Effect of auxotrophic mutation in Saccharomyces cerevisiae on the decay of intracellularly accumulated ?-lactamase during vegetative growth, encoded on YEp vector

1991 ◽  
Vol 13 (11) ◽  
pp. 815-820 ◽  
Author(s):  
Takao Igarashi ◽  
Kozo Koide ◽  
Kazuhisa Ohtaguchi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Auxotrophic Mutation

scholarly journals The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.

1995 ◽  
Vol 128 (4) ◽  
pp. 525-536 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Alpha Subunit ◽  
Protein Sequence Analysis ◽  
Exogenous Lipid ◽  
Microsomal Membranes ◽  
High Mannose

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.

scholarly journals Identification of Kel1p, a Kelch Domain-containing Protein Involved in Cell Fusion and Morphology in Saccharomyces cerevisiae

1998 ◽  
Vol 143 (2) ◽  
pp. 375-389 ◽  
Author(s):  
Jennifer Philips ◽  
Ira Herskowitz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Fusion ◽  
Vegetative Growth ◽  
Coiled Coils ◽  
Cell Integrity ◽  
Cell Morphogenesis ◽  
Kinase C ◽  
Kelch Domain ◽  
Drosophila Protein ◽  
Two Hybrid

We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I. Herskowitz. 1997. J. Cell Biol. 138:961–974). To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p. This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L. Cooley. 1993. Cell. 72:681– 693), and another domain predicted to form coiled coils. Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Δ and fps1Δ mutants. KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p. Its overexpression also suppressed the mating defect associated with activated Pkc1p. Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2. kel1Δ mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth. In contrast, kel2Δ mutants were not impaired in cell fusion or morphology. Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth. Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact. We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway.

Functional analysis of a duplicated linked pair of ribosomal protein genes in Saccharomyces cerevisiae

1990 ◽  
Vol 10 (11) ◽  
pp. 6097-6100
Author(s):  
D M Donovan ◽  
M P Remington ◽  
D A Stewart ◽  
J C Crouse ◽  
D J Miles ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Analysis ◽  
Growth Rate ◽  
Ribosomal Protein ◽  
Vegetative Growth ◽  
Maximum Growth ◽  
Gene Replacement ◽  
Maximum Growth Rate ◽  
Ribosomal Protein Genes ◽  
Genome Copy

Ribosomal protein genes RP28 and S16A (RP55) are closely linked. Another set of this pair of genes exists in the genome (copy 2), genetically unlinked to copy 1. By using gene replacement techniques, we have shown that RP28 from copy 1 is required for vegetative growth and that the cells need S16A from copy 2 to achieve maximum growth rate.

scholarly journals Binding interactions control SNARE specificity in vivo

2008 ◽  
Vol 183 (6) ◽  
pp. 1089-1100 ◽  
Author(s):  
Hui-Ju Yang ◽  
Hideki Nakanishi ◽  
Song Liu ◽  
James A. McNew ◽  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Vegetative Growth ◽  
Snare Complex ◽  
Mutant Form ◽  
Prospore Membrane ◽  
Complex Mutation ◽  
Ionic Layer

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

scholarly journals Mid2 Is a Putative Sensor for Cell Integrity Signaling in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (6) ◽  
pp. 3969-3976 ◽  
Author(s):  
Mathumathi Rajavel ◽  
Bevin Philip ◽  
Benjamin M. Buehrer ◽  
Beverly Errede ◽  
David E. Levin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Vegetative Growth ◽  
Elevated Temperatures ◽  
Transmembrane Domain ◽  
Wall Stress ◽  
Cell Lysis ◽  
Mitogen Activated Protein Kinase ◽  
Primary Role ◽  
Cell Integrity

ABSTRACTHcs77 is a putative cell surface sensor for cell integrity signaling inSaccharomyces cerevisiae. Its loss of function results in cell lysis during growth at elevated temperatures (e.g., 39°C) and impaired signaling to the Mpk1 mitogen-activated protein kinase in response to mild heat shock. We isolated theMID2gene as a dosage suppressor of the cell lysis defect of anhcs77null mutant.MID2encodes a putative membrane protein whose function is required for survival of pheromone treatment. Mid2 possesses properties similar to those of Hcs77, including a single transmembrane domain and a long region that is rich in seryl and threonyl residues. We demonstrate that Mid2 is required for cell integrity signaling in response to pheromone. Additionally, we show that Mid2 and Hcs77 serve a redundant but essential function as cell surface sensors for cell integrity signaling during vegetative growth. Both proteins are uniformly distributed through the plasma membrane and are highly O-mannosylated on their extracellular domains. Finally, we identified a yeast homolog ofMID2, designatedMTL1, which provides a partially redundant function withMID2for cell integrity signaling during vegetative growth at elevated temperature but not for survival of pheromone treatment. We conclude that Hcs77 is dedicated to signaling cell wall stress during vegetative growth and that Mid2 participates in this signaling, but its primary role is in signaling wall stress during pheromone-induced morphogenesis.

scholarly journals Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism.

1994 ◽  
Vol 127 (3) ◽  
pp. 751-762 ◽  
Author(s):  
D R Johnson ◽  
L J Knoll ◽  
D E Levin ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Carbon Source ◽  
Vegetative Growth ◽  
Fatty Acid Synthetase ◽  
Acid Activation ◽  
Beta Oxidation ◽  
Fatty Acid Activation ◽  
Long Chain

Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.

scholarly journals Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (1) ◽  
pp. 142-150 ◽  
Author(s):  
A Percival-Smith ◽  
J Segall
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Vegetative Growth ◽  
Cdna Probe ◽  
Cell Types ◽  
Sporulation Medium ◽  
Alpha Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Library ◽  
Vector Pbr322

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating a alpha cells and asporogenous alpha alpha cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the a alpha sporulation probe relative to the alpha alpha control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, a alpha, and alpha alpha cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in a alpha cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in a alpha cells in sporulation medium.

scholarly journals The Dual-Specificity Protein Phosphatase Yvh1p Acts Upstream of the Protein Kinase Mck1p in Promoting Spore Development in Saccharomyces cerevisiae

1999 ◽  
Vol 181 (17) ◽  
pp. 5219-5224 ◽  
Author(s):  
Alexander E. Beeser ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Vegetative Growth ◽  
Nitrogen Starvation ◽  
Developmental Pathway ◽  
Dual Specificity ◽  
Dual Specificity Protein Phosphatase ◽  
Specificity Protein ◽  
Spore Maturation

ABSTRACT Diploid Saccharomyces cerevisiae cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen. We show that yvh1 disruption causes a defect in spore maturation; overexpression of MCK1or IME1 suppresses this yvh1 phenotype. Whilemck1 mutations are epistatic to those in yvh1relative to spore maturation, overexpression of MCK1 does not suppress the yvh1 slow-vegetative-growth phenotype. We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth.

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