Ellison K S, Gwozd T, Prendergast J A, Paterson M C, Ellison M J
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1991 Dec 15;266(35):24116-20.
We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.
我们已经确定了来自酿酒酵母的细胞周期相关泛素结合酶CDC34（UBC 3）的一个温度敏感等位基因的基因序列。温度敏感性的基础是一个错义突变，导致在迄今鉴定的所有泛素结合酶中保守的一个残基处脯氨酸被丝氨酸取代。这一观察结果提出了一种可能性，即其他温度敏感的泛素结合酶可能以同样的方式产生。因此，我们在与DNA修复相关的泛素结合酶RAD6（UBC2）中进行了相应的取代，并研究了温度对这种改变的多肽的细胞增殖和DNA修复相关功能的影响。携带这种突变的酵母菌株在细胞增殖方面被证明对温度敏感，但在其他rad6突变体表现出的DNA损伤处理表型方面并非如此。在进一步研究该突变体表现出的增殖缺陷时，我们发现其他缺失该基因的rad6基因突变体在非允许温度下会发生细胞周期停滞，而工程化的温度敏感等位基因没有显示出细胞周期缺陷的迹象。从这些发现中，我们得出结论，RAD6的增殖功能可以细分为生长成分和细胞分裂周期成分，并且生长成分与RAD6的DNA修复功能无关。对这些结果的一个合理的解释是，在每种情况下不同的蛋白质被靶向进行泛素化。RAD6和CDC34保守的脯氨酸残基是所有泛素结合酶共有的一个转角基序的一部分。因此，位于转角处的脯氨酸的定点取代可能普遍适用于创建其他温度敏感的泛素结合酶，也可能适用于其他蛋白质。
