RRC ID 29171
Author Kanda T.
Title A ubiquitin-protein ligase (E3) mutation of Saccharomyces cerevisiae suppressed by co-overexpression of two ubiquitin-specific processing proteases.
Journal Genes Genet Syst
Abstract To isolate mutations related to the ubiquitin system, I constructed a plasmid carrying the YUH1 and UBP1 genes (genes of ubiquitin-specific processing proteases) whose expressions were under the control of the galactose-inducible GAL1-GAL10 promoter. Cells of a strain carrying the plasmid were mutagenized with ethyl methanesulfonate. One mutant, which showed galactose-dependent growth at a high temperature (37 degrees C), was isolated from about 380,000 mutagenized colonies. The mutation responsible for galactose-dependent growth at 37 degrees C was a single nuclear recessive mutation designated as uby1-1. UBP1 and YUH1 as well as the GAL1-GAL10 promoter are required to suppress uby1-1. At the restrictive temperature, a uby1-1 mutant did not arrest at a specific phase of the cell cycle, but still lost viability. Even at the permissive temperature (30 degrees C), the uby1-1 mutant grew somewhat slowly and showed pleiotropic phenotypes including hypersensitivity to stresses such as cadmium and canavanine, and sporulation defects. The genomic DNA fragments in a single-copy plasmid which complemented uby1-1 were isolated. Chromosomal mapping, sequencing and subcloning analyses indicated that the gene complementing uby1-1 is RSP5, which encodes a ubiquitin-protein ligase (E3) homologous to E6-AP (E6 associated protein). Deletion, complementation and linkage analyses revealed that UBY1 and RSP5 are the same gene. Therefore, the E3 protein encoded by RSP5 (UBY1) is required for vegetative growth, sporulation and stress response. The present procedure using suppression by co-overexpression of two cloned genes will be useful to isolate mutations of related genes and to analyze biochemical pathways and gene-interactions.
Volume 71(2)
Pages 75-83
Published 1996-4-1
DOI 10.1266/ggs.71.75
PMID 8752868
MeSH Base Sequence Chromosome Mapping Endopeptidases / genetics Endosomal Sorting Complexes Required for Transport Fungal Proteins / genetics Gene Expression Regulation, Fungal Genes, Suppressor Genetic Complementation Test Ligases / genetics* Molecular Sequence Data Mutation* Phenotype Promoter Regions, Genetic Saccharomyces cerevisiae / enzymology Saccharomyces cerevisiae / genetics* Saccharomyces cerevisiae Proteins* Ubiquitin-Protein Ligase Complexes* Ubiquitin-Protein Ligases Viral Proteins / chemistry Viral Proteins / genetics
IF 0.917
Times Cited 7
DNA material p1010 (RDB01670)