Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.
AUTOR(ES)
McDonough, M
RESUMO
Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176631Documentos Relacionados
- Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.
- The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae.
- The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme.
- Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.
- Degradation Signals Recognized by the Ubc6p-Ubc7p Ubiquitin-Conjugating Enzyme Pair