A minimal anaphase promoting complex/cyclosome (APC/C) in Trypanosoma brucei.
A minimal anaphase promoting complex/cyclosome (APC/C) in Trypanosoma brucei.
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DOI：
10.1371/journal.pone.0059258
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Wang CC
中科院分区：
文献类型：
--
作者：
Bessat M;Knudsen G;Burlingame AL;Wang CC
后期促进复合物/环体（APC/C）是一种多亚基E3泛素连接酶，通过靶向关键细胞周期调节因子进行蛋白水解破坏来启动染色体分离和有丝分裂退出。此前，在布氏锥虫基因组中鉴定出7个APC/C亚基同源物。在本研究中，我们在酵母互补研究中测试了其中的五个，发现它们都不能互补缺乏相应亚基的酵母突变体，这表明两个APC/C之间存在显着差异。有丝分裂检查点复合物（MCC）的亚单位同源物尚未在T。brucei，提示T.布鲁塞。我们进行了串联亲和纯化的蛋白质复合物含有APC 1融合蛋白表达的细胞中富集的细胞周期的不同阶段的前环形式T。并利用LC-MS/MS分析比较了它们的蛋白质谱。在整个细胞周期的蛋白质复合物中鉴定了七个推定的APC/C亚基以及三个另外的蛋白质，称为相关蛋白（AP）AP 1、AP 2和AP 3。这10种蛋白在整个细胞周期中的表达量相对不变，表明它们是APC/C的核心亚基。AP 1是APC 4的同源物。RNAi敲除APC 4和AP 3未显示出可检测的细胞表型，而AP 2敲除使细胞富集在G2/M期。去除AP 2的细胞显示稳定的有丝分裂周期蛋白B。在用蛋白酶体抑制剂MG 132处理的细胞中指示多聚泛素化细胞周期蛋白B的积累，证明蛋白酶体参与降解多聚泛素化细胞周期蛋白B。总之，在T. brucei没有连接到MCC样复合物，从而表明了独特的T.布氏杆菌APC/C.
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.
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