ROLE OF THE C-TERMINUS OF SACCHAROMYCES-CEREVISIAE UBIQUITIN-CONJUGATING ENZYME (RAD6) IN SUBSTRATE AND UBIQUITIN-PROTEIN-LIGASE (E3-R) INTERACTIONS

The product of the RAD6 (UBC2) ne of Saccharomyces cerevisiae is a ubi quitin-conjugating enzyme (Rad6) which is implicated in DNA repair, in duced mutagenesis, retrotransposition, sporulation and the degradation of proteins with destabilizing N-terminal amino acid residues. Deleti on of the 23-residue acidic C-terminus of Rad6 impairs sporulation and N-end rule protein degradation in vivo but does not affect other func tions such as DNA repair and induced mutagenesis. We have investigated the role of the C-terminus of Rad6 in in vitro interactions with vari ous substrates and with a putative ubiquitin-protein ligase, E3-R. The removal of the Rad6 C-terminus had significant different effects on e nzyme activity for individual substrates. Although the 23-residue trun cated Rad6-149 protein had markedly impaired activity for histone H2B and micrococcal nuclease, the activity for cytochrome c was the same a s that of the intact Rad6 protein. Similarly, truncation of Rad6 had n o effect on its activity for several poor substrates, namely, beta-cas ein, beta-lactoglobulin and oxidized RNase. E3-R stimulated the activi ties of both Rad6 and Rad6-149 for the latter three substrates to simi lar degrees. E3-R appears to act by enhancing the low intrinsic affini ty of Rad6 and Rad6-149 for these substrates. Thus Rad6 can act in thr ee different modes in vitro depending on the substrate, namely unassis ted C-terminus-dependent, unassisted C-terminus-independent and E3-R-a ssisted C-terminus-independent modes. We also examined the results of removing the C-terminal acidic region of Cdc34 (Ubc3), a ubiquitin-con jugating enzyme closely related to Rad6. Truncation of Cdc34 like that of Rad6 had no effect on activity for beta-casein, beta-lactoglobulin or oxidized RNase in the presence or absence of E3-R.