Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC
Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC
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DOI：
10.1093/emboj/20.18.5165
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发表时间：
2001-09-17
期刊：
影响因子：
11.4
通讯作者：
Seufert, W
中科院分区：
文献类型：
--
作者：
Schwab, M;Neutzner, M;Seufert, W
泛素介导的蛋白水解已经成为真核细胞中的关键调节机制。在细胞分裂过程中，称为后期促进复合物（APC）的多亚基泛素连接酶靶向关键调节蛋白，如securin和有丝分裂细胞周期蛋白，从而触发染色体分离和退出有丝分裂。先前在酿酒酵母中的研究鉴定了保守的WD 40蛋白Cdc 20和Hct 1（Cdh 1）作为APC的底物特异性激活剂，但其确切的作用机制仍不清楚。这项研究提供的证据表明，Hct 1作为一种底物受体，识别靶蛋白，并招募他们的APC泛素化和随后的蛋白水解。通过免疫共沉淀，我们发现Hct 1与有丝分裂细胞周期蛋白Clb 2和Clb 3以及polo相关激酶Cdc 5相互作用，而Cdc 20与securin Pds 1相互作用。未能与Hct 1相互作用导致Clb 2稳定。Hct 1衍生物的分析确定了C-box，这是Hct 1的APC关联所需的基序，并且在Cdc 20相关蛋白中保守。我们建议Cdc 20家族的蛋白质是泛素连接酶APC的底物识别亚基。
Ubiquitin-mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi-subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate-specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co-immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo-related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C-box, a motif required for APC association of Hct1 and conserved among Cdc20-related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.