Genetic Interactions Between a pep7 Mutation and the PEP12 and VPS45 Genes: Evidence for a Novel SNARE Component in Transport Between the Saccharomyces cerevisiae Golgi Complex and Endosome

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 467-478 ◽  
Author(s):  
Gene C Webb ◽  
Marloes Hoedt ◽  
Lynn J Poole ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Golgi Complex ◽  
Genetic Interactions ◽  
Snare Complex ◽  
Carboxypeptidase Y ◽  
Fusion Reactions ◽  
Vesicle Docking ◽  
Transport Vesicles ◽  
Allele Specific ◽  
Mutant Phenotypes

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn2+ sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

scholarly journals A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole

1997 ◽  
Vol 138 (3) ◽  
pp. 517-529 ◽  
Author(s):  
Tamara Darsow ◽  
Stephanie E. Rieder ◽  
Scott D. Emr
Keyword(s):  
Protein Transport ◽  
Temperature Shift ◽  
Subcellular Fractionation ◽  
Polyclonal Antiserum ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Fusion Reactions ◽  
Vesicle Docking ◽  
Transport Vesicle ◽  
Mutant Phenotypes

Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Δ mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Δ mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.

Pep3p/Pep5p Complex: A Putative Docking Factor at Multiple Steps of Vesicular Transport to the Vacuole of Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 105-122 ◽  
Author(s):  
Amit Srivastava ◽  
Carol A Woolford ◽  
Elizabeth W Jones
Keyword(s):  
Vacuolar Membrane ◽  
Snare Complex ◽  
Snare Proteins ◽  
Fusion Reactions ◽  
Hybrid Analysis ◽  
Transport Pathways ◽  
Transport Vesicles ◽  
Oligomeric Complex ◽  
Two Hybrid

Abstract Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3ts mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.

scholarly journals Formation and Turnover of NSF- and SNAP-containing “Fusion” Complexes Occur on Undocked, Clathrin-coated Vesicle–derived Membranes

1998 ◽  
Vol 9 (7) ◽  
pp. 1633-1647 ◽  
Author(s):  
Eileithyia Swanton ◽  
John Sheehan ◽  
Naomi Bishop ◽  
Stephen High ◽  
Philip Woodman
Keyword(s):  
Vesicular Transport ◽  
Snare Complex ◽  
Coated Vesicle ◽  
Transport Pathways ◽  
Vesicle Docking ◽  
Transport Vesicles ◽  
Transport Vesicle ◽  
Target Membrane ◽  
Vesicle Cycle

Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle–derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

scholarly journals Assembly of the ER to Golgi SNARE complex requires Uso1p.

1996 ◽  
Vol 132 (5) ◽  
pp. 755-767 ◽  
Author(s):  
S K Sapperstein ◽  
V V Lupashin ◽  
H D Schmitt ◽  
M G Waters
Keyword(s):  
Biochemical Analysis ◽  
Genetic Interactions ◽  
Snare Complex ◽  
Temperature Sensitive ◽  
Synthetic Lethal Interaction ◽  
Synthetic Lethal ◽  
Vesicle Docking ◽  
Temperature Sensitive Mutants ◽  
Golgi Transport ◽  
Temperature Sensitive Phenotype

Uso1p, a Saccharomyces cerevisiae protein required for ER to Golgi transport, is homologous to the mammalian intra-Golgi transport factor p115. We have used genetic and biochemical approaches to examine the function of Uso1p. The temperature-sensitive phenotype of the uso1-1 mutant can be suppressed by overexpression of each of the known ER to Golgi v-SNAREs (Bet1p, Bos1p, Sec22p, and Ykt6p). Overexpression of two of them, BET1p and Sec22p, can also suppress the lethality of delta uso1, indicating that the SNAREs function downstream of Uso1p. In addition, overexpression of the small GTP-binding protein Ypt1p, or of a gain if function mutant (SLY1-20) of the t-SNARE associated protein Sly1p, also confers temperature resistance. Uso1p and Ypt1p appear to function in the same process because they have a similar set of genetic interactions with the v-SNARE genes, they exhibit a synthetic lethal interaction, and they are able to suppress temperature sensitive mutants of one another when overexpressed. Uso1p acts upstream of, or in conjunction with, Ypt1p because overexpression of Ypt1p allows a delta uso1 strain to grow, whereas overexpression of Uso1p does not suppress a delta ypt1 strain. Finally, biochemical analysis indicates that Uso1p, like Ypt1p, is required for assembly of the v-SNARE/t-SNARE complex. The implications of these findings, with respect to the mechanism of vesicle docking, are discussed.

scholarly journals BET3 encodes a novel hydrophilic protein that acts in conjunction with yeast SNAREs.

1995 ◽  
Vol 6 (12) ◽  
pp. 1769-1780 ◽  
Author(s):  
G Rossi ◽  
K Kolstad ◽  
S Stone ◽  
F Palluault ◽  
S Ferro-Novick
Keyword(s):  
Integral Membrane Protein ◽  
Snare Complex ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Type Ii ◽  
Sensitive Mutant ◽  
Synthetic Lethal ◽  
New Genes ◽  
Golgi Transport ◽  
Transport Vesicles

Here we report the identification of BET3, a new member of a group of interacting genes whose products have been implicated in the targeting and fusion of endoplasmic reticulum (ER) to Golgi transport vesicles with their acceptor compartment. A temperature-sensitive mutant in bet3-1 was isolated in a synthetic lethal screen designed to identify new genes whose products may interact with BET1, a type II integral membrane protein that is required for ER to Golgi transport. At 37 degrees C, bet3-1 fails to transport invertase, alpha-factor, and carboxypeptidase Y from the ER to the Golgi complex. As a consequence, this mutant accumulates dilated ER and small vesicles. The SNARE complex, a docking/fusion complex, fails to form in this mutant. Furthermore, BET3 encodes an essential 22-kDa hydrophilic protein that is conserved in evolution, which is not a component of this complex. These findings support the hypothesis that Bet3p may act before the assembly of the SNARE complex.

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

1989 ◽  
Vol 109 (6) ◽  
pp. 2939-2950 ◽  
Author(s):  
A E Cleves ◽  
P J Novick ◽  
V A Bankaitis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Secretory Pathway ◽  
Genetic Data ◽  
Secretory Vesicle ◽  
Genetic Interactions ◽  
Spatial Regulation ◽  
Allele Specific ◽  
Regulation Of Secretion ◽  
High Degree

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.

scholarly journals Synthetic Genetic Interactions With Temperature-Sensitive Clathrin in Saccharomyces cerevisiae: Roles for Synaptojanin-Like Inp53p and Dynamin-Related Vps1p in Clathrin-Dependent Protein Sorting at the trans-Golgi Network

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 83-97
Author(s):  
Eric S Bensen ◽  
Giancarlo Costaguta ◽  
Gregory S Payne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Protein Sorting ◽  
Genetic Interactions ◽  
Carboxypeptidase Y ◽  
Temperature Sensitive ◽  
Trans Golgi Network ◽  
Vacuolar Protein ◽  
Golgi Network

Abstract Clathrin is involved in selective protein transport at the Golgi apparatus and the plasma membrane. To further understand the molecular mechanisms underlying clathrin-mediated protein transport pathways, we initiated a genetic screen for mutations that display synthetic growth defects when combined with a temperature-sensitive allele of the clathrin heavy chain gene (chc1-521) in Saccharomyces cerevisiae. Mutations, when present in cells with wild-type clathrin, were analyzed for effects on mating pheromone α-factor precursor maturation and sorting of the vacuolar protein carboxypeptidase Y as measures of protein sorting at the yeast trans-Golgi network (TGN) compartment. By these criteria, two classes of mutants were obtained, those with and those without defects in protein sorting at the TGN. One mutant with unaltered protein sorting at the TGN contains a mutation in PTC1, a type 2c serine/threonine phosphatase with widespread influences. The collection of mutants displaying TGN sorting defects includes members with mutations in previously identified vacuolar protein sorting genes (VPS), including the dynamin family member VPS1. Striking genetic interactions were observed by combining temperature-sensitive alleles of CHC1 and VPS1, supporting the model that Vps1p is involved in clathrin-mediated vesicle formation at the TGN. Also in the spectrum of mutants with TGN sorting defects are isolates with mutations in the following: RIC1, encoding a product originally proposed to participate in ribosome biogenesis; LUV1, encoding a product potentially involved in vacuole and microtubule organization; and INP53, encoding a synaptojanin-like inositol polyphosphate 5-phosphatase. Disruption of INP53, but not the related INP51 and INP52 genes, resulted in α-factor maturation defects and exacerbated α-factor maturation defects when combined with chc1-521. Our findings implicate a wide variety of proteins in clathrin-dependent processes and provide evidence for the selective involvement of Inp53p in clathrin-mediated protein sorting at the TGN.

scholarly journals Munc18a clusters SNARE-bearing liposomes prior to trans-SNARE zippering

2017 ◽  
Vol 474 (19) ◽  
pp. 3339-3354 ◽  
Author(s):  
Matthew Grant Arnold ◽  
Pratikshya Adhikari ◽  
Baobin Kang ◽  
Hao Xu (徐昊)
Keyword(s):  
Snare Complex ◽  
Binary Interaction ◽  
Fusion Reactions ◽  
Sm Proteins ◽  
Attachment Protein ◽  
Direct Role ◽  
Vesicle Docking ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Snare Complex Formation

Sec1–Munc18 (SM) proteins co-operate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here, we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs. We show that Munc18a plays a direct role in promoting proteoliposome clustering, underlying vesicle docking during exocytosis. In the three different fusion reactions examined, Munc18a-dependent clustering requires an intact N-terminal peptide (N-peptide) motif in syntaxin that mediates the binary interaction between syntaxin and Munc18a. Importantly, clustering is preserved under inhibitory conditions that abolish both trans-SNARE complex formation and lipid mixing, indicating that Munc18a promotes membrane clustering in a step that is independent of trans-SNARE zippering and activation.

PRAF1: a Golgi complex transmembrane protein that interacts with virusesThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

2006 ◽  
Vol 84 (6) ◽  
pp. 940-948 ◽  
Author(s):  
Shannon L. Compton ◽  
Ellen N. Behrend
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Transmembrane Protein ◽  
Snare Complex ◽  
Snare Proteins ◽  
Virus Life Cycle ◽  
Transport Vesicles ◽  
Health And Disease ◽  
Retroviral Assembly ◽  
Selection Of

Prenylated Rab acceptor domain family member 1 (PRAF1), a transmembrane protein whose precise function is unknown, localizes to the Golgi complex, post-Golgi vesicles, lipid rafts, endosomes, and the plasma membrane. VAMP2 and Rab3A are SNARE proteins that interact with PRAF1, and, as part of a SNARE complex, PRAF1 may function in the regulation of docking and fusion of transport vesicles both in the Golgi complex and at the plasma membrane. Alternately, PRAF1 may function as a sorting protein in the Golgi complex. In addition to interacting with SNARE proteins, PRAF1 interacts with rotaviral, retroviral, and herpes viral proteins. The function of viral protein interaction is unknown, but PRAF1 may enhance rotaviral and retroviral assembly. In contrast, PRAF1 may inhibit the herpes virus life cycle.

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