Wing S S, Jain P
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Biochem J. 1995 Jan 1;305 ( Pt 1)(Pt 1):125-32. doi: 10.1042/bj3050125.
Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4/UBC5. Its high expression in testis and ability to efficiently support conjugation to testis proteins suggest that this family of E2s may play a role in the proteolysis that occurs during spermatogenesis.
泛素结合酶（E2s）通过催化泛素与蛋白质底物的结合，在泛素介导的蛋白水解过程中发挥关键作用。我们之前报道了一种14 kDa结合酶[E2(14)k；Wing、Dumas和Banville（1992年）《生物化学杂志》267卷，6495 - 6501页]的cDNA克隆，该酶能有效支持网织红细胞提取物中的泛素化和蛋白质降解。令人惊讶的是，这种E2的结构与酿酒酵母DNA修复基因RAD6的相似性明显高于酿酒酵母UBC4/UBC5基因，后者是降解短命蛋白质以及支持酵母蛋白质大部分泛素化所必需的。这表明UBC4/UBC5的哺乳动物同源物仍有待鉴定。以源自酿酒酵母UBC4序列的寡核苷酸为引物，以大鼠肌肉cDNA为模板进行PCR反应，扩增出一个390 bp的DNA片段，该片段预测的氨基酸序列与酵母UBC4有83%的同一性。筛选大鼠睾丸cDNA文库鉴定出一个cDNA家族，该家族预测有两种非常相似的蛋白质，其碱性pI和分子量约为16,700 Da。亚型2E在大肠杆菌中表达并纯化至均一。它在体外能比E2(14)k更快地支持网织红细胞和睾丸蛋白质的泛素化，并产生更大的缀合物。对不同组织RNA的检测表明，这种类型的E2在广泛的组织中表达，但在睾丸中表达水平特别高。通过阴离子交换色谱对睾丸提取物进行分级分离，鉴定出几种假定的泛素蛋白连接酶活性，这种E2在促进泛素与蛋白质结合时可与它们相互作用。其中一种活性支持泛素与组蛋白H2A的结合，组蛋白H2A是泛素系统中通过非N端规则机制降解的底物。本文报道了酿酒酵母UBC4/UBC5首个明显的哺乳动物同源物的克隆。它在睾丸中的高表达以及有效支持与睾丸蛋白质结合的能力表明，这个E2家族可能在精子发生过程中发生的蛋白水解中发挥作用。
