Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions.

Abstract

The RAD6 gene ofSaccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B. We have now examined whether the E2 activity of RAD6 is involved in its various biological functions. Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine. The mutant proteins were overproduced in yeast cells and purified to near homogeneity. We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity. The rad6Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele. We suggest that the biological functions of RAD6 require its E2 activity.

Specific complex formation between yeast RAD6 and RAD18 proteins: a potential mechanism for targeting RAD6 ubiquitin-conjugating activity to DNA damage sites.

“…The protein encoded by the rad6 Ala-88 allele, which resembles the rad6A mutant in UV sensitivity, is devoid of ubiquitin-conjugating activity (Sung et al 1990). In this study we found that the same amount of RAD 18 can be coprecipitated from cell extract with rad6 Ala-88 mutant protein as with wild-type RAD6 protein by anti-RAD6 immunobeads (Fig.…”

“…Previously, we evaluated the biological role of the RAD6 ubiquitin-conjugating function by mutating Cys-88 in RAD6, the site of thioester formation with ubiquitin during its conjugation to substrates (Sung et al 1990;Hershko 1991), to other residues, including alanine. The protein encoded by the rad6 Ala-88 allele, which resembles the rad6A mutant in UV sensitivity, is devoid of ubiquitin-conjugating activity (Sung et al 1990).…”

“…Interestingly, a fiftyfold overexpression of tad6 Ala-88 protein by use of the A D C I promoter in plasmid pR648 (Sung et al 1990) in the RAD + strain renders cells highly sensitive to UV light (Fig. 5B).…”

“…In this assay RAD6 protein (25 and 50 pmoles; Sung et al 1988) and immunopurified RAD18 protein (9 and 18 pmoles) were electrophoresed in denaturing polyacrylamide gels, transferred onto nitrocellulose, and, after renaturing treatment, incubated with 3~p-labeled linear double-stranded (ds) M13 DNA or ss M13 DNA generated from the double-stranded form by thermal denaturation. After extensive washing, the nitrocellulose filters were subjected to autoradiography to detect DNA binding.…”

Modification of Ran GTPase-activating Protein by the Small Ubiquitin-related Modifier SUMO-1 Requires Ubc9, an E2-type Ubiquitin-conjugating Enzyme Homologue

“…Sequence comparison of HsUbc9 with known E2-type UBCs suggests that the cysteine residue at position 93 is the active site for thiolester conjugation (19), a conclusion that is supported by genetic reconstitution analysis of Ubc9 mutants (12,13). This prediction was tested by comparing the electrophoretic migration of 35 S-labeled recombinant wild-type (W) or mutant (M) HsUbc9 translated in reticulocyte lysates supplemented with buffer control (Fig.…”