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

1999 ◽  
Vol 10 (10) ◽  
pp. 3317-3329 ◽  
Author(s):  
Dong-Wook Kim ◽  
Michael Sacher ◽  
Al Scarpa ◽  
Anne Marie Quinn ◽  
Susan Ferro-Novick
Keyword(s):  
Temperature Sensitive ◽  
Physical Interaction ◽  
Synthetic Lethal ◽  
Vesicle Docking ◽  
Vesicular Traffic ◽  
Golgi Transport ◽  
Synthetic Lethal Interactions ◽  
Golgi Protein ◽  
Late Stages ◽  
Suppressor Analysis

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 thesec34-2 mutant has resulted in the identification of theSEC34 structural gene and a novel gene calledGRP1. GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160. Although GRP1 is not essential for growth, the grp1Δ 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 suppressessec35-1. SEC34 encodes a hydrophilic protein of ∼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 ∼480 kDa. These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic.

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 Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C.

1995 ◽  
Vol 6 (7) ◽  
pp. 793-807 ◽  
Author(s):  
P B Meluh ◽  
D Koshland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Synthetic Lethal ◽  
Cis Acting ◽  
Centromere Protein ◽  
Synthetic Lethal Interactions ◽  
Dna Complex ◽  
Two Temperature ◽  
High Instability ◽  
Temperature Sensitive Phenotype

The MIF2 gene of Saccharomyces cerevisiae has been implicated in mitosis. Here we provide genetic evidence that MIF2 encodes a centromere protein. Specifically, we found that mutations in MIF2 stabilize dicentric minichromosomes and confer high instability (i.e., a synthetic acentric phenotype) to chromosomes that bear a cis-acting mutation in element I of the yeast centromeric DNA (CDEI). Similarly, we observed synthetic phenotypes between mutations in MIF2 and trans-acting mutations in three known yeast centromere protein genes-CEP1/CBF1/CPF1, NDC10/CBF2, and CEP3/CBF3B. In addition, the mif2 temperature-sensitive phenotype can be partially rescued by increased dosage of CEP1. Synthetic lethal interactions between a cep1 null mutation and mutations in either NDC10 or CEP3 were also detected. Taken together, these data suggest that the Mif2 protein interacts with Cep1p at the centromere and that the yeast centromere indeed exists as a higher order protein-DNA complex. The Mif2 and Cep1 proteins contain motifs of known transcription factors, suggesting that assembly of the yeast centromere is analogous to that of eukaryotic enhancers and origins of replication. We also show that the predicted Mif2 protein shares two short regions of homology with the mammalian centromere Ag CENP-C and that two temperature-sensitive mutations in MIF2 lie within these regions. These results provide evidence for structural conservation between yeast and mammalian centromeres.

scholarly journals Sec3p is involved in secretion and morphogenesis in Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (4) ◽  
pp. 647-662 ◽  
Author(s):  
F P Finger ◽  
P Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Temperature Sensitive ◽  
Synthetic Lethal ◽  
Growth Defects ◽  
Actin Structure ◽  
Synthetically Lethal ◽  
Bud Site ◽  
Slow Growing ◽  
Late Stages ◽  
Synthetic Growth

Two new temperature-sensitive alleles of SEC3, 1 of 10 late-acting SEC genes required for targeting or fusion of post-Golgi secretory vesicles to the plasma membrane in Saccharomyces cerevisiae, were isolated in a screen for temperature-sensitive secretory mutants that are synthetically lethal with sec4-8. The new sec3 alleles affect early as well as late stages of secretion. Cloning and sequencing of the SEC3 gene revealed that it is identical to profilin synthetic lethal 1 (PSL1). The SEC3 gene is not essential because cells depleted of Sec3p are viable although slow growing and temperature sensitive. All of the sec3 alleles genetically interact with a profilin mutation, pfy1-111. The SEC3 gene in high copy suppresses pfy1-111 and sec5-24 and causes synthetic growth defects with ypt1, sec8-9, sec10-2, and sec15-1. Actin structure is only perturbed in conditions of chronic loss of Sec3p function, implying that Sec3p does not directly regulate actin. All alleles of sec3 cause bud site selection defects in homozygous diploids, as do sec4-8 and sec9-4. This suggests that SEC gene products are involved in determining the bud site and is consistent with a role for Sec3p in determining the correct site of exocytosis.

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 Identification of a Second Myosin-II in Schizosaccharomyces pombe:

1997 ◽  
Vol 8 (12) ◽  
pp. 2693-2705 ◽  
Author(s):  
Magdalena Bezanilla ◽  
Susan L. Forsburg ◽  
Thomas D. Pollard
Keyword(s):  
Catalytic Domain ◽  
Myosin Ii ◽  
Growth Conditions ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Binding Motifs ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions ◽  
Morphological Defects ◽  
Cold Sensitive

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. We clonedmyp2+ , a myosin-II inSchizosaccharomyces pombe, conditionally required for cytokinesis. myp2+ , the second myosin-II identified in S. pombe, does not completely overlap in function with myo2+ . The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2+ is not lethal but under nutrient limiting conditions cells lackingmyp2+ function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Additionally,Δmyp2 cells are cold sensitive in high salt, failing to form colonies at 17°C. Thus, myp2+ is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Additionally, we constructed double mutants of Δmyp2 with temperature-sensitive mutant strains defective in cytokinesis. We observed synthetic lethal interactions between Δmyp2 and three alleles ofcdc11ts, as well as more modest synthetic interactions with cdc14ts and cdc16ts, implicatingmyp2+ function for efficient cytokinesis under normal conditions.

scholarly journals Interactions between Centromere Complexes in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (12) ◽  
pp. 4931-4946 ◽  
Author(s):  
Vladimir S. Nekrasov ◽  
Melanie A. Smith ◽  
Sew Peak-Chew ◽  
John V. Kilmartin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Immunoprecipitation ◽  
Complex Structure ◽  
Protein A ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
The Other ◽  
Temperature Sensitive ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions

We have purified two new complexes from Saccharomyces cerevisiae, one containing the centromere component Mtw1p together with Nnf1p, Nsl1p, and Dsn1p, which we call the Mtw1p complex, and the other containing Spc105p and Ydr532p, which we call the Spc105p complex. Further purifications using Dsn1p tagged with protein A show, in addition to the other components of the Mtw1p complex, the two components of the Spc105p complex and the four components of the previously described Ndc80p complex, suggesting that all three complexes are closely associated. Fluorescence microscopy and immunoelectron microscopy show that Nnf1p, Nsl1p, Dsn1p, Spc105p, and Ydr532p all localize to the nuclear side of the spindle pole body and along short spindles. Chromatin immunoprecipitation assays show that all five proteins are associated with centromere DNA. Homologues of Nsl1p and Spc105p in Schizosaccharomyces pombe also localize to the centromere. Temperature-sensitive mutations of Nsl1p, Dsn1p, and Spc105p all cause defects in chromosome segregation. Synthetic-lethal interactions are found between temperature-sensitive mutations in proteins from all three complexes, in agreement with their close physical association. These results show an increasingly complex structure for the S. cerevisiae centromere and a probable conservation of structure between parts of the centromeres of S. cerevisiae and S. pombe.

NBD-labeled phosphatidylcholine enters the yeast vacuole via the pre-vacuolar compartment

2002 ◽  
Vol 115 (13) ◽  
pp. 2725-2733 ◽  
Author(s):  
Pamela K. Hanson ◽  
Althea M. Grant ◽  
J. Wylie Nichols
Keyword(s):  
Plasma Membrane ◽  
Low Temperature ◽  
Head Group ◽  
Temperature Sensitive ◽  
Vesicular Traffic ◽  
Yeast Vacuole ◽  
Golgi Transport ◽  
Vacuolar Compartment ◽  
Wild Type Yeast ◽  
Similar Enrichment

At low temperature, the short-chain fluorescent-labeled phospholipids,1-myristoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl]-phosphatidylcholine (M-C6-NBD-PC) and its phosphatidylethanolamine analog, M-C6-NBD-PE, are internalized by flip across the plasma membrane of S. cerevisiae and show similar enrichment in intracellular membranes including the mitochondria and nuclear envelope/ER. At higher temperatures (24-37°C), or if low temperature internalization is followed by warming, M-C6-NBD-PC, but not M-C6-NBD-PE, is trafficked to the lumen of the vacuole. Sorting of M-C6-NBD-PC to the vacuole is blocked by energy-depletion and by null mutations in the VPS4 and VPS28 genes required for vesicular traffic from the pre-vacuolar compartment (PVC) to the vacuole. This sorting is not blocked by a temperature-sensitive mutation in SEC12,which inhibits ER to Golgi transport, a null mutation in VPS8, which inhibits Golgi to PVC transport, or temperature-sensitive and null mutations in END4, which inhibit endocytosis from the plasma membrane. Monomethylation or dimethylation of the primary amine head-group of M-C6-NBD-PE is sufficient for sorting to the yeast vacuole in both wild-type yeast and in strains defective in the phosphatidylethanolamine methylation pathway. These data indicate that methylation of M-C6-NBD-PE produces the crucial structural component required to sort these phospholipid analogues to the vacuole via the PVC.

scholarly journals Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae

1993 ◽  
Vol 122 (3) ◽  
pp. 635-644 ◽  
Author(s):  
DA Holtzman ◽  
S Yang ◽  
DG Drubin
Keyword(s):  
Focal Adhesions ◽  
Null Alleles ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Synthetic Lethal ◽  
Amino Acid Region ◽  
New Genes ◽  
Membrane Cytoskeleton ◽  
Synthetic Lethal Interactions

Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects. Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton. The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions. Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton. In addition, synthetic-lethal interactions were observed for double-mutants containing null alleles of SLA2 and SAC6. In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.

Synthetic Lethal Interactions in Cancer Therapy

2017 ◽  
Vol 17 (4) ◽  
pp. 304-310 ◽  
Author(s):  
Xinwei Geng ◽  
Xiaohui Wang ◽  
Dan Zhu ◽  
Songmin Ying
Keyword(s):  
Cancer Therapy ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions
Sign in / Sign up

Export Citation Format

Share Document