Raboy B, Kulka R G
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel.
Eur J Biochem. 1994 Apr 1;221(1):247-51. doi: 10.1111/j.1432-1033.1994.tb18735.x.
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.
酿酒酵母RAD6（UBC2）基因的产物是一种泛素结合酶（Rad6），它与DNA修复、诱导突变、逆转座、孢子形成以及具有不稳定N端氨基酸残基的蛋白质降解有关。删除Rad6的23个残基酸性C末端会损害体内的孢子形成和N端规则蛋白降解，但不影响其他功能，如DNA修复和诱导突变。我们研究了Rad6的C末端在体外与各种底物以及假定的泛素-蛋白连接酶E3-R相互作用中的作用。去除Rad6的C末端对单个底物的酶活性有显著不同的影响。虽然截短23个残基的Rad6-149蛋白对组蛋白H₂B和微球菌核酸酶的活性明显受损，但对细胞色素c的活性与完整的Rad6蛋白相同。同样，Rad6的截短对其对几种较差底物（即β-酪蛋白、β-乳球蛋白和氧化核糖核酸酶）的活性没有影响。E3-R对后三种底物刺激Rad6和Rad6-149的活性程度相似。E3-R似乎通过增强Rad6和Rad6-149对这些底物的低内在亲和力来发挥作用。因此，Rad6在体外可以根据底物以三种不同模式起作用，即无辅助的C末端依赖性、无辅助的C末端非依赖性和E3-R辅助的C末端非依赖性模式。我们还研究了去除与Rad6密切相关的泛素结合酶Cdc34（Ubc3）的C末端酸性区域的结果。与Rad6一样，Cdc34的截短在有或没有E3-R的情况下对β-酪蛋白、β-乳球蛋白或氧化核糖核酸酶的活性没有影响。
