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Genetics, Vol. 176, 181-192, May 2007, Copyright © 2007
doi:10.1534/genetics.106.060640

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Modulation of Ubc4p/Ubc5p-Mediated Stress Responses by the RING-Finger-Dependent Ubiquitin-Protein Ligase Not4p in Saccharomyces cerevisiae

Klaas W. Mulder*, Akiko Inagaki*, Elisabetta Cameroni{dagger}, Florence Mousson*, G. Sebastiaan Winkler*, Claudio De Virgilio{dagger}, Martine A. Collart{dagger} and H. Th. Marc Timmers*,1

* Department of Physiological Chemistry, University Medical Centre Utrecht, 3584 CG Utrecht, The Netherlands and {dagger} Department of Microbiology and Molecular Medicine, CMU, CH-1211 Geneva 4, Switzerland

1 Corresponding author: Department of Physiological Chemistry, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.
E-mail: h.t.m.timmers{at}umcutrecht.nl

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 not4{Delta} 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.




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E. Lenssen, N. Azzouz, A. Michel, E. Landrieux, and M. A. Collart
The Ccr4-Not Complex Regulates Skn7 through Srb10 Kinase
Eukaryot. Cell, December 1, 2007; 6(12): 2251 - 2259.
[Abstract] [Full Text] [PDF]


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