The EMBO Journal vol.11 no.10 pp.3609-3617, 1992

Bosi

membrane protein required for ER to Golgi transport in yeast, co-purifies with the carrier vesicles and with Bet1p and the ER membrane p, a

Anna P.Newman1, Mary E.Groesch and Susan Ferro-Novick2 Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA 'Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA 'Corresponding author Communicated by P.De Camilli

BOSI and BETI are required for transport from the ER to the Golgi complex in yeast and genetically interact with each other and a subset of the other genes, whose products function at this stage of the secretory pathway. In a previous study, we reported that BOSI encodes a putative 27 kDa membrane protein. Here we show that BET] is structurally similar to the synaptobrevins and identical to the SLY12 gene product. Overexpression of SLY12 compensates for the loss of function of the ras-like GTP-binding protein Yptl. Both Boslp and Betlp are cytoplasmically oriented membrane proteins. Boslp co-purifies with the ER to Golgi transport vesicles and co-fractionates with Betlp and the ER membrane. Key words: membrane proteins/yeast transport

Introduction The movement of secretory proteins from the lumen of the endoplasmic reticulum (ER) to the cell surface is dependent on a series of vesicular transport events (Palade, 1975). Proteins that traverse this pathway are packaged into vesicles that mediate membrane traffic from one part of the cell to another. These vesicles contain GTP-binding proteins (Goud et al., 1988; Serafini et al., 1991a) and, in some cases, a non-clathrin coat (Duden et al., 1991; Serafini et al., 1991b). Integral membrane components that interact with the coat structure or GTP-binding proteins have not been identified. A set of four proteins (COPs): a-COP (160 kDa), $-COP (110 kDa), -y-COP (98 kDa) and b-COP (61 kDa), comprise the non-clathrin coat of Golgi transport vesicles (Serafini et al., 199 lb). These proteins also form a 'coatomer' complex in the cytoplasm that may represent an unassembled precursor of the coat (Waters et al., 1991). One member, fl-COP, is homologous to the amino-terminal domain of fi-adaptin (Duden et al., 1991). Since the adaptins mediate the association of clathrin with membranes (Keen, 1990), fl-COP may play a similar role in non-clathrin coated vesicles. Brefeldin A, a drug that blocks secretion (Lippincott-Schwartz et al., 1989), displaces f-COP from Golgi membranes (Donaldson et al., 1990) and prevents the assembly of non-clathrin coated vesicles (Orci et al., 1991). Low molecular weight GTP-binding proteins have been proposed to maintain the specificity of vesicular traffic by directing the transport vesicle to its appropriate target Oxford University Press

membrane (Bourne, 1988). In order to accomplish this task, a soluble form of the protein is post-translationally modified in the cytosol (Rossi et al., 1991) prior to its membrane attachment. It has been hypothesized that the GTP-binding protein binds to a transport vesicle to deliver this compartment to an acceptor membrane before it is returned to the cytosol to attach to another vesicle. By this model, the unidirectionality of the cycle is ensured by the sequential binding and hydrolysis of GTP. Transport between the ER and Golgi complex has been studied using several complementary approaches. For example, cell-free systems have permitted the reconstitution of this event in vitro, both in mammalian cells and in yeast

(Beckers et al., 1987; Baker et al., 1988; Ruohola et al., 1988). In addition, the yeast in vitro transport assay has facilitated the isolation of a functional vesicular compartment that mediates transit at this stage of the secretory pathway (Groesch et al., 1990; Rexach and Schekman, 1991). This intermediate contains the components that direct ER to Golgi traffic, as well as transported proteins. Mutational analysis in the yeast Saccharomyces cerevisiae has defined nine SEC and two BET genes, whose products participate in ER to Golgi transport (Novick et al., 1981; Newman and Ferro-Novick, 1987). The corresponding mutants have been referred to as ER-accumulating, based on their morphology at the restrictive temperature (Novick et al., 1981; Newman and Ferro-Novick, 1987). Of those genes analysed at the molecular and biochemical level, some, such as SEC12, have been shown to encode membrane proteins (Nakano et al., 1988). Others such as SEC18 and SEC23, encode hydrophilic proteins that may be associated with the periphery of intracellular membranes (Eakle et al., 1988; Hicke and Schekman, 1989). An additional yeast gene identified several years ago, YPTI, is also required for ER to Golgi transport (Schmitt et al., 1988; Segev et al., 1988; Bacon et al., 1989; Baker et al., 1990). Since YPTI is a ras-like gene, this finding correlates well with data from yeast in vitro studies showing a requirement for proteins that bind GTP (Baker et al., 1988; Ruohola et al., 1988). Finally, SARI and BOSI, initially identified as suppressors of ER-accumulating mutants (Nakano and Muramatsu, 1989; Newman et al., 1990) have now been shown to be required for transport at this stage of the pathway (Nakano and Muramatsu, 1989; Shim et al., 1991). The SARI gene product is also a GTP-binding protein. Genetic and biochemical data has defined subclasses of genes whose products appear to function together to execute a common step in ER to Golgi transport (Bacon et al., 1989; Clary et al., 1990; Kaiser and Schekman, 1990; Newman et al., 1990; Rossi et al., 1991). Our studies have revealed that BET], BOSI and SEC22 comprise one of these groups (Newman et al., 1990). BOSI and SEC22 encode small proteins that are hydrophilic at their amino-termini with a stretch of hydrophobic amino acids at their carboxy-termini (Shim et al., 1991; Newman et al., 1992). Here we report 3609

A.P.Newman, M.E.Groesch and S.Ferro-Novick

that BETI is the same as SLY12, a multicopy suppressor gene that can compensate for the loss of YPTI. In addition, we show that BosIp and BetIp are membrane proteins. BosIp co-purifies with the ER to Golgi transport vesicles and with BetIp and the ER membrane.

Results The BET1 gene encodes a putative membrane protein that is structurally similar to the synaptobrevins and identical to SLY12 BOSI, a gene identified by its ability to suppress the bet] mutant (Newman et al., 1990) encodes a putative membrane protein (Shim et al., 1991). In a previous study, we reported the DNA sequence of BOSI (Shim et al., 1991). To begin to elucidate the nature of the interactions between BOSI and BET], we also sequenced BET]. Previously, we described the isolation of a 2.2 kb insert that integrates at the BET] locus and fully complements the growth defect of the bet]-] mutant (Newman et al., 1990). A Bgll site within this gene was also found. DNA sequence analysis has revealed a single open reading frame that spans this BglH site. The identification of an intron upstream of this open reading frame and downstream of the first four amino acids of the predicted Betl protein (Betlp) is described in Materials and methods. A comparison of Betlp with other sequences in the National Biomedical Research Foundation data bank, has revealed that BET] is identical to SLY12 (suppressor of loss of YPTI function), a multicopy gene that suppresses the growth defect of yeast cells that are depleted of the Yptl protein (Dascher et al., 1991). Prior to this analysis, a requirement for SLYl2 in ER to Golgi transport has not been demonstrated. Furthermore, antibody to Betlp raised in this study has enabled us to test predictions that can be made from the DNA sequence. The relevant features of this sequence are briefly discussed below. The sequence of BET] predicts a protein (Betlp) of 142 amino acids that lacks a signal sequence and is rich in serines at its amino-terminus (21 % of the first 58 amino acids and 42% of the amino acids between 27 and 50). Hydropathy analysis (Kyte and Doolittle, 1982) has revealed that Betlp is hydrophilic and contains a carboxy-terminal hydrophobic stretch of 19 amino acids that may span the membrane. The BETI sequence also contains three potential N-linked glycosylation sites. However, since Betlp does not appear to be sensitive to digestion with Endo H (not shown), it is unlikely that these sites are utilized. In overall structure, Betlp possesses features in common with the synaptobrevins, a family of proteins found on synaptic vesicles (Baumert et al., 1989; Sudhof et al., 1989). The synaptobrevins are low molecular weight proteins composed of three domains: an initial region rich in prolines (bovine and Torpedo sequences) or asparagines (Drosophila), a hydrophilic middle segment and a hydrophobic carboxy-terminal transmembrane anchor. The hydrophilic core of the synaptobrevins is the most highly conserved region of the protein and it has strong az-helical potential. Similarly, computer analysis of Betlp (Devereux et al., 1984) has demonstrated that the middle region of this protein (amino acids -50-115) has significant at-helical potential. A sequence of four amino acids (KLKR), which is conserved among the synaptobrevins, is also present in Betlp at the appropriate distance from the hydrophobic stretch of amino

3610

Fig. 1. Antibody prepared to a BetI -TrpE hybrid protein recognizes a protein species of 18 kDa that sediments with yeast membranes. (A) Wild type yeast cells transformed with a high copy vector (SFNY 59) or with a high copy vector containing the BETJ structural gene (SFNY 60) were lysed with glass beads. Briefly, cells (100 OD599 nm units) grown overnight in minimal medium were resuspended in 1% SDS (1 OD599 nm units/l10 gl) containing 1 x protease inhibitor cocktail (Ruohola et al., 1988) and then lysed in the presence of glass beads. The lysate was immediately heated to 100°C for 7 min and the cell debris was removed during centrifugation in a clinical table top centrifuge. The supernatant was mixed with Laemmli sample buffer and subjected to electrophoresis (12.5% SDS-PAGE). Samples containing 1 (lanes 1 and 6), 1/2 (lanes 2 and 7) or 1/4 (lanes 3 and 8) OD599 nm units of SFNY 60, or 3 (lanes 4 and 9) or 1 (lanes 5 and 10) OD599 nm units of SFNY 59 were immunoblotted with anti-Betl antibody (lanes 1-5) or pre-immune serum (lanes 6-10). (B) Yeast cells (500 OD599 nm units of NY579) were grown to early exponential phase at 25°C in YPD medium, converted to spheroplasts and lysed in 5 ml of 0.8 M sorbitol-TEA (10 mM triethanolamine, pH 7.2, 1 mM EDTA) containing protease inhibitor cocktail. The sample was homogenized (20 strokes) with a Wheaton tissue grinder (type A) and unbroken cells were removed during a 3 min spin at 450 g. The supernatant from this spin was centrifuged for 1 h at 100 000 g to generate soluble and insoluble fractions. The 450 g supernatant (lysate, lane 1), soluble (100 000 g sup, lane 2) and insoluble (100 000 g pellet, lane 3) fractions were boiled in SDS and subjected to Western blot analysis using a 1:1500 dilution of anti-BetI antibody.

acids (15 amino acids). Thus, BET] encodes a putative membrane protein that shares structural properties with a protein that is found on synaptic vesicles. Antibody to the BET1 gene product recognizes an 18 kDa protein that is associated with membranes The nucleotide sequence of BET] predicts a protein of 18 kDa. Polyclonal antiserum raised to a Betl -TrpE fusion protein, recognizes a band of 18 kDa that was overproduced in a strain that contains BET] on a multicopy plasmid (Figure 1A, compare lanes 4 and 5 with lanes 1, 2 and 3). This

Bet 1p and Bos 1p co-localize to the ER membrane Table I. Distribution of Boslp and Betip following extractions

Control 1 M NaCl l% Triton X-100 0.2 M Na2CO3 (pH 11.4)

Bos Ip Sup (%)

Pellet (%)

BetIp Sup (%)

Pellet (%)

15 13 92 18

85 87 8 82

1 3 89 13

99 97 11 87

Extractions were performed as described in Materials and methods. The results are expressed as percentages of the total c.p.m. in the supernatant and pellet.

Table II. Distribution of Boslp, Betlp and marker enzymes following centrifugation of the lysate at 450 g, 10 000 g and 100 000 g

SI P1 S2 P2 S3 P3 (%) (%) (%) (%) (%) (%) Total protein 97 5 85 23 76 NADPH-cytochrome c reductase (ER) 85 10 27 57 12 GDPase (Golgi) 102 8 60 53 18 a-mannosidase (vacuole) 94 7 39 42 26 ATPase (PM) 69 17 72 24 57 Mif2p (mitochondria) 80 6 7 82 3 Boslp 99 5 48 42 15 Betlp 90 6 48 45 n.d.

16 13 44 13 4 2 16 n.d.

Wild type yeast cells (200 OD599 nm units of SFNY26-6A) were grown to early exponential phase at 25°C in YPD medium, converted to spheroplasts and lysed in 8 ml of 0.8 M sorbitol-TEA (10 mM triethanolamine, pH 7.2, 1 mM EDTA) containing a protease inhibitor cocktail (Ruohola et al., 1988). The sample was homogenized with 20 strokes in a Wheaton tissue grinder (type A) and the lysate was separated into supernatant (SI) and pellet (P1) fractions during a 3 min spin at 450 g. The SI was spun at 10 000 g for 10 min to yield a second supernatant (S2) and pellet (P2). The S2 was further centrifuged at 100 000 g for 1 h to form the 100 000 g supematant (S3) and 100 000 g pellet (P3). The results are expressed as percentages of the amount in the lysate. For Betlp, the determination of the percentage of the total found in the SI and P1 is from a separate experiment and the results of the 10 000 g spin are expressed as percentages of the amount in the SI fraction. Boslp, Betlp and Mif2p were quantified by Western blot analysis using anti-Bos1 (1:2000 dilution), anti-Betl (1:1500) or a 1:1000 dilution of anti-Mif2 antibody. Enzyme assays were performed as described in Materials and methods. Most of the Betlp residing in the S2 fraction was degraded during, or subsequent to, the spin at 100 000 g. For this reason, we were unable to estimate (n.d. or not determined) the Betlp remaining in the S3 and P3 fractions. Although one-fourth of the vanadate-sensitive ATPase was found in the P2, about half the enzyme activity appeared to be soluble (S3). Since the plasma membrane ATPase is an integral membrane protein, this activity may be the consequence of non-specific ATPases or phosphatases that interfere with the assay. This distribution of the vanadate-sensitive ATPase activity has been seen before in other fractionation studies (Walworth et al., 1989).

protein species was not detected by preimmune serum (Figure IA, lanes 6, 7, 8, 9 and 10). A hydrophobic stretch of 19 amino acids at the carboxyterminus of this protein suggests that Betlp may be bound to membranes. To address this question, a simple fractionation experiment was performed. Since Betlp was found to be susceptible to proteolysis in a wild type yeast lysate, this experiment was performed in a strain (NY579) that has reduced protease activity. In the protocol we employed, spheroplasts formed by enzymatic removal of the yeast cell wall were gently lysed. The lysate, prepared as described

in the legend to Figure lB (lane 1), was centrifuged for 1 h at 100 000 g. Subsequent to this spin, the 18 kDa Betlp protein was found entirely in the membrane fraction (compare lanes 2 and 3). A 14 kDa species was also observed; this band was probably a proteolysis product of Betlp, since it was overproduced in a strain harboring BETJ on a multicopy plasmid (not shown). Similar results were previously obtained for Boslp (Shim et al., 1991). The nature of the association of Bos lp and Betip with membranes We have examined the nature of the association of Boslp and Betlp with membranes by assessing the ability of different reagents to extract these proteins from the particulate fraction. Detergents, such as Triton X-100, release proteins from membranes by solubilizing the bilayer, while peripherally associated proteins are extracted by high concentrations of salt or high pH (Howell and Palade, 1982). Extraction studies were performed by incubating samples in one of four reagents [0.8 M sorbitol -TEA (control); 1 M NaCl, 0.8 M sorbitol-TEA; 1% TX 100, 0.8 M sorbitolTEA or 0.2 M Na2CO3 in water] for 15 min on ice. At the end of this incubation, the samples were centrifuged for 1 h at 100 000 g and the distribution of Boslp and Betlp in the supernatant and pellet fractions was determined by Western blot analysis. Although the majority of both Boslp and Betlp were solubilized by Triton X-100, reagents such as NaCl or Na2CO3 did not release substantial amounts of these proteins from membranes (Table I). Thus, by these criteria, BosIp and Betlp behave like membrane proteins and appear to be anchored to the lipid bilayer by hydrophobic interactions.

Boslp and Betip co-fractionate with the ER membrane Since Boslp and BetIp are membrane proteins required for ER to Golgi transport, they may reside on the ER, Golgi or both compartments. To address this question, the intracellular location of these proteins was determined through subcellular fractionation studies. In the protocol we employed, a homogenate was first spun for 3 min at 450 g. This spin removes unlysed cells without an appreciable loss in total protein or Boslp and Betlp (Table II). The 450 g supernatant (S1) was further centrifuged at 10 000 g and the pellet (P2) from this centrifugation step was subfractionated on a sucrose density gradient. The distribution of organelles in each fraction was assessed by assaying marker enzymes. These studies have revealed that GDPase, a Golgi marker protein (Abeijon et al., 1989) and the vacuolar enzyme a-mannosidase (Opheim, 1978) were present to approximately the same extent in the supernatant and pellet fractions of the 10 000 g spin (Table II). The enzyme NADPH-cytochrome c reductase (Kreibich et al., 1973), which is an established marker for the ER membrane in yeast fractionation studies (Walworth et al., 1989), was found preferentially in the P2 along with nearly all the mitochondrial Mif2 protein (Pollock et al., 1988). The majority of the plasma membrane vanadate-sensitive ATPase (Bowman and Slayman, 1979), as well as total cellular protein, remained in the supematant. Under these conditions, approximately half the Boslp and Betlp were in the P2 fraction. These data demonstrate that Boslp and Betlp are associated with at least one structure that partially pellets

3611

A.P.Newman, M.E.Groesch and S.Ferro-Novick

at relatively low speeds. Both the ER and the Golgi complex

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Fraction Fig. 2. Boslp and Betlp co-fractionate with each other and an ER marker on a 30-55% sucrose step gradient. Yeast cells (300-400 OD units of SFNY26-6A or NY579) were grown overnight at 25°C in YPD medium, converted to spheroplasts and lysed in 0.8 M sorbitol-TEA (10 mM triethanolamine, pH 7.2, 1 mM EDTA) containing 1 mM PMSF and protease inhibitor cocktail. The sample was homogenized (20 strokes) with a Wheaton tissue grinder (type A) and unbroken cells were removed during a 3 min spin at 450 g. The supernatant (SI) was spun at 10 000 g for 10 min to generate S2 and P2 fractions. The P2 pellet was resuspended in 2 ml of 55% sucrose (wt/wt) and homogenized (4 strokes) with a 2 ml Wheaton tissue grinder (Wheaton Scientific). This material was placed at the bottom of an SW41 ultracentrifuge tube (Beckman Instruments) and overlaid with sucrose solutions as follows: 1 ml of 50%, 1.5 ml each of 47.5%, 45%, 42.5% and 40%, 1 ml each of 37.5%, 35% and 30%. (All sucrose solutions were buffered with 10 mM HEPES, pH 7.2). The gradient was centrifuged at 4°C for 16 h at 170 000 g and fractionated from the bottom. The pellet (fraction 1) was resuspended in the same volume as the other fractions. Fraction 1 contains the resuspended pellet and fraction 2 is the densest fraction of the gradient. Enzyme assays and Western blots were performed as described in the Materials and methods. The units of activity for each marker enzyme are as follows: NADPH-cytochrome c reductase (expressed as the rate of increase in the A550 of the reaction); GDPase (nmol of phosphate produced per min per fraction); mannosidase (j.mol of p-nitrophenyl a-D-mannopyranoside hydrolysed nm

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fulfill this criterion. In order to determine the organelle with which Boslp and Betlp are associated, the membrane fraction was analysed further on a sucrose density gradient. This was done by resuspending the 10 000 g pellet in 55 % sucrose and layering the sample at the bottom of a 30-55% step gradient. The gradient was centrifuged for 16 h and then fractionated. Intracellular organelles were followed by enzyme assays and the location of BosIp and BetIp was assessed by quantification of Western blots. The peaks of Bosip and BetIp were found to coincide precisely with one another and with the ER membrane (Figure 2A and B). These coincident peaks were well separated from the mitochondria and vacuole (Figure 2C apd B). However, the plasma membrane also peaked in the same fraction (not shown); and the GDPase activity was bimodal with a minor dense fraction that partially overlapped with the ER enzyme. To separate Boslp and Betlp from these other membranes, a modified version of this gradient was employed. The protocol used was the same as described above, except the P2 was resuspended in 60% sucrose and placed at the bottom of a 30-60% step gradient. On this gradient, the ER membrane separated into two peaks (Figure 3A). Boslp and Betlp co-fractionated (Figure 3C) with the denser fraction (Figure 3A and B) and were well separated from the plasma membrane (Figure 3A and D). Better resolution of Bos Ip and the dense fraction of GDPase was also observed on these gradients (Figure 4). When the 10 000 g supernatant was spun at 100 000 g for 1 h, nearly all the GDPase activity pelleted (P3; Table II). Analysis of the P3 on an equilibrium sucrose density gradient revealed that the membrane-bound BosIp co-fractionated with the ER membrane and was well separated from most of the GDPase activity that resided in this fraction (Figure 5). The same results were obtained when Betlp was analysed (not shown). Therefore these fractionation studies demonstrate that Boslp and BetIp co-localize to a compartment of the ER membrane that is dense- and not with the GDPase containing Golgi complex. Bos ip and Bet ip reside on the cytoplasmic face of the ER membrane To assess the topology of Boslp and Betlp with respect to the ER, protease protection assays were performed with membrane fractions isolated from either wild type or secl8 mutant cells (Novick et al., 1981). In secl8, transport from the ER to the Golgi complex is blocked at 37°C and precursors to secreted proteins accumulate within the lumen of the ER. These accumulated proteins provide markers that can be used to assess the integrity of this organelle during protease treatment. In the experiment shown in Figure 6, we measured the sensitivity of Boslp and Betlp to trypsin. As a control, the accessibility of accumulated invertase to its substrate was determined subsequent to protease treatment. A 2-fold stimulation in the activity of this enzyme was observed when Triton X-100 was added to the trypsintreated membranes, indicating that a substantial fraction of the ER membrane (-50%) was tightly sealed following treatment with protease. Under these conditions, Boslp and Betlp were completely digested by trypsin in the absence (compare lanes 2 with 4 and 6 with 8) or presence (compare lanes 1 with 3 and 5 with 7) of detergent. In subsequent experiments, the transported proteins a-factor and CPY were used as lumenal marker proteins. Approximately 80 % of the

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Fig. 3. Boslp and Betlp co-fractionate with a subcompartment of the ER membrane on a 30-60% sucrose step gradient. The protocol followed was the same as described in the legend to Figure 2, except the material from a P2 pellet was resuspended in 60% (w/w) sucrose and overlaid as follows: 1 ml of 55%, 1.5 ml each of 50%, 47.5%, 45% and 40%, and 1 ml each of 37.5%, 35% and 30%. The data in A and B of this figure were obtained from a separate gradient than that shown in C and D. The yields of several marker proteins were estimated from these gradients. Approximately 84% of the NADPH-cytochrome c reductase, 98% of the plasma membrane ATPase and 69% of the Betlp loaded onto the gradient was recovered. The units of activity for each enzyme marker are as follows: PM-ATPase (nmol of phosphate produced per min per fraction); NADPH -cytochrome c reductase (expressed as the rate of increase in the A550 nm of the reaction). The amounts of BosIp and BetIp in each fraction was quantified by densitometric scanning of Western blots. The scales reported for the quantification of these proteins in this figure are not the same as different densitometers were used for this analysis.

800

accumulated proteins were protected from digestion under conditions where BosIp and BetIp were completely degraded (not shown). These findings indicate that Boslp and Betlp reside on the cytoplasmic surface of the ER membrane.

Bos lp co-purifies with the camer vesicles that mediate ER to Golgi transport Cytoplasmically oriented membrane proteins that reside on the ER may also be constituents of the ER to Golgi transport vesicles. ER to Golgi carrier vesicles are normally present in yeast cells in low amounts and blocking transport at this stage of the pathway leads to only a modest accumulation of this intermediate (Novick et al., 1981; Kaiser and Schekman, 1990). Therefore, to trap a large number of vesicular carriers, we have used an assay that reconstitutes ER to Golgi transport. The ability to form this compartment in vitro (Groesch et al., 1990; Rexach and Schekman, 1991) has permitted the isolation and characterization of these vesicles. ER to Golgi transport has been reconstituted in yeast using cells permeabilized by hypo-osmotic or freeze -thaw lysis (Baker et al., 1988; Ruohola et al., 1988). Transport is assessed in these assays by monitoring the processing of a radiolabelled precursor of the secreted pheromone a-factor. Prepro-ax-factor, translated in a yeast lysate (19 kDa), is posttranslationally translocated into the lumen of the ER where it is converted to a 26 kDa species. Conversion of 26 kDa

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pro-a-factor to a high molecular weight form accompanies transport to the Golgi complex. In our assay system (Ruohola et al., 1988), the ER is retained within permeabilized yeast cells (PYC) and conversion of the 26 kDa form to the high molecular weight species is dependent upon the addition of exogenous acceptor Golgi membranes, cytosol and ATP. Although the ER remains within the PYC during the reaction, a portion of the 26 kDa core-glycosylated form of pro-a-factor is 3613

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Fraction Fig. 5. The Boslp that remains in the 10 000 g supernatant also co-fractionates with the ER membrane and not with the Golgi marker enzyme, GDPase. The P3 from 600 OD units of SFNY26-6A was layered on the bottom of a 30-60% sucrose gradient, centrifuged at 4°C for 16 h at 170 000 g and fractionated from the bottom. Fraction 1 contains the resuspended pellet, and fraction 2 is the densest fraction of the gradient.

subsequently found outside the cells (Ruohola et al., 1988). That this species of pro-a-factor is contained within a population of vesicles en route to the Golgi complex has been shown in several ways (Groesch et al., 1990). The carrier vesicles released from the PYC migrate as a single peak on a sucrose density gradient (Groesch et al., 1990) and are biochemically distinct from the ER, implying that they are formed by an authentic budding process and not by non-specific fragmentation of the donor compartment. This hypothesis is supported by a requirement for cytosol and ATP, which was previously documented for intra-Golgi transport in mammalian in vitro systems (Balch et al., 1984) and anticipated from in vivo studies of yeast and mammalian cells (Jamieson and Palade, 1968; Novick et al., 1981). The vesicles released from the PYC are also functional for transport.

In this study, we first formed carrier vesicles in vitro and then analysed the membranes released from the PYC on a 20-50% sucrose density gradient. The data shown in Figure 7 demonstrate that the Boslp released from the cells co-migrates with the pro-a-factor containing vesicles (Figure 7A), indicating that the membranes containing Boslp are the same density as the carrier vesicles. The data described above suggest that Boslp is a vesicle constituent. If this is true, the release of BosIp from the PYC should require cytosol and ATP and display the same kinetics of release from the PYC as pro-ca-factor. The data shown in Figure 8 indicate that approximately equivalent amounts of Bos Ip and pro-ae-factor were released from the PYC under conditions in which a resident protein of the ER was retained 3614

Fig. 6. BosIp and BetIp reside on the cytoplasmic surface of the ER membrane. The protocol described in the legend to Figure 2 was used to generate the P2 pellet. This pellet was resuspended in one third of the initial volume of 0.8 M Sorbitol-TEA and the material was incubated with (lanes 3, 4, 7 and 8) or without (lanes 1, 2, 5 and 6) trypsin (final concentration of 50 ttg/ml) for 1 h on ice. This incubation was performed in the presence (lanes 1, 3, 5 and 7) or absence (lanes 2, 4, 6 and 8) of 0.1% Triton X-100 and was terminated by the addition of trypsin inhibitor (final concentration: 1 mg/mi) during a 5 min incubation on ice. Samples were resuspended in 1 x Laemmli sample buffer, heated to 100°C and subjected to Western blot analysis. A parallel experiment performed with the secl8 mutant strain yielded identical results and enabled us to assess the latency of the ER membrane.

(Groesch et al., 1990). Thus, sorting appears to occur in vitro and some proteins remain in the ER while others leave the donor cells. Cytosolic factors were also required for this event, since heat-treated cytosol failed to support the departure of Boslp from the PYC (Figure 8). In addition, the release of Boslp from the cells was stimulated by the presence of ATP (Figure 7B). When the kinetics of transport was examined during a 60 min incubation, we observed that like the pro-a-factor containing vesicles, approximately onethird of the ATP-dependent release of Boslp occurred during the first 20 min of the reaction. Therefore, based on several criteria, it appears that BosIp is a component of the ER to Golgi transport vesicles.

Discussion BOS] was identified by its ability to suppress the bet] secretory mutant (Newman et al., 1990). The BET] and BOSI genes have now been found to share features in common. Each contains an intron and encodes a small hydrophilic protein containing a hydrophobic carboxyterminal stretch of amino acids that are potentially membrane spanning (Shim et al., 1991). In addition, Betlp is similar in structure to synaptobrevin, an integral membrane protein

associated with the cytoplasmic surface of synaptic vesicles. Biochemical data support the hypothesis that Boslp and Betlp are membrane proteins. Both proteins are extracted from membranes with detergent, but not by treatment with high concentrations of salt or sodium carbonate (Table I). Since Boslp and Betlp each lack an apparent signal sequence, the hydrophobic tails of these proteins may serve as signals for insertion and as membrane anchors. Fractionation studies have revealed that BosIp and BetIp co-localize to a dense compartment of the ER membrane and not with the lighter fraction (Figure 3). Separation of the yeast ER into light and dense fractions has been previously reported (Marriott and Tanner, 1979; Sanderson and Meyer, 1991). In one study, the rough ER marker ribophorin I fractionated with the dense ER and shifted to the lighter fraction when the membranes were stripped of ribosomes (Sanderson and Meyer, 1991). A similar shift in density was

Bet 1p and Bos 1p co-localize to the ER membrane

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Fraction

Fig. 7. Boslp co-fractionates with the pro-a-factor containing vesicles 20-50% sucrose density gradient. (A) The distribution of Boslp in the membranes released from the PYC during the transport assay was compared to that of the pro-a-factor containing vesicles on a sucrose density gradient. In these experiments, two sets of reactions were performed to monitor the release of Boslp and pro-a-factor. In the first set (six reactions), radiolabelled pro-a-factor was translocated into donor ER membranes and released from the PYC into vesicles in an ATP-dependent fashion as described before (Ruohola et al., 1988; Groesch et al., 1990). A parallel set of reactions (96) were also performed and pooled to monitor the release of Boslp. In this second set, no marker protein, such as radiolabelled pro-ca-factor, was introduced into the donor compartment. At the end of each set of assays, the donor PYC were pelleted and the supernatants from these reactions were sedimented during a spin at 120 000 g for 1 h. The pelleted membranes were resuspended in transport buffer that contains osmotic support (see Ruohola et al., 1988) and then layered onto two parallel 20-50% (w/w) sucrose gradients that were centrifuged for 21 h at 120 000 g. The gradients were fractionated from the top and the samples from the first set of reactions were processed as described previously (see Groesch et al., 1990). The gradient, containing the membranes that were released from the PYC in 96 reactions, was subjected to Western blot analysis and quantified as before (Ruohola et al., 1988). Approximately 85% of the Boslp loaded onto the gradient was recovered. In (B) samples were depleted of ATP with apyrase as described before (Ruohola et al., 1988; Groesch et al., 1990). SFNY60 was used for the experiment shown in (A). In addition, assays were also performed with donor PYC prepared from our standard wild type strain (SFNY26-6A). The results obtained were independent of the strain that was used in these studies. on a

observed for the ER enzyme NADPH -cytochrome c reductase (Marriott and Tanner, 1979). We have also found that Boslp co-purifies with the pro-a-factor containing vesicles on sucrose gradients and is released from the PYC in a time and cytosol-dependent fashion (Figures 7 and 8). Efficient release of Boslp from the PYC requires ATP (Figure 8). Our data imply that Boslp and Betlp reside on

Fig. 8. Equivalent amounts of Bosip and pro-ca-factor are released from the PYC in a cytosol-dependent manner. Assays were performed as described in the legend to Figure 7, except that 10 reactions were used to monitor the release of Bos Ip and four were done to assess the transport of pro-ca-factor (i.e. ConA precipitable counts). In the cytosol-dependence experiment, the cytosolic fraction used in the assay was heat-treated as described before (Groesch et al., 1990).

the cytoplasmic surface of a subcompartment of the ER membranes. In addition, Boslp appears to be a constituent of the ER to Golgi transport vesicles. We are currently determining if Betlp also resides on this membrane. Neither Boslp nor Betlp are found in association with the GDPase containing Golgi compartment (Figures 2, 4 and 5). Thus, when the transport vesicles fuse with the Golgi, Boslp is either recycled back to the ER or rapidly degraded. That BET] is identical to SLY12, a multicopy suppressor gene that can compensate for the loss of YPTI function (Dascher et al., 1991), implies that BET] and YPTI perform related functions. It was observed that cells overexpressing SLY12 (BET]) survive when YPTI is disrupted (Dascher et al., 1991; Y.Jiang and S.Ferro-Novick, unpublished observations); however, they grow slowly. This finding suggests that although related, the roles of BET] and YPTI in ER to Golgi transport may not be interchangeable. We speculate that Yptl interacts with Betlp on the surface of membranes to modulate the activity of Betlp. When Betlp is present in sufficient concentration, this regulation may become unnecessary. Alternatively, BET] and YPTI may act on parallel pathways and the YPTI-dependent pathway is dispensable when BET] is overproduced. BOSI and BET] interact genetically with additional genes required for transport from the ER to the Golgi complex. More specifically, we have shown that the growth and secretion defect of the sec22-3 mutant is suppressed by overproduction of either BET] or BOSI (Newman et al., 1990). In addition, the ER-accumulating mutant, sec21-1, is suppressed to a more limited degree by overproduction of BET] or SEC22 (Newman et al., 1990, 1992). Another indication that BET] genetically interacts with SEC22 is the observation that haploid bet] sec22 double mutants are lethal. Double mutants of bet] and sec2] were also found to be growth impaired, again suggesting an interaction between BET] and SEC21 (Newman et al., 1990). These data indicate that in addition to BOSI, BET] genetically interacts with SEC22 and SEC21. Recently, we have found that SEC22 is identical to SLY2 (Newman et al., 1992). Like SLY12 (BET]), SLY2 is a multicopy suppressor gene that can compensate for the loss of YPTI function and encodes a protein that is structurally similar to synaptobrevin (Dascher et al., 1991). The Sec22 3615

A.P.Newman, M.E.Groesch and S.Ferro-Novick

protein may act late in ER to Golgi transport. This question was addressed in a study in which the ER-accumulating sec mutants were screened for the build up of a putative 50 nm vesicular transport intermediate (Kaiser and Schekman, 1990). Since the sec22 mutant was found to contain such vesicles, it was concluded that Sec22 functions in the targeting or fusion of vesicles with the Golgi apparatus. The bet] mutant and BosIp-depleted cells also accumulate 50 nm vesicles (Newman and Ferro-Novick, 1987; Shim et al., 1991). Therefore, Sec22p, Boslp and Betlp may function together to execute a late step in ER to Golgi traffic. Although BOSI and BETI display strong genetic interactions, we have found that overexpression of BOSI cannot alleviate the lethality that is associated with the disruption of BET] (Newman et al., 1990). This finding suggests that the function of Boslp and Betlp are distinct and noninterchangeable. Since the products of BOSI and BET] interact genetically and co-fractionate to the cytoplasmic surface of the ER membrane, these proteins could physically

interact with each other as components of a membrane-bound

complex. The assembly and function of this complex may be dependent upon the productive interaction of Boslp, BetIp and additional members such as Sec22p. A special system of membrane-associated proteins has been proposed to form a matrix that binds the COPs (Duden et al., 1991; Serafini et al., 1991b). Since this matrix must reside on the donor compartment and vesicles, Boslp and possibly Betlp may be components of such a membrane complex. Clearly, one of the first steps in understanding how a membrane protein functions is to determine the organelle(s) with which it is associated. However, once this task has been accomplished, many questions remain to be answered. Since both Boslp and Betlp are cytoplasmically oriented, we are currently using the in vitro transport assay (Ruohola et al., 1988) to assess the effect of antibodies directed against these proteins. Information from such experiments will complement our

data on the intracellular localization of these

proteins and should enable us to define the nature of the interactions displayed among Boslp, Betlp and a subset of the other gene products required for ER to Golgi transport.

Materials and methods Yeast strains and genetic techniques The yeast strains used in this study were as follows: SFNY 26-6A (MAToh his4-619); NY13 (MATa ura3-52); NY579 (MA Ta leu2-3,112 ura3-52, pep4::URA3); DBY1087 (MATa rna2 rna8 ural; from D.Botstein). SFNY60 was constructed by transforming NY13 with pFN100 (containing BET] cloned into the 2 lsm vector pCGS40) and plasmid pAN109 (BOSI cloned into pCGS40) was transformed into NY13 to generate SFNY61. SFNYS9 is NY13 with pCGS40 (Goff et al., 1984). Strains were grown in YP medium [1% yeast extract (Difco Laboratories, Detroit, MI), 2% Bacto-Peptone] containing 2% glucose, or in minimal medium with 2% glucose and the appropriate amino acids, as described before (Newman and

Ferro-Novick, 1987).

Nucleic acid techniques, DNA sequence analysis and computer

analysis

The isolation and amplification of plasmids were performed as described in Newman et al. (1990). The Sequenasetm DNA sequencing kit (United States Biochemicals) was used to sequence BET] by the dideoxy chain termination method (Sanger et al., 1977). The sequencing reactions were each conducted in the presence of 5 ACi of [35S]dATP obtained from Amersham International (Arlington Heights, IL). Single-stranded DNA templates consisted of M13 phage derivatives (mpl8 or mpl9; Dale et al., 1985) into which restriction fragments of plasmid pAN102 (Newman et al., 1990) had been subcloned. The sequence of Betlp was compared with those in the National Biomedical Research Foundation Library as described

3616

before (Shim et al., 1991). Analysis of protein secondary structure was performed using the software package of the Genetics Computer Group

(Devereux

et

al., 1984).

Preparation of RNA and reverse transcription Total cellular RNA was obtained from S. cerevisiae by glass bead lysis in the presence of phenol and chloroform by the protocol of Carlson and Botstein (1982), with the addition of 1% SDS to the lysis buffer. The oligonucleotides synthesized (Genosys Biotechnologies, The Woodlands, TX) for reverse transcription and PCR were as follows: 5' oligonucleotide containing HindIu restriction site: 5'-AAGCTTTGATTGAAGAGCCTCACA-3'; 3' oligonucleotide containing Sall restriction site: 5'-GTCGACATTCTCTGACCCATAGCT-3'. Reverse transcriptions were performed using 10 ltg of RNA and 200 ng of the 3' oligonucleotide in 20 ytl of buffer containing 100 mM Tris (pH 8.3), 75 mM KCI, 10 mM DTT, 3 mM MgCl2 and 525 ptM dNTPs. Following 2 min at 65'C, 30 U of RNasin (Promega) and 50 U of AMV reverse transcriptase (Boehringer Manheim) were added, and the sample was incubated at 42°C for 1 h.

Polymerase chain reaction BETI was hypothesized to have an intron because the sequence does not predict a methionine at the 5' end of the open reading frame. Since BETI did not contain the consensus 5' site (GTATGT) for RNA splicing, the polymerase chain reaction (PCR) was used (as described below) to determine this site. In order to do this, two oligonucleotides, one upstream and one downstream of the putative intron, were synthesized (5' oligonucleotide containing HindlIl restriction site: 5'-AAGCTTTGATTGAAGAGCCTCACA-3'; 3' oligonucleotide containing Sall restriction site: 5'-GTCGACATTCTCTGACCCATAGCT-3') and a sample containing poly(A+) mRNA was reverse-transcribed using the 3' oligonucleotide as a primer. The resulting cDNA, as well as a sample containing genomic DNA, were then amplified using PCR. The product resulting from PCR amplification of genomic DNA was 370 bp, which is precisely the size predicted from the DNA sequence. In contrast, the predominant product was 130 bp smaller when PCR was used to amplify the reverse-transcribed mRNA. Both the 370 bp and the 240 bp bands were cut with ScaI, a restriction endonuclease site contained within the BET] coding region. This resulted in the anticipated decrease in molecular weight, verifying the identity of the observed bands. When mRNA from an rna2 rna8 splice-defective mutant (Rosbasch et al., 1981) was amplified, the resulting product was - 130 bp larger than that obtained from wild type mRNA and co-migrated with the band observed when genomic DNA was amplified. This result was consistent with results of Northern blot analysis performed on mRNA that was isolated from wild type and the rna2 rna8 mutant. Given the approximate size of the intron, the most likely 5' splice site is GTATGA, which contains five of the six consensus base pairs and whose initial nucleotide is located 106 bp upstream of the TACTAAC box. This prediction was confirmed by directly sequencing the 240 bp PCR product. PCR was performed using 10 jd of a reverse transcription reaction of 1 itg of genomic DNA, as well as 200 ng each of the 5' and 3' oligonucleotides, in 100 ytl of buffer containing 10 mM Tris (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 200 AM dNTPs. The initial amplification cycle consisted of denaturation at 95'C for S min, annealing at 60'C for 10 min and extension at 72'C for 2 min. 2 U of Taq DNA polymerase (Perkin Elmer) were added to each sample during the 60'C incubation. The samples were then subjected to hree cycles of denaturation at 92'C (1 min), annealing at 60°C (1 min) and extension at 72'C (2 min). An additional 27 cycles of amplification were conducted in two stages (92'C for 1 min and 650C for 2 min). A final 10 min extension was performed at 72'C. In order to sequence the 240 bp PCR product, the appropriate band was first excised and purified from an agarose gel. The 5' oligonucleotide was labelled at its 5' end with [-y-32P]ATP by the forward reaction of T4 polynucleotide kinase. Coupled amplification and sequencing was then performed as described by Ruano and Kidd (1990).

Production of antibody to a Bet 1- TrpE fusion protein Plasmid pATH 1 (provided by T.Koerner and A.Tzagaloff) contains the inducible TRPE gene. In order to fuse a portion of BET] to TRPE, plasmid pAN 102, containing BET], was digested with RsrII and filled in with Escherichia coli DNA polymerase I Klenow fragment (Boehringer Mannheim), then digested with BgllI. The resulting 0.29 kb fragment was ligated into the BamHI and SnaI sites of pATH I 1. The fusion protein, which contained amino acids 23- 118 of the Bet 1 protein, was used to transform DH-l cells. DH-l cells containing the plasmid were grown at 37°C in M9 CA medium supplemented with ampicillin (100 /g/ml) and tryptophan (40 jsg/ml). An overnight culture was diluted 1:10 into the same medium, grown to an OD599 nm of 1.0 and diluted 1:20 into 100 ml of medium without tryptophan. Following an additional 2 h at 37'C, indoleacrylic acid

Bet1 p and Bos 1p co-localize to the ER membrane was added to a final concentration of 20 jg/ml and growth was continued for 4 h. The Bet 1-TrpE fusion protein and anti-BetI antibody were prepared as described before (Shim et al., 1991).

Assays and other procedures The in vitro transport assay was performed as described previously (Ruohola et al., 1988; Groesch et al., 1990). The ER marker enzyme, NADPH -cytochrome c reductase, was assayed by the method of Kreibich et al. (1973). Vanadate-sensitive plasma membrane ATPase was assayed as described by Bowman and Slayman (1979) and GDPase was measured according to the method of Abeijon et al. (1989). The method of Tulsiani et al. (1977), with the modifications of Opheim (1978), was used to measure a-mannosidase. Western blots were performed and quantified as described before (Ruohola et al., 1988). Antibodies were used at the following dilutions: anti-Bosl (1:2000), anti-Betl (1:1500) and anti-Mif2 antibody (1:1000). Extraction studies Cells (NY 579) were grown, converted to spheroplasts and lysed as described above. Extractions were performed using one of two procedures. In the first, a P2 pellet (prepared as described in the legend to Figure 2) was resuspended in one of the following buffers: 0.8 M sorbitol -TEA (control); 1 M NaCl, 0.8 M Sorbitol-TEA; 1% TX 100, 0.8 M sorbitol-TEA; 0.2 M Na2CO3 in water. Samples were incubated for 15 min on ice, then ultracentrifuged at 100 000 g for 1 h to separate the soluble from the insoluble material and the pellets were resuspended in an equal volume of lysis buffer. All samples were boiled in SDS, electrophoresed (15% SDS-PAGE) and subjected to Western blot analysis. The second method was the same except that instead of a P2 fraction, a (2 x) 450 g supernatant was used and mixed 1: 1 with 2 x extraction buffer. Both methods yielded similar results.

Acknowledgements We thank A.West, B.Pollock and A.Horwich for poly(A+) RNA and antiMif2 antibody, D.Botstein for strains, P.Brenwald and G.Ruano for advice on PCR amplification, E.Ullu for advice on the analysis of RNA and J.Shim for his assistance in the preparation of antibody. L.Stone assisted with the computer analysis of Betlp and J.Graf and M.Clague provided technical assistance. We also thank P.De Camilli for stimulating discussions on the structure of the synaptobrevins. This work was supported by grants awarded to S.F-N. from the National Institutes of Health (1 ROI GM45431), (CA 46128) and the Mathers Foundation. A.Newman received salary support from a National Science Foundation predoctoral fellowship and a National Institutes of Health Grant (CA 46128) awarded to S.F-N.

References Abeijon,C., Orlean,P., Robbins,P.W. and Hirschberg,C.B. (1989) Proc. Natl. Acad. Sci. USA, 86, 6935-6939. Bacon,R.A., Salminen,A., Ruohola,H., Novick,P. and Ferro-Novick,S. (1989) J. Cell Biol., 109, 1015-1022. Baker,D., Hicke,L., Rexach,M., Schleyer,M. and Schekman,R. (1988) Cell, 54, 335-344. Baker,D., Wuestehube,L., Schekman,R., Botstein,D. and Segev,N. (1990) Proc. Natl. Acad. Sci. USA, 87, 355-359. Balch,W.E., Dunphy,W.G., Braell,W.A. and Rothman,J.E. (1984) Cell, 39, 405-416. Baumert,M., Maycox,P.R., Navone,F., De Camilli,P. and Jahn,R. (1989) EMBO J., 8, 379-384. Beckers,C.J.M., Keller,D.S. and Balch,W.E. (1987) Cell, 50, 523-534. Bourne,H.R. (1988) Cell, 53, 669-671. Bowman,B.J. and Slayman,C.W. (1979) J. Biol. Chem., 254, 2928 - 2934. Carlson,M. and Botstein,D. (1982) Cell, 28, 145-154. Clary,D.O., Griff,I.C. and Rothman,J.E. (1990) Cell, 61, 709-721. Dale,R., McClure,B. and Houghins,J. (1985) Plasmid, 13, 31-40. Dascher,C., Ossig,R., Gallwitz,D. and Schmitt,H.D. (1991) Mol. Cell. Biol., 11, 872-885. Devereux,J., Haberli,P. and Smithies,O. (1984) Nucleic Acids Res., 12, 387-395. Donaldson,J.G., Lippincott-Schwartz,J., Bloom,G.S., Kreis,T.E. and Klausner,R.D. (1990) J. Cell Biol., 111, 2295-2306. Duden,R., Griffiths,G., Frank,R., Argosand,P. and Kreis,T.E. (1991) Cell,

Goud,B., Salminen,A., Walworth,N.C. and Novick,P. (1988) Cell, 53, 753 -768. Groesch,M., Ruohola,H., Bacon,R., Rossi,G. and Ferro-Novick,S. (1990) J. Cell Biol., 111, 45-53. Hicke,L. and Schekman,R. (1989) EMBO J., 8, 1677-1684. Howell,K.E. and Palade,G.E. (1982) J. Cell Biol., 92, 822-832. Jamieson,J. and Palade,G. (1968) J. Cell Biol., 39, 589-603. Kaiser,C.A. and Schekman,R. (1990) Cell, 61, 723-733. Keen,J.H. (1990) Annu. Rev. Biochem., 59, 415-438. Kreibich,G., Debey,P. and Sabatini,D.D. (1973) J. Cell Biol., 58, 436-462. Kyte,J. and Doolittle,R.F. (1982) J. Mol. Biol., 157, 105-132. Lippincott-Schwartz,J., Yuan,L.C., Bonifacino,J.S. and Klausner,R.D. (1989) Cell, 56, 801-813. Marriott,M. and Tanner,W. (1979) J. Bacteriol., 139, 565-572. Nakano,A. and Muramatsu,M. (1989) J. Cell Biol., 109, 2677-2691. Nakano,A., Brada,D. and Schekman,R. (1988) J. Cell Biol., 107, 851-863. Newman,A. and Ferro-Novick,S. (1987) J. Cell Biol., 105, 1587-1594. Newman,A., Shim,J. and Ferro-Novick,S. (1990) Mol. Cell. Biol., 10, 3405-3414. Newman,A., Graf,J., Mancini,P., Rossi,G., Lian,J.P. and Ferro-Novick,S. (1992) Mol. Cell. Biol., 12, 3663-3664. Novick,P., Ferro,S. and Schekman,R. (1981) Cell, 25, 461-469. Opheim,D.J. (1978) Biochim. Biophys. Acta, 524, 121-130. Orci,L., Tagaya,M., Amherdt,M., Perrelet,A., Donaldson,J.G., LippincottSchwartz,J., Klausner,R.D. and Rothman,J.E. (1991) Cell, 64, 1183-1195. Palade,G. (1975) Science, 189, 347-358. Pollock,R.A., Hartl,F.-U., Cheng,M.Y., Ostermann,J., Horwich,A. and Neupert,W. (1988) EMBO J., 7, 3493-3500. Rexach,M.F. and Schekman,R.W. (1991) J. Cell Biol., 114, 219-320. Rosbasch,M., Harris,P.K.W., Woodford,J.L. and Teem,J.L. (1981) Cell, 24, 679-686. Rossi,G., Jiang,Y., Newman,A.P. and Ferro-Novick,S. (1991) Nature, 351, 158- 161. Ruano,G. and Kidd,K.K. (1990) Am. J. Human Genetics, 47, A116. Ruohola,H., Kabcenell,A. and Ferro-Novick,S. (1988) J. Cell Biol., 107, 1465-1476. Sanderson,C.M. and Meyer,D.I. (1991) J. Biol. Chem., 266, 13423- 13430. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Schmitt,H., Puzicha,M. and Gallwitz,D. (1988) Cell, 53, 635-647. Segev,N., Mulholland,J. and Botstein,D. (1988) Cell, 52, 915-924. Serafini,T., Orci,L., Amherdt,M., Brunner,M., Kahn,R.A. and Rothman,J.E. (1991a) Cell, 67, 239-253. Serafini,T., Stenbeck,G., Brecht,A., Lottspeich,F., Orci,L., Rothman,J.E. and Wieland,F.T. (1991b) Nature, 349, 215-220. Shim,J., Newman,A.P. and Ferro-Novick,S. (1991) J. Cell Biol., 113, 55-64.

Sudhof,T.C., Baumert,M., Perin,M.S. and Jahn,R. (1989) Neuron, 2, 1475-1481. Tulsiani,E.R.P., Opheim,D.J. and Touser,O. (1977) J. Biol. Chem., 252, 3227 -3233.

Walworth,N.C., Goud,B., Ruohola,H. and Novick,P.J. (1989) Methods Cell Biol., 31, 335-354. Waters,M.G., Serafini,T. and Rothman,J.E. (199 1) Nature, 349, 248-25 1. Received on

MaN' 25,

1992

64, 649-665. Eakle,K.A., Bernstein,M. and Emr,S.D. (1988) Mol. Cell. Biol., 8, 4098-4109.

Goff,C.G., Moir,D.T., Kohno,T., Gravius,T.C., Smith,R.A., Yamasaki,E. and Tauntin-Rigby,A. (1984) Gene, 27, 35-46.

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