Gregori L, Poosch M S, Cousins G, Chau V
Department of Pharmacology, School of Medicine, Wayne State University, Detroit, Michigan 48201.
J Biol Chem. 1990 May 25;265(15):8354-7.
The proteolytic targeting function of ubiquitin was investigated by a combination of site-specific mutagenesis and covalent modification. Lys48 was replaced by a cysteine via mutagenesis of a synthetic ubiquitin gene to generate the mutant Ub-C48. The single cysteine residue in Ub-C48 can be converted into a lysine analog by modification with the sulfhydryl-specific reagent, aminoethyl-8 (N-(iodoethyl)trifluoroacetamide). The resulting protein, Ub-(S-aminoethyl)C48, is equivalent to a wild type ubiquitin except for the substitution of a sulfur atom at the gamma carbon of Lys48. We have tested the ability of these two modified ubiquitins to target the degradation of an engineered beta-galactosidase substrate protein in ubiquitin-depleted reticulocyte lysates. Ub-C48 was unable to stimulate the degradation of this protein substrate although a monoubiquitinated beta-galactosidase was formed. In contrast, Ub-(S-aminoethyl)C48 appears to be as effective as wild type ubiquitin in targeting this substrate protein's degradation as well as the formation of multiply ubiquitinated beta-galactosidase intermediates. In conjunction with the cysteine substitution and modification, we have also examined the effects of blocking the amino groups in ubiquitin with reductive methylation. The methylation of either Lys48 in ubiquitin or its S-aminoethylcysteine counterpart abolished its proteolytic function while the blockage of the remaining six lysines in Ub-(S-aminoethyl)C48 did not alter its competence. Thus, of the seven lysine residues in ubiquitin, only Lys48 is essential. These results established unambiguously that a uniform multiubiquitin chain with ubiquitin-ubiquitin linkage solely at Lys48 is sufficient to target the degradation of a substrate protein in ubiquitin-mediated proteolysis.
通过位点特异性诱变和共价修饰相结合的方法，对泛素的蛋白水解靶向功能进行了研究。通过对合成泛素基因进行诱变，将赖氨酸48替换为半胱氨酸，以产生突变体Ub - C48。Ub - C48中的单个半胱氨酸残基可以通过用巯基特异性试剂氨基乙基 - 8（N -（碘乙基）三氟乙酰胺）进行修饰，转化为赖氨酸类似物。所得蛋白质Ub -（S - 氨基乙基）C48，除了在赖氨酸48的γ碳处取代了一个硫原子外，与野生型泛素相当。我们测试了这两种修饰后的泛素在泛素缺失的网织红细胞裂解物中靶向降解工程化β - 半乳糖苷酶底物蛋白的能力。尽管形成了单泛素化的β - 半乳糖苷酶，但Ub - C48无法刺激该蛋白底物的降解。相比之下，Ub -（S - 氨基乙基）C48在靶向该底物蛋白的降解以及形成多泛素化的β - 半乳糖苷酶中间体方面似乎与野生型泛素一样有效。结合半胱氨酸替换和修饰，我们还研究了用还原甲基化阻断泛素中氨基的影响。泛素中赖氨酸48或其S - 氨基乙基半胱氨酸对应物的甲基化消除了其蛋白水解功能，而Ub -（S - 氨基乙基）C48中其余六个赖氨酸的阻断并未改变其功能。因此，在泛素的七个赖氨酸残基中，只有赖氨酸48是必需的。这些结果明确地确定，仅在赖氨酸48处具有泛素 - 泛素连接的均匀多泛素链足以在泛素介导的蛋白水解中靶向底物蛋白的降解。
