Role of the C‐terminus of Saccharomyces cerevisiae ubiquitin‐conjugating enzyme (Rad6) in substrate and ubiquitin‐protein‐ligase (E3‐R) interactions

Bilha RABOY, Richard G. KULKA*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

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, β‐casein, β‐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 β‐casein, β‐lactoglobulin or oxidized RNase in the presence or absence of E3‐R.

Original languageEnglish
Pages (from-to)247-251
Number of pages5
JournalEuropean Journal of Biochemistry
Volume221
Issue number1
DOIs
StatePublished - Apr 1994

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