Document Detail

Engineering of the yeast ubiquitin ligase Rsp5: isolation of a new variant that induces constitutive inactivation of the general amino acid permease Gap1.
MedLine Citation:
PMID:  19054125     Owner:  NLM     Status:  MEDLINE    
Rsp5 is an essential ubiquitin-protein ligase in Saccharomyces cerevisiae. We found previously that the Ala401Glu rsp5 mutant is hypersensitive to various stresses that induce protein misfolding, suggesting that Rsp5 is a key enzyme for yeast cell growth under stress conditions. To isolate new Rsp5 variants as suppressors of the A401E mutant, PCR random mutagenesis was used in the rsp5(A401E) gene, and the mutagenized plasmid library was introduced into rsp5(A401E) cells. As a phenotypic suppressor of rsp5(A401E) cells, we isolated a quadruple variant (Thr357Ala/Glu401Gly/Lys764Glu/Glu767Gly) on a minimal medium containing the toxic proline analogue azetidine-2-carboxylate (AZC). Site-directed mutagenesis experiments showed that the rsp5(T357A/K764E) cells were much more tolerant to AZC than the wild-type cells, due to the smaller amounts of intracellular AZC. However, the T357A/K764E variant Rsp5 did not reverse the hypersensitivity of rsp5(A401E) cells to other stresses such as high growth temperature, ethanol, and freezing treatment. Interestingly, immunoblot and localization analyses indicated that the general amino acid permease Gap1, which is involved in AZC uptake, was absent on the plasma membrane and degraded in the vacuole of rsp5(T357A/K764E) cells before the addition of ammonium ions. These results suggest that the T357A/K764E variant Rsp5 induces constitutive inactivation of Gap1.
Yutaka Haitani; Maiko Nakata; Toshiya Sasaki; Akiko Uchida; Hiroshi Takagi
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-11-15
Journal Detail:
Title:  FEMS yeast research     Volume:  9     ISSN:  1567-1364     ISO Abbreviation:  FEMS Yeast Res.     Publication Date:  2009 Feb 
Date Detail:
Created Date:  2009-01-09     Completed Date:  2009-03-06     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  101085384     Medline TA:  FEMS Yeast Res     Country:  England    
Other Details:
Languages:  eng     Pagination:  73-86     Citation Subset:  IM    
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, Japan.
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MeSH Terms
Amino Acid Transport Systems / antagonists & inhibitors*
Antifungal Agents / pharmacology
Azetidinecarboxylic Acid / analogs & derivatives,  pharmacology
Drug Resistance, Fungal
Endosomal Sorting Complexes Required for Transport
Genetic Complementation Test
Mutagenesis, Site-Directed
Mutant Proteins / genetics*,  metabolism*
Mutation, Missense
Polymerase Chain Reaction / methods
Saccharomyces cerevisiae / enzymology*,  genetics
Saccharomyces cerevisiae Proteins / antagonists & inhibitors*,  genetics*,  metabolism*
Ubiquitin-Protein Ligase Complexes / genetics*,  metabolism*
Reg. No./Substance:
0/Amino Acid Transport Systems; 0/Antifungal Agents; 0/Endosomal Sorting Complexes Required for Transport; 0/GAP1 protein, S cerevisiae; 0/Mutant Proteins; 0/Saccharomyces cerevisiae Proteins; 127310-57-0/azetidine-2,4-dicarboxylic acid; 2517-04-6/Azetidinecarboxylic Acid; EC protein, S cerevisiae; EC Ligase Complexes

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