The diversity of Rab proteins in vesicle transport.

Rab proteins have been primarily implicated in vesicle docking as regulators of SNARE pairing. Recent findings, however, indicate that their function in vesicle trafficking can go beyond this role, and a number of proteins, unrelated to each other, have been identified as putative Rab effectors. Although the GTPase switch of Rab proteins is highly conserved, functional mechanisms may be highly diversified among members of the Rab family.

Spatial regulation of the exocyst complex by Rho1 GTPase.

Spatial regulation of membrane traffic is fundamental to many biological processes, including epithelial cell polarization and neuronal synaptogenesis. The multiprotein exocyst complex is localized to sites of polarized exocytosis, and is required for vesicle targeting and docking at specific domains of the plasma membrane. One component of the complex, Sec3, is thought to be a spatial landmark for polarized exocytosis. We have searched for proteins that regulate the polarized localization of the exocyst in the budding yeast Saccharomyces cerevisiae. Here we report that certain rho1 mutant alleles specifically affect the localization of the exocyst proteins. Sec3 interacts directly with Rho1 in its GTP-bound form, and functional Rho1 is needed both to establish and to maintain the polarized localization of Sec3. Sec3 is not the only mediator of the effect of Rho1 on the exocyst, because some members of the complex are correctly targeted independently of the interaction between Rho1 and Sec3. These results reveal the action of parallel pathways for the polarized localization of the exocytic machinery, both of which are under the control of Rho1, a master regulator of cell polarity.

Protein complexes in transport vesicle targeting.

The transport of material between membrane-bounded organelles in eukaryotic cells requires the accurate delivery of different classes of carrier vesicles to specific target compartments. Recent studies indicate that different targeting reactions involve distinct protein complexes that act to mark the target organelle for incoming vesicles. This review focuses on the proteins and protein complexes that have been implicated in various targeting reactions.

Export of major cell surface proteins is blocked in yeast secretory mutants.

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.

Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle.

SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast. Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane. The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl. Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane. Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p. However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes. Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable. During the course of these studies we have found that two activities associated with the yeast Golgi apparatus, Kex2 endopeptidase and GDPase, are in separable subcompartments.

Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.

The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast.

SEC15 function is required at a late stage of the yeast secretory pathway. Duplication of the gene encoding the ras-like, GTP-binding protein, Sec4, can suppress the partial loss of function resulting from the sec15-l mutation, but cannot suppress disruption of sec15. Analysis of the SEC15 gene predicts a hydrophilic protein product of 105 kD. Anti-Sec15 antibody recognizes a protein of 116-kD apparent molecular mass which is associated with a microsomal fraction of yeast in a strongly pH dependent fashion. Overproduction of Sec15 protein interferes with the secretory pathway, resulting in the formation of a cluster of secretory vesicles, and a patch of Sec15 protein revealed by immunofluorescence. The sec4-8 and sec2-4l mutations, but not mutations in other SEC genes, prevent formation of the Sec15 protein patch. We propose that Sec15 protein responds to the function of the Sec4 protein to control vesicular traffic.

Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain.

SEC2 function is required at the post-Golgi apparatus stage of the yeast secretory pathway. The SEC2 sequence encodes a protein product of 759 amino acids containing an amino terminal region that is predicted to be in an alpha-helical, coiled-coil conformation. Two temperature-sensitive alleles, sec2-41 and sec2-59, encode proteins truncated by opal stop codons and are suppressible by an opal tRNA suppressor. Deletion analysis indicates that removal of the carboxyl terminal 251 amino acids has no apparent phenotype, while truncation of 368 amino acids causes temperature sensitivity. The amino terminal half of the protein, containing the putative coiled-coil domain, is essential at all temperatures. Sec2 protein is found predominantly in the soluble fraction and displays a native molecular mass of greater than 500 kD. All phenotypes of the temperature-sensitive sec2 alleles are partially suppressed by duplication of the SEC4 gene, but the lethality of a sec2 disruption is not suppressed. The sec2-41 mutation exhibits synthetic lethality with the same subset of the late acting sec mutants as does sec4-8 and sec15-1. The Sec2 protein may function in conjunction with the Sec4 and Sec15 proteins to control vesicular traffic.

Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis.

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. We propose that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane.

Ordering the final events in yeast exocytosis.

In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

The role of Myo2, a yeast class V myosin, in vesicular transport.

Previous studies have shown that temperature-sensitive, myo2-66 yeast arrest as large, unbudded cells that accumulate vesicles within their cytoplasm (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). In this study we show that myo2-66 is synthetically lethal in combination with a subset of the late-acting sec mutations. Thin section electron microscopy shows that the post-Golgi blocked secretory mutants, sec1-1 and sec6-4, rapidly accumulate vesicles in the bud, upon brief incubations at the restrictive temperature. In contrast, myo2-66 cells accumulate vesicles predominantly in the mother cell. Double mutant analysis also places Myo2 function in a post-Golgi stage of the secretory pathway. Despite the accumulation of vesicles in myo2-66 cells, pulse-chase studies show that the transit times of several secreted proteins, including invertase and alpha factor, as well as the vacuolar proteins, carboxy-peptidase Y and alkaline phosphatase, are normal. Therefore the vesicles which accumulate in this mutant may function on an exocytic pathway that transports a set of cargo proteins that is distinct from those analyzed. Our observations are consistent with a role for Myo2 in transporting a class of secretory vesicles from the mother cell along actin cables into the bud.

Purification and characterization of constitutive secretory vesicles from yeast.

We have developed a purification procedure for the isolation of constitutive post-Golgi secretory vesicles from Saccharomyces cerevisiae. Although the post-Golgi stage of the secretion pathway is normally very rapid, we have used a temperature-sensitive secretory mutant, sec 6-4, to greatly expand the population of secretory vesicles. Following invertase as a marker, intact vesicles are enriched 36-fold from the crude lysate. The final preparation contains few contaminants as assessed by morphologic and biochemical examination. Three proteins (110, 40-45, and 18 kD) co-purify with the vesicle marker enzyme invertase. Metabolic labeling experiments indicate that these vesicle-associated proteins are synthesized during the period of vesicle accumulation. They are not apparent in the corresponding fractions from wild-type cells. Analysis of these proteins indicates that the 110-kD protein is a major glycoprotein residing in the vesicle lumen, while the 40-45- and 18-kD proteins are not glycosylated and are firmly associated with the vesicle membrane, each with at least one domain exposed on the cytoplasmic surface.

Yeast secretory mutants that block the formation of active cell surface enzymes.

Yeast cells secrete a variety of glycosylated proteins. At least two of these proteins, invertase and acid phosphatase, fail to be secreted in a new class of mutants that are temperature-sensitive for growth. Unlike the yeast secretory mutants previously described (class A sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell., 21:205-420), class B sec mutants (sec 53, sec 59) fail to produce active secretory enzymes at the restrictive temperature (37 degrees C). sec 53 and sec 59 appear to be defective in reactions associated with the endoplasmic reticulum. Although protein synthesis continues at a nearly normal rate for 2 h at 37 degrees C, incorporation of [3H]mannose into glycoprotein is reduced. Immunoreactive polypeptide forms of invertase accumulate within the cell which have mobilities on SDS PAGE consistent with incomplete glycosylation: sec 53 produces little or no glycosylated invertase, and sec 59 accumulates forms containing 0-3 of the 9-10 N-linked oligosaccharide chains that are normally added to the protein. In addition to secreted enzymes, maturation of the vacuolar glycoprotein carboxypeptidase Y, incorporation of the plasma membrane sulfate permease activity, and secretion of the major cell wall proteins are blocked at 37 degrees C.

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion.

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.

Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles.

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

The role of the COOH terminus of Sec2p in the transport of post-Golgi vesicles.

Sec2p is required for the polarized transport of secretory vesicles in S. cerevisiae. The Sec2p NH(2) terminus encodes an exchange factor for the Rab protein Sec4p. Sec2p associates with vesicles and in Sec2p COOH-terminal mutants Sec4p and vesicles no longer accumulate at bud tips. Thus, the Sec2p COOH terminus functions in targeting vesicles, however, the mechanism of function is unknown. We found comparable exchange activity for truncated and full-length Sec2 proteins, implying that the COOH terminus does not alter the exchange rate. Full-length Sec2-GFP, similar to Sec4p, concentrates at bud tips. A COOH-terminal 58-amino acid domain is necessary but not sufficient for localization. Sec2p localization depends on actin, Myo2p and Sec1p, Sec6p, and Sec9p function. Full-length, but not COOH-terminally truncated Sec2 proteins are enriched on membranes. Membrane association of full-length Sec2p is reduced in sec6-4 and sec9-4 backgrounds at 37 degrees C but unaffected at 25 degrees C. Taken together, these data correlate loss of localization of Sec2 proteins with reduced membrane association. In addition, Sec2p membrane attachment is substantially Sec4p independent, supporting the notion that Sec2p interacts with membranes via an unidentified Sec2p receptor, which would increase the accessibility of Sec2p exchange activity for Sec4p.

Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function.

The budding mode of Saccharomyces cerevisiae cell growth demands that a high degree of secretory polarity be established and directed toward the emerging bud. We report here our demonstration that mutations in SAC1, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing sec14 lethalities associated with yeast Golgi defects. Moreover, these sac1 suppressor properties also extended to sec6 and sec9 secretory vesicle defects. The genetic data are consistent with the notion that SAC1p modulates both secretory pathway and actin cytoskeleton function. On this basis, we suggest that SAC1p may represent one aspect of the mechanism whereby secretory and cytoskeletal activities are coordinated, so that proper spatial regulation of secretion might be achieved.

The yeast class V myosins, Myo2p and Myo4p, are nonprocessive actin-based motors.

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 microm/s for Myo2p and 1.1 microm/s for Myo4p. Myo2p motility is Ca(2+) insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca(2+), unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.

The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants.

The YPT1 gene encodes a raslike, GTP-binding protein that is essential for growth of yeast cells. We show here that mutations in the ypt1 gene disrupt transport of carboxypeptidase Y to the vacuole in vivo and transport of pro-alpha-factor to a site of extensive glycosylation in the Golgi apparatus in vitro. Two different ypt1 mutations result in loss of function of the Golgi complex without affecting the activity of the endoplasmic reticulum or soluble components required for in vitro transport. The function of the mutant Golgi apparatus can be restored by preincubation with wild-type cytosol. The transport defect observed in vitro cannot be overcome by addition of Ca++ to the reaction mixture. We have also established genetic interactions between ypt1 and a subset of the other genes required for transport to and through the Golgi apparatus.

Sec1p binds to SNARE complexes and concentrates at sites of secretion.

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.