CHARACTERIZATION OF NOVEL YEAST RAD6 (UBC2) UBIQUITIN-CONJUGATING ENZYME MUTANTS CONSTRUCTED BY CHARGE-TO-ALANINE SCANNING MUTAGENESIS

Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiq uitin-conjugating (E2) enzyme is required for cellular processes as di verse as DNA repair, selective proteolysis, and normal growth. For mos t RAD6-dependent functions, the relevant in vivo targets, as well as t he mechanisms and cofactors that govern RAD6 substrate selectivity, ar e unknown. We have explored the utility of ''charge-to-alanine'' scann ing mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitinatio n but that are nevertheless severely handicapped with respect to sever al in vivo functions. Five of the nine mutants we generated show defec ts in their in vivo functions, but almost all of the most severely aff ected mutants displayed unfacilitated ubiquitin-conjugating activity i n vitro. We suggest that E2 mutants obtained by this approach are like ly to be defective with respect to interaction with other, trans-actin g factors required for their intracellular activity or substrate selec tivity and therefore will be useful for further genetic and biochemica l studies of ubiquitin-conjugating enzyme function.