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 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 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.

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.

Genetic and biochemical studies of asparagine-linked oligosaccharide assembly

1982 ◽  
Vol 300 (1099) ◽  
pp. 207-223 ◽  
Keyword(s):  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Oligosaccharide Processing ◽  
Biochemical Studies ◽  
The Common ◽  
Specific Lipid ◽  
Temperature Sensitive Phenotype

The formation of N -glycosidic linkages of eukaryotic glycoproteins involves the assembly of a specific lipid-linked precursor oligosaccharide in the endoplasmic reticulum. This oligosaccharide is transferred from the lipid carrier to appropriate asparagine residues during protein synthesis. The protein-linked oligosaccharide then undergoes processing reactions that include both removal and addition of carbohydrate residues. In this paper we report recent studies from our laboratory on the synthesis of asparagine-linked oligosaccharides. In the first part we describe the isolation and characterization of temperature-sensitive mutants of yeast blocked at specific stages in the assembly of the lipid-linked oligosaccharide. In addition, we are using these mutants to clone the genes for the enzymes in this pathway by complementation of the temperature-sensitive phenotype. The second part deals with the topography of asparagine-linked oligosaccharide assembly. Our studies on the transmembrane movement of sugar residues during the assembly of secreted glycoproteins from cytoplasmic precursors are presented. Finally, experiments on the control of protein-linked oligosaccharide processing are described. Recent data are presented on the problem of how specific oligosaccharides are assembled from the common precursors at individual sites on glycoproteins.

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 Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis

1999 ◽  
Vol 19 (6) ◽  
pp. 4480-4494 ◽  
Author(s):  
Vardit Lustgarten ◽  
Jeffrey E. Gerst
Keyword(s):  
Secretory Pathway ◽  
Snare Complex ◽  
Negative Regulator ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Interacting Protein ◽  
Cell Extracts ◽  
Temperature Sensitive Phenotype ◽  
Novel Protein

ABSTRACT We have screened for proteins that interact with v-SNAREs of the late secretory pathway in the yeast Saccharomyces cerevisiae. A novel protein, designated Vsm1, binds tightly to the Snc2 v-SNARE in the two-hybrid system and can be coimmunoprecipitated with Snc1 or Snc2 from solubilized yeast cell extracts. Disruption of the VSM1 gene results in an increase of proteins secreted into the medium but does not affect the processing or secretion of invertase. In contrast,VSM1 overexpression in cells which bear a temperature-sensitive mutation in the Sec9 t-SNARE (sec9-4cells) results in the accumulation of non-invertase-containing low-density secretory vesicles, inhibits cell growth and the secretion of proteins into the medium, and blocks rescue of the temperature-sensitive phenotype by SNC1 overexpression. Yet, VSM1 overexpression does not affect yeast bearing asec9-7 allele which, in contrast to sec9-4, encodes a t-SNARE protein capable of forming a stable SNARE complex in vitro at restrictive temperatures. On the basis of these results, we propose that Vsm1 is a novel v-SNARE-interacting protein that appears to act as negative regulator of constitutive exocytosis. Moreover, this regulation appears specific to one of two parallel exocytic paths which are operant in yeast cells.

scholarly journals SL-Cloud: A Computational Resource to Support Synthetic Lethal Interaction Discovery

2021 ◽  
Author(s):  
Bahar Tercan ◽  
Guangrong Qin ◽  
Taekkyun Kim ◽  
Boris Aguilar ◽  
Christopher J. Kemp ◽  
...  
Keyword(s):  
Genetic Interactions ◽  
Computational Resource ◽  
Synthetic Lethal Interaction ◽  
Synthetic Lethal ◽  
Damage Repair ◽  
Interaction Partners ◽  
Synthetic Lethal Interactions ◽  
Multiple Prediction ◽  
Context Specific ◽  
Repair Genes

Synthetic lethal interactions (SLIs), genetic interactions whereby the simultaneous inactivation of two genes leads to a lethal phenotype, are promising targets for therapeutic intervention in cancer. We present SL-Cloud, an integrated resource and framework to facilitate prediction of context-specific synthetic lethal interactions using cloud-based technologies. This resource addresses two main challenges related to SLI inference, namely, the need to wrangle and preprocess large multi-omic datasets and the ability to integrate multiple prediction approaches, each of which comes with its own assumptions. We demonstrate the utility of this resource by using a set of DNA damage repair genes as the basis for predicting potential synthetic lethal interaction partners using multiple computational strategies. Context specific SLI potential can also be studied using the framework. The SL-Cloud computational resource demonstrates a variety of use cases and demonstrates the utility of this approach for customizable and extensible in silico inference of SLIs.

scholarly journals Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum.

1993 ◽  
Vol 4 (11) ◽  
pp. 1145-1159 ◽  
Author(s):  
M A Scidmore ◽  
H H Okamura ◽  
M D Rose
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Integral Membrane Protein ◽  
Genetic Interactions ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Synthetic Lethal ◽  
Hsp70 Family ◽  
Dnaj Protein

KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family. Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion. Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation. In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p. First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations. Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1. Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels. Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E. coli HSP70, DnaK, and DnaJ. We propose that the interaction between these two proteins is critical to their function in protein translocation.

scholarly journals Temperature-sensitive mutants ofPhysarum polycephalum– expression of mutations in amoebae and plasmodia

1979 ◽  
Vol 34 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Tim Burland ◽  
Jennifer Dee
Keyword(s):  
Temperature Sensitivity ◽  
Growth Temperature ◽  
Physarum Polycephalum ◽  
Temperature Sensitive ◽  
Optimum Growth ◽  
Efficient Procedure ◽  
Temperature Sensitive Mutants ◽  
Temperature Sensitive Phenotype ◽  
Laborious Method

SUMMARYOver 100 temperature-sensitive mutants ofmt-h (apogamic) strains ofPhysarum polycephalumwere isolated either by testing clones of mutagenized amoebae (ATS mutants) or by the more laborious method of testing plasmodia derived from such clones (PTS mutants). When amoebae and plasmodia of each mutant were tested for growth temperature sensitivity on different media (to give optimum growth of each phase), only 21% of 73 ATS mutants and 32% of 31 PTS mutants appeared to be temperature-sensitive in both phases, suggesting that the majority of mutants are phase-specific, as concluded from several similar studies by previous authors. When the mutants were tested on a third medium which allows growth of both amoebae and plasmodia, many of the mutants no longer had a temperature-sensitive phenotype in either phase. Among the remainder, 51% of ATS mutants and 67% of PTS mutants were temperature-sensitive in both phases. It was suggested that certain media have a remedial effect on some temperature-sensitive mutants so that the phenotype is apparently normal. Thus, the proportion of phase-specific mutants may be over-estimated if tests of temperature-sensitivity are done on the different media commonly used for culture of amoebae and plasmodia respectively. It was concluded that the most efficient procedure for isolation of temperature-sensitive mutants expressed in plasmodia is to screen clones of amoebae on a medium resembling as closely as possible that which is to be used for testing plasmodia.

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