High-Copy Suppressor Analysis Reveals a Physical Interaction between Sec34p and Sec35p, a Protein Implicated in Vesicle Docking

A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport. A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER. Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function. In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi. This screen revealed that SEC34 specifically suppresses sec35-1. SEC34 encodes a hydrophilic protein of approximately 100 kDa. Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble. Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.     

The Sec34/Sec35p complex,a Ypt1p effector required for retrograde intra-Golgi trafficking,interacts with Golgi SNAREs and COPI vesicle coat proteins

The Sec34/35 complex was identified as one of the evolutionarily conserved protein complexes that regulates a cis-Golgi step in intracellular vesicular transport. We have identified three new proteins that associate with Sec35p and Sec34p in yeast cytosol. Mutations in these Sec34/35 complex subunits result in defects in basic Golgi functions, including glycosylation of secretory proteins, protein sorting, and retention of Golgi resident proteins. Furthermore, the Sec34/35 complex interacts genetically and physically with the Rab protein Ypt1p, intra-Golgi SNARE molecules, as well as with Golgi vesicle coat complex COPI. We propose that the Sec34/35 protein complex acts as a tether that connects cis-Golgi membranes and COPI-coated, retrogradely targeted intra-Golgi vesicles.     

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1 , a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the γ-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21 , we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20 .     

Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay.     

Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.     

Identification of Sec36p,Sec37p,and Sec38p: components of yeast complex that contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.     

Sgf1p, a New Component of the Sec34p/Sec35p Complex

Here we report the identification of SGF1 as a high-copy suppressor of the sec35–1 mutant. SGF1 encodes an essential hydrophilic protein of ∼ 100 kDa. Using the yeast two-hybrid system and coprecipitation studies, we demonstrate that Sgf1p is a new subunit of the multiprotein Sec34p/Sec35p complex. Reduced levels of Sgf1p lead to the accumulation of a variety of membranes as well as a kinetic block in endoplasmic reticulum to Golgi traffic. Immunofluorescence studies demonstrate that Sec34p is found throughout the Golgi, with a high concentration on early Golgi. Although an earlier study suggested that Sec34p (Grd20p) is not required for protein secretion, we show here that the sec34–2 and sec35–1 mutations lead to a pleiotropic block in the secretion of all proteins into the growth medium.     

The Rab GTPase Ypt1p and tethering factors couple protein sorting at the ER to vesicle targeting to the Golgi apparatus

GPI-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event can be reproduced in vitro. When extracts from a uso1 mutant were used, the sorting of GPI-anchored proteins from other secretory proteins was defective. Complementation with purified Uso1p restored sorting. The Rab GTPase Ypt1p and the tethering factors Sec34p and Sec35p, but not Bet3p, a member of the TRAPP complex, were also required for protein sorting upon ER exit. Therefore, the Ypt1p tethering complex couples protein sorting in the ER to vesicle targeting to the Golgi apparatus. Sorting of GPI-anchored proteins from other secretory proteins was also observed in vivo. The sorting defect observed in vitro with uso1 and ypt1 mutants was reproduced in vivo.     

SED4 encodes a yeast endoplasmic reticulum protein that binds Sec16p and participates in vesicle formation

SEC16 is required for transport vesicle budding from the ER in Saccharomyces cerevisiae, and encodes a large hydrophilic protein found on the ER membrane and as part of the coat of transport vesicles. In a screen to find functionally related genes, we isolated SED4 as a dosage- dependent suppressor of temperature-sensitive SEC16 mutations. Sed4p is an integral ER membrane protein whose cytosolic domain binds to the COOH-terminal domain of Sec16p as shown by two-hybrid assay and coprecipitation. The interaction between Sed4p and Sec16p probably occurs before budding is complete, because Sed4p is not found in budded vesicles. Deletion of SED4 decreases the rate of ER to Golgi transport, and exacerbates mutations defective in vesicle formation, but not those that affect later steps in the secretory pathway. Thus, Sed4p is important, but not necessary, for vesicle formation at the ER. Sec12p, a close homologue of Sed4p, also acts early in the assembly of transport vesicles. However, SEC12 performs a different function than SED4 since Sec12p does not bind Sec16p, and genetic tests show that SEC12 and SED4 are not functionally interchangeable. The importance of Sed4p for vesicle formation is underlined by the isolation of a phenotypically silent mutation, sar1-5, that produces a strong ER to Golgi transport defect when combined with sed4 mutations. Extensive genetic interactions between SAR1, SED4, and SEC16 show close functional links between these proteins and imply that they might function together as a multisubunit complex on the ER membrane.     

Sec35p,a Novel Peripheral Membrane Protein,Is Required for ER to Golgi Vesicle Docking

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.Protein transport through the secretory pathway occurs via transport vesicles under the direction of a large set of protein components (Rothman, 1994). The process can be divided into three stages: (a) vesicle budding, (b) vesicle docking, and (c) membrane fusion, with distinct sets of proteins mediating each phase. The budding step involves recruitment of coat proteins to the membrane and culminates with the release of coated vesicles (Schekman and Orci, 1996). The docking reaction is likely to require a set of integral membrane proteins on the vesicle and target membranes, termed v-SNAREs¹ and t-SNAREs (vesicle- and target membrane-soluble N-ethylmaleimide–sensitive fusion protein [NSF] attachment protein [SNAP] receptors, respectively), that are thought to confer specificity through their pair-wise interactions (Söllner et al., 1993b ). Small GTP-binding proteins of the rab family also assist in the docking process (Ferro-Novick and Novick, 1993), but their precise function is not known. The fusion step ensues after docking and results in the delivery of the vesicular cargo to the next compartment in the secretory pathway.Vesicular transport from the ER to the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively characterized. Transport vesicle budding involves the assembly of the COPII coat, composed of the Sec13p/Sec31p (Pryer et al., 1993; Salama et al., 1993; Barlowe et al., 1994) and Sec23p/Sec24p heterodimers (Hicke and Schekman, 1989; Hicke et al., 1992), under the direction of an integral membrane protein, Sec12p (Nakano et al., 1988; Barlowe and Schekman, 1993), a small GTPase, Sar1p (Nakano and Muramatsu, 1989), and a multidomain protein, Sec16p (Espenshade et al., 1995; Shaywitz et al., 1997). Docking is thought to require a tethering event mediated by Uso1p (Cao et al., 1998), the yeast homologue of mammalian p115 (Barroso et al., 1995; Sapperstein et al., 1995), followed by or concurrent with the interaction of a set of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p (Newman and Ferro-Novick, 1987; Newman et al., 1990; Ossig et al., 1991; Shim et al., 1991; Søgaard et al., 1994) and perhaps Ykt6p (Søgaard et al., 1994; McNew et al., 1997), with the cognate t-SNARE on the Golgi, Sed5p (Hardwick and Pelham, 1992). For some time it was thought that fusion may be initiated by disassembly of the v/t-SNARE complex (Söllner et al., 1993a ) by yeast SNAP, Sec17p, (Griff et al., 1992) and NSF, Sec18p (Eakle et al., 1988; Wilson et al., 1989). However, this concept has been challenged by studies with a yeast system that reconstitutes homotypic vacuolar fusion, which suggests the action of Sec18p is before vesicle docking (Mayer et al., 1996; Mayer and Wickner, 1997). In addition, a prefusion role for NSF has been supported by the recent finding that liposomes bearing SNAREs alone can fuse in the absence of NSF (Weber et al., 1998).Several proteins involved in the regulation of yeast ER to Golgi v/t-SNARE complex assembly have been identified, including Ypt1p, Uso1p, and Sly1p. Ypt1p is a member of the rab family of small GTP-binding proteins that have been identified as important components of almost every stage in the secretory pathway (Ferro-Novick and Novick, 1993). Hydrolysis of GTP by rab-like proteins has been hypothesized to provide the regulatory switch that controls the fidelity of vesicular transport (Bourne, 1988). A second protein, Uso1p (Nakajima et al., 1991), appears to function in the same pathway as Ypt1p (Sapperstein et al., 1996), and both proteins have been demonstrated to be essential for SNARE complex assembly (Søgaard et al., 1994; Sapperstein et al., 1996; Lupashin and Waters, 1997). The third protein, Sly1p, is associated with the t-SNARE Sed5p (Søgaard et al., 1994). SLY1 is an essential gene in yeast (Dascher et al., 1991; Ossig et al., 1991), and Sly1p is required for ER to Golgi transport in vitro (Lupashin et al., 1996) and in vivo (Ossig et al., 1991). However, several lines of evidence, particularly from Sly1p homologues in other organisms, indicate that Sly1p may also function as a negative regulator of v/t-SNARE complex assembly, perhaps by preventing the association of the v- and t-SNAREs (Hosono et al., 1992; Pevsner et al., 1994; Schulze et al., 1994). A dominant allele of SLY1, termed SLY1-20, is capable of suppressing mutations in YPT1 and USO1, including complete deletions (Dascher et al., 1991; Sapperstein et al., 1996). Thus, in the presence of Sly1-20p, two components required for SNARE complex assembly are no longer essential. We have proposed a model (Sapperstein et al., 1996; Lupashin and Waters, 1997) in which Ypt1p and Uso1p function to relieve the inhibitory action of Sly1p on SNARE complex assembly. In this model Sly1-20p can be thought of as a noninhibitory form of SLY1 that renders Ypt1p and Uso1p superfluous.We believe that the ability of SLY1-20 to suppress defects in upstream docking regulators can be used to identify additional components involved in the regulation of vesicular docking. We have undertaken a genetic screen (to be presented elsewhere) to isolate novel components in this pathway which, when mutated, depend upon Sly1-20p for viability. In the course of this work, we discovered that two recently identified mutants, sec34 and sec35, can be suppressed by SLY1-20 and thus satisfy the criterion of our screen. These mutants were isolated in a novel screen to identify components involved in transport at any step between the ER and the trans-Golgi network (i.e., the Kex2p compartment) in yeast (Wuestehube et al., 1996). Both sec34 and sec35 accumulate the core-glycosylated form of secretory proteins at the nonpermissive temperature, indicating a block in ER to Golgi transport. Furthermore, electron microscopy indicated that both sec34 and sec35 accumulate numerous vesicles upon shift to the restrictive temperature (Wuestehube et al., 1996), a hallmark of genes whose protein products are involved in the docking or fusion phase of transport (Kaiser and Schekman, 1990). In this report we describe the cloning of SEC35 and analysis of its genetic interactions with other secretory genes. Strong genetic interaction between SEC35 and SLY1, YPT1, and USO1 suggests that Sec35p may function in vesicle docking. To test this possibility, we devised an in vitro transport assay that depends on the addition of purified Sec35p and Uso1p. Vesicles synthesized in the absence of functional Sec35p do not fuse with the Golgi compartment and remain as freely diffusible intermediates. Upon addition of Sec35p and Uso1p, vesicles dock to the Golgi and proceed to membrane fusion. Requirements for Sec35p at the vesicle docking step correlates our genetic experiments with the biochemically distinguishable steps of vesicle docking and membrane fusion.     

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.     

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.     

Smy2p participates in COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex

The coat protein complex II (COPII) is essential for vesicle formation from the endoplasmic reticulum (ER) and is composed of two heterodimeric subcomplexes, Sec23p/Sec24p and Sec13p/Sec31p, and the small guanosine triphosphatase Sar1p. In an effort to identify novel factors that may participate in COPII vesicle formation, we isolated SMY2 , a yeast gene encoding a protein of unknown function, as a multicopy suppressor of the temperature-sensitive sec24-20 mutant. We found that even a low-copy expression of SMY2 was sufficient for the suppression of the sec24-20 phenotypes, and the chromosomal deletion of SMY2 led to a severe growth defect in the sec24-20 background. In addition, SMY2 exhibited genetic interactions with several other genes involved in the ER-to-Golgi transport. Subcellular fractionation analysis showed that Smy2p was a peripheral membrane protein fractionating together with COPII components. However, Smy2p was not loaded onto COPII vesicles generated in vitro . Interestingly, coimmunoprecipitation between Smy2p and the Sec23p/Sec24p subcomplex was specifically observed in sec23-1 and sec24-20 backgrounds, suggesting that this interaction was a prerequisite for the suppression of the sec24-20 phenotypes by overexpression of SMY2 . We propose that Smy2p is located on the surface of the ER and facilitates COPII vesicle formation through the interaction with Sec23p/Sec24p subcomplex.     

Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature- sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.     

Sec7p directs the transitions required for yeast Golgi biogenesis

Endoplasmic reticulum (ER)-to-Golgi traffic in yeast proceeds by the maturation of membrane compartments from post-ER vesicles to intermediate small vesicle tubular clusters (VTCs) to Golgi nodular membrane networks (Morin-Ganet et al., Traffic 2000; 1: 56–68). The balance between ER and Golgi compartments is maintained by COPII- and COPI-mediated anterograde and retrograde traffic, which are dependent on Sec7p and ARF function. The sec7-4 temperature-sensitive allele is a mutation in the highly conserved Sec7 domain (Sec7d) found in all ARF-guanine nucleotide exchange factor proteins. Post-ER trafficking is rapidly inactivated in sec7-4 mutant yeast at the restrictive temperature. This conditional defect prevented the normal production of VTCs and instead generated Golgi-like tubes emanating from the ER exit sites. These tubes progressively developed into stacked cisternae defining the landmark sec7 mutant phenotype. Consistent with the in vivo results, a Sec7d peptide inhibited ER-to-Golgi transport and displaced Sec7p from its membrane anchor in vitro . The similarities in the consequences of inactivating Sec7p or ARFs in vivo was revealed by genetic disruption of yeast ARFs or by addition of brefeldin A (BFA) to whole cells. These treatments, as in sec7-4 yeast, affected the morphology of membrane compartments in the ER-Golgi transition. Further evidence for Sec7p involvement in the transition for Golgi biogenesis was revealed by in vitro binding between distinct domains of Sec7p with ARFs, COPI and COPII coat proteins. These results suggest that Sec7p coordinates membrane transitions in Golgi biogenesis by directing and scaffolding the binding and disassembly of coat protein complexes to membranes, both at the VTC transition from ER exit sites to form Golgi elements and for later events in Golgi maturation.     

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.     

Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.     

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.     

Ypt32 recruits the Sec4p guanine nucleotide exchange factor,Sec2p,to secretory vesicles; evidence for a Rab cascade in yeast

SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.     

The Sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.

SEC13 encodes a 33 kDa protein that participates in vesicle budding from the endoplasmic reticulum (ER). In order to purify a functional form of Sec13p, a SEC13-dihydrofolate reductase (mouse) fusion gene (SEC13:DHFR) was constructed that complements both sec13 temperature sensitive and null mutations. Methotrexate-agarose affinity chromatography facilitated the purification of two forms of the Sec13-dhfrp fusion protein: a monomeric form and a high molecular weight complex. The complex form consists of two subunits: Sec13-dhfrp and a 150 kDa protein (p150). Native immunoprecipitation experiments confirm that Sec13p exists in a complex with p150 in wild type cells. Functional analysis supports a role for both subunits in protein transport. Vesicle budding from the ER in a cell-free reaction is inhibited by Fab antibody fragments directed against either Sec13p or p150. The purified Sec13-dhfrp/p150 complex, but not the Sec13-dhfrp monomer, in combination with two other pure protein fractions (Sar1p and a Sec23/Sec24 protein complex) satisfies the requirement for cytosol in a cell-free vesicle budding reaction. The vesicles formed with the purified protein fractions are competent to fuse with the Golgi and are biochemically distinct from the ER membrane fraction from which they derive.