Modulation of Ubc4p/Ubc5p-mediated stress responses by the RING-finger-dependent ubiquitin-protein ligase Not4p in Saccharomyces cerevisiae.
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.
Mulder
Klaas W
KW
Department of Physiological Chemistry, University Medical Centre Utrecht, Utrecht, The Netherlands.
Inagaki
Akiko
A
Cameroni
Elisabetta
E
Mousson
Florence
F
Winkler
G Sebastiaan
GS
De Virgilio
Claudio
C
Collart
Martine A
MA
Timmers
H Th Marc
HT
D000222:Adaptation, Physiological / Q000187:drug effects*; D000483:Alleles; D000595:Amino Acid Sequence; D005800:Genes, Fungal; D018869:Heat-Shock Response / Q000187:drug effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D006921:Hygromycin B / Q000494:pharmacology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000302:isolation & purification; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*
D050505:Mutant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D006921:Hygromycin B; C489270:UBC5 protein, S cerevisiae; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C086923:MOT2 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; C078657:CCR4 protein, S cerevisiae; D012260:Ribonucleases; D006918:Hydroxyurea