Van Nocker S, Vierstra R D
Department of Horticulture, University of Wisconsin, Madison 53706.
Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):10297-301. doi: 10.1073/pnas.88.22.10297.
Recent evidence indicates that the commitment to degrade cellular proteins by the ubiquitin proteolytic pathway is dependent on the covalent attachment of multiubiquitin chains to the target protein [Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K. & Varshavsky, A. (1989) Science 243, 1576-1583]. We have isolated a 20-kDa ubiquitin carrier protein [E2(20 kDa)] from wheat by using ubiquitin covalent affinity chromatography and anion-exchange HPLC that catalyzes multiubiquitin chain formation in vitro. This reaction is blocked by the addition of a mutant ubiquitin in which arginine has been substituted for lysine at residue 48, demonstrating that the coupling of ubiquitin to ubiquitin is likely to be through an isopeptide linkage between the C-terminal glycine and Lys48 of ubiquitin. By immunoscreening a wheat cDNA expression library with anti-E2(20 kDa) antibodies, a cDNA encoding the complete protein was isolated. The clone (designated UBC7) was confirmed as encoding E2(20 kDa) by comparison of the derived amino acid sequence with peptide sequences of E2(20 kDa) tryptic fragments. The encoded protein contains a single cysteine at position 91, which is presumably the active site, and has regions of amino acid sequence similarity to other known E2s from plants and yeast. Expression of this cDNA in Escherichia coli produced an active E2 capable of catalyzing multiubiquitin chain formation in vitro. By virtue of its activity, E2(20 kDa) may have a pivotal role in protein degradation by the ubiquitin-dependent proteolytic pathway.
最近的证据表明，通过泛素蛋白水解途径降解细胞蛋白质的过程依赖于多聚泛素链与靶蛋白的共价连接[Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K. & Varshavsky, A. (1989) Science 243, 1576 - 1583]。我们通过泛素共价亲和层析和阴离子交换高效液相色谱从小麦中分离出一种20 kDa的泛素载体蛋白[E2(20 kDa)]，它在体外催化多聚泛素链的形成。加入一种突变泛素后该反应被阻断，此突变泛素中第48位的赖氨酸被精氨酸取代，这表明泛素与泛素的偶联可能是通过泛素C末端甘氨酸与赖氨酸48之间的异肽键。用抗E2(20 kDa)抗体对小麦cDNA表达文库进行免疫筛选，分离出一个编码完整蛋白的cDNA。通过将推导的氨基酸序列与E2(20 kDa)胰蛋白酶片段的肽序列进行比较，确认该克隆（命名为UBC7）编码E2(20 kDa)。编码的蛋白在第91位含有一个半胱氨酸，推测这是活性位点，并且其氨基酸序列区域与来自植物和酵母的其他已知E2具有相似性。该cDNA在大肠杆菌中的表达产生了一种能够在体外催化多聚泛素链形成的活性E2。凭借其活性，E2(20 kDa)可能在泛素依赖性蛋白水解途径的蛋白质降解中起关键作用。
