the Sec22p-GST was recruited to synthetic COPII vesicles from an unbound pool of hybrid protein rather than from the surface of a donor liposome. Budding reactions were conducted with or without nucleotide using liposomes containing surface-bound Sec22p-GST or pure lipo- somes supplemented with hybrid protein after comple- tion of the budding reaction. COPII vesicles were iso- lated from these four incubations and evaluated by immunoblot of Sec22p-GST and by colloidal gold stain- ing of coat subunits (Figure 3C). Sec22p-GST was enriched only in the presence of nucleotide and only when it was bound to starting liposomes. We conclude that Sec22p-GST is concentrated by capture of membrane- bound hybrid protein into synthetic COPII vesicles. Finally, we compared the enrichment of Sec22p-GST with another v-snare hybrid, GST-Bos1p, and used GST alone and hybrids representing resident ER proteins, Sec12p-GST and Ufe1p-GST, as controls of sort- ing. Glut-PE liposomes containing bound GST or a mix- ture of Ufe1p-GST and Sec22p-GST or GST-Bos1p and Sec12p-GST were incubated with or without GMP-PNP, and synthetic COPII vesicles were isolated by density and velocity sedimentation. Quantitative immunoblot of SDS-PAGE samples showed a consistent ca. 3-fold en- richment of the GST-Bos1p with no enrichment of GST, Sec12p-GST, or Ufe1p-GST control proteins (Figure 4). Figure 3. Concentration of Sec22p-GST into Synthetic COPII Vesicles (A) Separation of synthetic COPII vesicles from Sec22p-GST donor liposomes by equilibrium sucrose density gradient centrifugation. Sec22p-GST-loaded liposomes were used for COPII budding in the presence or absence of GMP-PNP. Upper panel, distribution of lipids in the gradient. Lower panels, distribution of Sec22p-GST detected by immunoblot. (B) Quantitative analysis of Sec22p-GST in the purified vesicle fraction. GST hybrid proteins in Sec22p-GST-loaded liposomes and in the vesicle fraction were separated by SDS-PAGE and detected by immunoblot (upper panel). After detection of Sec22p-GST, coat proteins were stained by colloidal gold. The signal of Sec13p is shown (lower panel). The amount of fluorescent lipids in the vesicle fraction GMP-PNP is the same as in liposomes 1. The loaded volumes of liposomes in different lanes are multiplied by the 1 lane as indicated. (C) Recovery of Sec22p-GST in synthetic COPII vesicles requires liposome-bound hybrid protein. A budding reaction was performed with liposomes not containing Sec22p-GST, and then the reaction was further incubated with Sec22p-GST (after budding). Sec22p- GST-bound liposomes served as a control (before budding). Upper panel, Sec22p-GST detected by immunoblot. Lower panel, Sec24p signal by colloidal gold staining. from uncoated and large liposomes by equilibrium sedimentation on a sucrose density gradient. The distribution of Sec22p-GST was analyzed by immunoblot of an SDS-PAGE, and of NBD phospholipids marking the liposome by measurement of fluorescence (Figure 3A). The dense peak of phospholipids (fractions 11 14) corresponds to the position of synthetic COPII vesicles. Over 50% of the Sec22p-GST cosedimented in these

4 Molecular Cell 706 coat on the liposome may provide a multivalent binding surface for the SNAREs. Similar weak interactions between COPII subunits and the FF motif in p24 and ERGIC-53 may allow substantial enrichment of these proteins within COPII vesicles (Kappeler et al., 1997; Dominguez et al., 1998). Although several relevant features of vesicle budding and cargo capture are reproduced in our synthetic reaction, the efficiency of sorting is not what can be achieved with intact ER membrane and pure COPII proteins. For example, COPII vesicles formed from native ER membranes are over 100-fold enriched in Sec22p relative to phospholipid (K. M., unpublished data). One reason for the discrepancy may be that the synthetic reaction employs a 5-fold higher concentration of COPII proteins so as to optimize the enclosure of phospholipids (Matsuoka et al., 1998). A typical synthetic budding reaction vesiculates 25% 30% of the total phospholipid whereas optimum budding of protein cargo from ER membrane frac- Figure 4. v-snare-gst Hybrid Proteins, but Not GST and Fusion Proteins of GST and ER Resident Proteins, Are Enriched in the tions captures 1% of the total phospholipid (Matsuoka Synthetic COPII Vesicle Fraction et al., 1998; K. M., unpublished data). Previously we (A) Enrichment of Sec22p-GST, but not of Ufe1p-GST, from lipo- speculated that Sec16p, which is not present in our somes that contain both proteins. Liposomes containing both reconstitution, may facilitate the nucleation of a coat Ufe1p-GST and Sec22p-GST were subjected to a COPII budding and reduce the concentration of COPII proteins needed reaction as in Figure 3. The liposome lane and the vesicle fraction GMP-PNP lane contain equal amounts of fluorescent lipids, and the to bud a vesicle (Matsuoka et al., 1998). In contrast to vesicle fraction and GMP-PNP lanes contain equal volumes the synthetic reaction, Sec16p is retained on the cyto- of the fraction. Liposome, protein-loaded liposomes used for the plasmic face of the ER membranes, where it participates budding reaction. The band above Ufe1-GST in the vesicle frac- in the packaging of v-snare proteins into COPII vesition GMP-PNP lane is an unrelated protein that cross-reacts with cles (Espenshade et al., 1995; Campbell and Schekman, antibody to GST. 1997). Sec16p interacts through distinct domains with (B) Specific enrichment of GST-Bos1p. GST-Bos1p and Sec12p- GST were bound to the same liposomes, and the budding reaction Sec23p, Sec24p, and Sec31p (Espenshade et al., 1995; was carried out as in (A). Gimeno et al., 1996; Shaywitz et al., 1997); thus, it could (C) Quantitative evaluation of the enrichment of each protein in the create a polyvalent network of COPII subunits and signifvesicle fraction. Protein enrichment from the donor liposomes based icantly improve the capture of cargo molecules relative on fluorescent lipids in liposomes and vesicles is shown. Bars indi- to phospholipid. Furthermore, Sec16p or other proteins cate means, and the error bars indicate SEM. Numbers above bar may contribute to an organized transitional zone of the indicate the numbers of independent experiments. ER in which cargo molecules may be concentrated and from which resident proteins may be excluded (Orci et We conclude that the reconstituted sorting reaction is al., 1991; Kuehn and Schekman, 1997; Tang et al., 1997; protein selective. Aridor et al., 1998). Control proteins, such as GST, Sec12p-GST, and Discussion Ufe1p-GST, are neither enriched nor depleted in synthetic COPII vesicles. Here again, the optimized packag- The cytoplasmically exposed domains of the v-snares, ing of phospholipid may carry nonspecific membrane Sec22p and Bos1p, interact with subunits of the COPII constituents at their prevailing concentration into budcoat and are clustered into COPII vesicles formed from ding vesicles. However, other factors such as the length synthetic liposomes. In contrast, GST, the fusion partner of the membrane anchor domain or extrinsic proteins we used to tether v-snare hybrids to liposomes, and may serve to enhance the retention of ER resident prothe cytoplasmic domain of ER resident proteins, Sec12p teins (Sato et al., 1996; Campbell and Schekman, 1997; and Ufe1p, are not enriched within synthetic COPII vesi- Fielder and Rothman, 1997; Honsho et al., 1998). The cles. In direct binding experiments using pure proteins influence of intrinsic and extrinsic factors on the fidelity without a membrane, GST-Bos1p binds well to Sar1p- of sorting may now be explored with the basic sorting GMP-PNP and Sec23/24p whereas Sec22p-GST binds and budding machinery in hand. poorly (Springer and Schekman, 1998). Similarly, Sec22p- GST did not recruit COPII proteins to liposomes when Experimental Procedures the hybrid protein was bound to the liposomes made of PC, PE, and Glut-PE (K. M. and R. S., unpublished data). Preparation of Glut-PE In spite of this, the two SNAREs are enriched to similar Reduced glutathione (1 g) was mixed with 2.3 ml 50 mm phosphate extents in the liposome budding experiments described buffer (ph 7.5) and 770 l 1,4-butanediol diglycidyl ether (Aldrich), adjusted to ph 7.5 with 2 N NaOH, and incubated at 37 C for 24 hr here. Perhaps the recruitment of coat subunits to the under Ar. The reaction was applied to an AG-1 resin (Bio-Rad) colliposome surface promoted by the presence of acidic umn (30 ml) in the OH form, and the resin was washed with 150 ml phospholipids enhances a weak interaction between H 2 O. Bound materials were eluted with 0.1 N HCl into Sec22p and COPII. Alternatively, polymerization of the ml fractions. Fractions containing ninhydrin-positive materials were

5 Reconstitution of Membrane Protein Sorting ). In some cases, budding reactions were performed in the absence of a GST hybrid protein. In such cases, 0.5 g of GST hybrid protein was added after a normal 30 min budding reaction, and incubation continued at 30 C for 30 min. Coated vesicles were separated by equilibrium sucrose density gradient centrifugation (Matsuoka et al., 1998). Pooled high-density lipid peak fractions (200 l) (Matsuoka et al., 1998) were mixed with 900 l B88 and loaded on a 900 l linear 8%-15% Ficoll 400 gradient in B88 on the top of a shelf of 200 l B88, 2.2 M sucrose. The resulting gradient was centrifuged in a Beckman TLS55 rotor at 55,000 rpm for 90 min and separated into 22 fractions. The fluorescence peak fraction was used as the vesicle fraction. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane. GST hybrid proteins on the membrane were decorated by affinity-purified rabbit anti-gst anti- body and alkaline-phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad). Alkaline-phosphatase activity on the membrane was de- tected using the Vistra ECF substrate (Amersham). The fluorescence product was detected and quantified by a STORM 860 image ana- lyzer. After detection of the fluorescence image, coat proteins on the membrane were detected by colloidal gold staining (PROTOGOLD; Research Diagnostics). Acknowledgments We thank Robert Lesch for COPII proteins, Sebastian Springer for v-snare-gst fusion proteins, Martin Latterich for a cloned Ufe1 gene, and David Madden for the construction of the Ufe1-GST expression plasmid. This work was supported by the HHMI (R. S.). K. M. is a visiting scholar from the Graduate School of Bioagricultural Sciences, Nagoya University. pooled and concentrated by rotary evaporation to ca. 8 ml. The concentrated materials were mixed with 25 mg DOPE (Avanti) dissolved in 30 ml tetrahydrofrane/h 2 O (2:1 by volume), the ph was adjusted to 9.0 with NaOH, and conjugation was performed at 37 C for 40 hr under Ar. After addition of concentrated HCl to adjust the ph to around 1, solvents were partially removed by rotary evaporation. Partially dried material ( 10 ml) was mixed with 10 ml H 2 O/MeOH/ concentrated HCl (100:100:2 by volume), and the lipids were extracted with CHCl 3 (20 ml 3 extractions). The pooled CHCl 3 phase was concentrated by rotary evaporation. Dried materials were dissolved in 40 ml CHCl 3 /MeOH (1:1 by volume) and applied to a silica gel column (10 ml; Aldrich #28,862-4), which was washed with 20 ml CHCl 3 /MeOH. Bound materials were eluted with MeOH (6 6 ml) followed by 20 6 ml of MeOH/H 2 O (9:1 by volume). Fractions containing Glut-PE (major spot on TLC plate) were pooled, concentrated, and spotted to a preparative silica gel TLC plate (Aldrich). The plate was developed with an alkaline solvent system (Munnik et al., 1994). Lipids in the plate were stained by water, and the lipid spot at Rf 0.3 was scraped from the plate. Lipids were extracted from the silica matrix with MeOH/H 2 O, concentrated by rotary evaporation, and stored at 20 C under Ar. A typical yield was mg of Glut-PE. The purity of the lipids was confirmed by separation on oxalatecontaining silica gel plates using the acidic and the alkaline solvent systems (Munnik et al., 1994). The lipid sample was mixed with a liquid matrix composed of thioglycerol/glycerol/m-nitrobenzyl alco- hol (1:1:1 by volume) with or without a drop of 0.1 N-NaI or KI solution. FAB-MS spectra were measured using a JMS-700 instru- ment (JEOL, Akishima, Japan) with the positive mode (Barber et al., 1981). Proteins COPII coat proteins, GST, Sec22p-GST, and GST-Bos1p were prepared Received August 17, 1998; revised September 16, as described (Barlowe et al., 1994; Matsuoka et al., 1998; Springer and Schekman, 1998). Sec12p-GST was prepared as fol- References lows. A DNA fragment encoding the cytoplasmic domain of Sec12p and a fragment from petgexct (Sharrocks, 1994) encoding GST Aridor, M., Weissman, J., Bannykh, S., Nuoffer, C., and Balch, W. were placed in tandem behind the Gal1 promoter in an expression (1998). Cargo selection by the COPII budding machinery during vector, YCpLGN78T (Y. Ohya, personal communication). The re- export from the ER. J. Cell Biol. 141, sulting plasmid was introduced into S. cerevisiae RSY 445 (gal2, Barber, M., Bordori, R.S., Sedgwick, R.D., and Tyler, A.N. (1981). leu2-3,112, ura3-52, trp1-289, his4-579, prb1, pep4::ura3, MAT ), Fast atom bombardment of solid (F. A. B.): a new ion source for and fusion protein expression was induced by galactose. Expressed mass spectrometry. J. Chem. Soc. Chem. Comm., protein was purified as described (Smith and Johnson, 1988). Purified Sec12p-GST was active in promoting GDP/GTP exchange on Barlowe, C., and Schekman, R. (1993). SEC12 encodes a guanine Sar1p. A Ufe1p-GST expression plasmid was constructed by infrom the ER. Nature 365, nucleotide exchange factor essential for transport vesicle budding serting a DNA fragment encoding the cytoplasmic domain of Ufe1p into petgexct (Sharrocks, 1994). Fusion protein was expressed Barlowe, C., d Enfert, C., and Schekman, R. (1993). Purification and in E. coli (Sharrocks, 1994) and purified as described (Smith and characterization of Sar1p, a small GTP-binding protein required for Johnson, 1988). Affinity-purified anti-gst antibody was prepared transport vesicle formation from the endoplasmic reticulum. J. Biol. from anti-gst-sec22p antiserum (Bednarek et al., 1995) using immobilized Chem. 268, GST. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Sa- lama, N., Rexach, M.F., Ravazzola, M., Amherdt, M., and Schekman, Liposome Budding Assay R. (1994). COPII: a membrane coat formed by Sec proteins that drive Liposomes were prepared from a modified formulation in which 3.5 vesicle budding from the endoplasmic reticulum. Cell 77, mol% of Glut-PE was substituted for a corresponding fraction of Bednarek, S.Y., Ravazzola, M., Hosobuchi, M., Amherdt, M., Perre- DOPE in a lipid mixture described previously (Matsuoka et al., 1998). let, A., Schekman, R., and Orci, L. (1995). COPI- and COPII-coated Other lipids were identical to those used for the morphological analy- vesicles bud directly from the endoplasmic reticulum in yeast. Cell sis (Matsuoka et al., 1998). Liposomes were prepared by extrusion 83, through a polycarbonate filter (400 nm pore size). Unless otherwise Campbell, J.L., and Schekman, R.S. (1997). Selective packaging of noted, extruded liposomes corresponding to 108 g phospholipids cargo molecules into endoplasmic reticulum-derived COPII vesiwere incubated with 2 g GST hybrid protein in B88 (20 mm HEPEScles. Proc. Natl. Acad. Sci. USA 94, KOH [ph 6.8], 0.15 M KOAc, 0.25 M sorbitol, and 5 mm Mg(OAc) 2 ) Cosson, P., and Letourner, F. (1994). Coatomer interaction with diin a 150 l reaction at 37 C for 30 min. The reaction was applied to lysine endoplasmic reticulum retention motifs. Science 263, 1629 a Sepharose CL 2B column (1.5 ml bed vol; Pharmacia), and the liposomes were eluted with B88 in fractions of 100 l each. Fractions of the lipid peak were pooled, and the concentration of the lipids d Enfert, C., Barlowe, C., Nishikawa, S., Nakano, A., and Schekman, was measured by monitoring NBD fluorescence. In some cases, R. (1991). Structural and functional dissection of a membrane glyco- proteins in the fractions, corresponding to 1 g phospholipid, were protein required for vesicle budding from the endoplasmic reticulum. separated by SDS-PAGE, stained by SYPRO RED (Molecular Mol. Cell. Biol. 11, Probes), and detected by a STORM 860 image analyzer (Molecular Dominguez, M., Dejgaard, K., Fukkekrug, J., Dahn, S., Frazel, A., Dynamics). Protein-loaded liposomes, corresponding to 27 g Paccaud, J.-P., Thomas, D., Bergeron, J., and Nilsson, T. (1998). phospholipids, were incubated with COPII proteins in a 300 l reac- gp25l/emp24/p24 protein family members of the cis-golgi network tion for 30 min at 30 C at5 COPII, as described (Matsuoka et al., bind both COPI and II coatomer. J. Cell Biol. 140,

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