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) gene of Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme (Rad6) which is implicated in DNA repair, induced mutagenesis, retrotransposition, sporulation and the degradation of proteins with destabilizing N-terminal amino acid residues. Deletion of the 23-residue acidic C-terminus of Rad6 impairs sporulation and N-end rule protein degradation in vivo but does not affect other functions such as DNA repair and induced mutagenesis. We have investigated the role of the C-terminus of Rad6 in in vitro interactions with various substrates and with a putative ubiquitin-protein ligase, E3-R. The removal of the Rad6 C-terminus had significant different effects on enzyme activity for individual substrates. Although the 23-residue truncated Rad6-149 protein had markedly impaired activity for histone H2B and micrococcal nuclease, the activity for cytochrome c was the same as that of the intact Rad6 protein. Similarly, truncation of Rad6 had no effect on its activity for several poor substrates, namely, beta-casein, beta-lactoglobulin and oxidized RNase. E3-R stimulated the activities of both Rad6 and Rad6-149 for the latter three substrates to similar degrees. E3-R appears to act by enhancing the low intrinsic affinity of Rad6 and Rad6-149 for these substrates. Thus Rad6 can act in three different modes in vitro depending on the substrate, namely unassisted C-terminus-dependent, unassisted C-terminus-independent and E3-R-assisted C-terminus-independent modes. We also examined the results of removing the C-terminal acidic region of Cdc34 (Ubc3), a ubiquitin-conjugating 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.
Raboy
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Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Kulka
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RG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004260:DNA Repair; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D013172:Spores, Fungal / Q000502:physiology; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases