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PMID: 17513889 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Modulation of Ubc4p/Ubc5p-mediated stress responses by the RING-finger-dependent ubiquitin-protein ligase Not4p in Saccharomyces cerevisiae.

Genetics ·Vol. 176 ·No. 1 ·2007-05-00 ·Pages 181-92

Mulder KW, Inagaki A, Cameroni E, Mousson F, Winkler GS, De Virgilio C, Collart MA, Timmers HT

Abstract

The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitin-protein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4Delta and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-amino-acid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.

MeSH Terms
Adaptation, Physiological/drug effects Alleles Amino Acid Sequence Genes, Fungal Heat-Shock Response/drug effects Humans Hydroxyurea/pharmacology Hygromycin B/pharmacology Molecular Sequence Data Mutant Proteins/isolation & purification Mutation/genetics Protein Binding/drug effects Repressor Proteins Ribonucleases/metabolism Saccharomyces cerevisiae/cytology,drug effects,genetics,metabolism Saccharomyces cerevisiae Proteins/metabolism Ubiquitin/metabolism Ubiquitin-Conjugating Enzymes/metabolism Ubiquitin-Protein Ligases/chemistry,metabolism
Chemicals
Mutant Proteins Repressor Proteins Saccharomyces cerevisiae Proteins Ubiquitin Hygromycin B UBC5 protein, S cerevisiae Ubc4 protein, S cerevisiae Ubiquitin-Conjugating Enzymes MOT2 protein, S cerevisiae Ubiquitin-Protein Ligases CCR4 protein, S cerevisiae Ribonucleases Hydroxyurea
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Mulder Klaas W
Department of Physiological Chemistry, University Medical Centre Utrecht, Utrecht, The Netherlands.
Inagaki Akiko
Cameroni Elisabetta
Mousson Florence
Winkler G Sebastiaan
De Virgilio Claudio
Collart Martine A
Timmers H Th Marc
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Article Info
Journal
Genetics
Abbr.
Genetics
ISSN
0016-6731
DOI
10.1534/genetics.106.060640
Published
2007-05-00
Pages
181-92
Language
English
Region
United States
NLM ID
0374636
PMCID
PMC1893070
Subset
IM
External Links
PubMed source DOI: 10.1534/genetics.106.060640 PMC Full Text View journal details
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