Mulder Klaas W, Inagaki Akiko, Cameroni Elisabetta, Mousson Florence, Winkler G Sebastiaan, De Virgilio Claudio, Collart Martine A, Timmers H Th Marc
Department of Physiological Chemistry, University Medical Centre Utrecht, Utrecht, The Netherlands.
Genetics. 2007 May;176(1):181-92. doi: 10.1534/genetics.106.060640.
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.
Ccr4-Not复合物由九个亚基组成，在酿酒酵母中作为mRNA生物合成的调节因子发挥作用。酵母NOT4的人类直系同源物CNOT4在重组体外系统中表现出依赖UbcH5B的泛素蛋白连接酶（E3连接酶）活性。然而，尚未确定这种酶活性在体内的作用。对酵母Not4p的RING结构域进行定点诱变，确定了与Ubc4p和Ubc5p（UbcH5B的酵母直系同源物）相互作用所需的残基。随后用纯化的Ccr4-Not复合物进行的体外试验显示了Not4p介导的E3连接酶活性，该活性依赖于与Ubc4p的相互作用。为了研究这种活性在体内的相关性，我们使用not4Delta和not4L35A等位基因进行了合成遗传阵列（SGA）分析。这表明Not4p的RING结构域参与转录、泛素化和DNA损伤反应。此外，我们发现UBC4缺失与编码Not4p的RING结构域单氨基酸取代的突变体之间存在表型重叠。总之，我们的结果表明，Not4p在体内通过调节Ubc4p/Ubc5p介导的应激反应发挥E3连接酶的功能。
