Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

A protein interaction map for cell polarity development.

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

Testing the 3Q:1R "rule": mutational analysis of the ionic "zero" layer in the yeast exocytic SNARE complex reveals no requirement for arginine.

The crystal structure of the synaptic SNARE complex reveals a parallel four-helix coiled-coil arrangement; buried in the hydrophobic core of the complex is an unusual ionic layer composed of three glutamines and one arginine, each provided by a separate alpha-helix. The presence of glutamine or arginine residues in this position is highly conserved across the t- and v-SNARE families, and it was recently suggested that a 3Q:1R ratio is likely to be a general feature common to all SNARE complexes. In this study, we have used genetic and biochemical assays to test this prediction with the yeast exocytic SNARE complex. We have determined that the relative position of Qs and Rs within the layer is not critical for biological activity and that Q-to-R substitutions in the layer reduce complex stability and result in lethal or conditional lethal growth defects. Surprisingly, SNARE complexes composed of four glutamines are fully functional for assembly in vitro and exocytic function in vivo. We conclude that the 3Q:1R layer composition is not required within the yeast exocytic SNARE complex because complexes containing four Q residues in the ionic layer appear by all criteria to be functionally equivalent. The unexpected flexibility of this layer suggests that there is no strict requirement for the 3Q:1R combination and that the SNARE complexes at other stages of transport may be composed entirely of Q-SNAREs or other noncanonical combinations.

Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.

The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity.

Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.

Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.

Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex.

In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.

Formation of a yeast SNARE complex is accompanied by significant structural changes.

The evolutionarily conserved SNARE (SNAP receptor) proteins and their complexes are key players in the docking and fusion of secretory vesicles with their target membrane. Biophysical techniques were used to characterize structural and energetic properties of the cytoplasmic domains of the yeast SNAREs Snc1 and Sso1, of the SNAP-25-like domain of Sec9, and of the Sso1:Sec9 and Sso1:Sec9:Snc1 complexes. Individually, all three SNAREs are monomeric; Sso1 shows significant secondary structure while Snc1 and Sec9 are largely unstructured. Ternary SNARE complex formation (KD <50 nM) is accompanied by a more than two-fold increase in secondary structure. This binding induced structure, the large increase in thermal stability, and the self-association of the ternary complex represent conserved properties of SNAREs that are probably important in vesicle docking and fusion.

Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast.

SEC4 is an essential gene encoding a small GTPase that is involved in Golgi to cell surface transport in Saccharomyces cerevisiae and is a paradigm for studies on the mode of action of Rab proteins. We describe here the features of interaction of Sec4p with the accessory protein Dss4p. Dss4p is found both on membranes and in the cytosol; however, it is the membrane fraction that is complexed to Sec4p. Dss4p, like its mammalian counterpart, Mss4, binds zinc, and disruption of the zinc-binding site disrupts the ability of the protein to interact with Sec4p. DSS4 overexpression can rescue the lethal phenotype of two alleles of SEC4, corresponding to dominant mutations of Ras. We demonstrate that this suppression is due to the ability of Dss4p to form a tight complex with the mutant forms of Sec4p and hence sequester the mutant protein from its inhibitory effect. These results imply an in vivo role for Dss4p as a guanine nucleotide dissociation stimulator. In vitro the protein has the ability to stimulate the dissociation rate of both GDP and GTP from Sec4p. We examined the relationship of GDI1 and DSS4 with SEC4 both genetically and biochemically. These results exclude a role for DSS4 in the recruitment of Sec4p/GDI onto membranes.

Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9.

SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells. The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied. We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17. Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar. The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1. However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1. The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion. In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo. As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro. In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro. Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro. Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter. Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis.

To identify potential Sec4 effectors, we isolated high copy suppressors of a Sec4 effector domain mutant. The most potent of these was found to be SEC9, a gene required for post-Golgi transport. The sole essential domain of Sec9 has significant sequence similarity to the neuronal protein SNAP-25, a component of the SNARE complex, that is implicated in vesicle targeting and fusion. Analogous to SNAP-25, Sec9 is bound to the yeast plasma membrane and is absent from post-Golgi vesicles. Furthermore, Sec9 is physically associated with two proteins that are homologous to components of the neuronal SNARE complex. Our results identify Sec9 as the yeast cognate of SNAP-25 and suggest that SNARE complexes acting at specific stages of vesicular transport serve as the ultimate targets of regulation by members of the Sec4/Ypt1/Rab family of GTPases.

Genetic and biochemical analysis of the fission yeast ribonucleoprotein particle containing a homolog of Srp54p.

Mammalian signal recognition particle (SRP), a complex of six polypeptides and one 7SL RNA molecule, is required for targeting nascent presecretory proteins to the endoplasmic reticulum (ER). Earlier work identified a Schizosaccharomyces pombe homolog of human SRP RNA and showed that it is a component of a particle similar in size and biochemical properties to mammalian SRP. The recent cloning of the gene encoding a fission yeast protein homologous to Srp54p has made possible further characterization of the subunit structure, subcellular distribution, and assembly of fission yeast SRP. S. pombe SRP RNA and Srp54p co-sediment on a sucrose velocity gradient and coimmunoprecipitate, indicating that they reside in the same complex. In vitro assays demonstrate that fission yeast Srp54p binds under stringent conditions to E. coli SRP RNA, which consists essentially of domain IV, but not to the full-length cognate RNA nor to an RNA in which domain III has been deleted in an effort to mirror the structure of bacterial homologs. Moreover, the association of S. pombe Srp54p with SRP RNA in vivo is disrupted by conditional mutations not only in domain IV, which contains its binding site, but in domains I and III, suggesting that the particle may assemble cooperatively. The growth defects conferred by mutations throughout SRP RNA can be suppressed by overexpression of Srp54p, and the degree to which growth is restored correlates inversely with the severity of the reduction in protein binding. Conditional mutations in SRP RNA also reduce its sedimentation with the ribosome/membrane pellet during cell fractionation. Finally, immunoprecipitation under native conditions of an SRP-enriched fraction from [35S]-labeled fission yeast cells suggests that five additional polypeptides are complexed with Srp54p; each of these proteins is similar in size to a constituent of mammalian SRP, implying that the subunit structure of this ribonucleoprotein is conserved over vast evolutionary distances.

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast.

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-alpha-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.

Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4.

The genes SEC4 and YPT1 encode Ras-related GTP-binding proteins in the yeast Saccharomyces cerevisiae. Ypt1 is necessary for vesicular transport from the endoplasmic reticulum to the Golgi, whereas Sec4 is required for fusion of post-Golgi secretory vesicles to the plasma membrane. Recently, three structural domains have been proposed to specify the stage in cellular transport at which members of the Sec4/Ypt1/Rab family act: the effector domain, the C-terminal hypervariable region, and a region corresponding to loop 7 in the structure of p21ras (ref. 8). Here we use Sec4/Ypt1 chimaeras to show that these three regions cooperate to specify Ypt1 function and that the C-terminal hypervariable region is needed for Ypt1 localization to the Golgi. Unexpectedly, we found that a single chimaera can function as either Ypt1 or Sec4 without missorting carboxypeptidase Y or invertase.

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA.

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.

The cycle of SEC4 function in vesicular transport.

Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.

Distinct and specific GAP activities in rat pancreas act on the yeast GTP-binding proteins Ypt1 and Sec4.

Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide-binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyces cerevisiae. Ypt1, another ras-like 23 kDa guanine nucleotide-binding protein in yeast has been found to be involved in ER-Golgi transport. A mammalian homologue of Ypt1 called rab1 has also been identified. Recent studies using purified Sec4 protein have identified a component of yeast lystate that specifically stimulates the hydrolysis of GTP bound to Sec4. In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E. coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas. Our studies showed that 65% of Sec4-GAP activity was associated with the 150,000 x g pancreatic particulate fraction with approximately 35% being found in the cytosol. On the other hand, more than 95% of Ypt1-GAP activity was found to associate with the particulate fraction. Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs. The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.