An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei
An easily dissociated 26 S proteasome catalyzes an essential ubiquitin-mediated protein degradation pathway in Trypanosoma brucei
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DOI：
10.1074/jbc.m109029200
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发表时间：
2002-05-03
影响因子：
4.8
通讯作者：
Wang, CC
中科院分区：
文献类型：
--
作者：
Li, ZY;Zou, CB;Wang, CC
26 S蛋白酶体是20 S蛋白酶体和19 S调节单元之间的复合物，催化真核生物中未折叠和泛素化蛋白质的ATP依赖性降解。我们以前在布氏锥虫中鉴定过20 S蛋白酶体和活化的20 S蛋白酶体，但未鉴定出26 S蛋白酶体.然而，T.通过细胞裂解物水解酪蛋白，这一过程需要ATP但被lactacystin抑制，以及完整细胞中泛素化蛋白质的lactacystin敏感性周转，提示了布鲁氏菌。T.克隆并表达了编码6个蛋白酶体ATP酶同源物（Rpt）的布鲁氏菌cDNA。六个T中的五个。除Rpt 2外，布氏Rpt cDNA能够在功能上互补酿酒酵母的相应rpt缺失突变体。免疫印迹显示T. Rpt蛋白的布氏裂解物，其通过凝胶过滤与酵母19 S蛋白酶体复合物共分级分离并定位在甘油梯度的19 S级分中。所有的Rpt和推定的19 S非ATP酶（Rpn）蛋白从T。通过单个抗Rpt抗体检测布鲁氏菌裂解物。治疗T.具有化学交联剂的布氏细胞导致20 S蛋白酶体与在甘油梯度中沉降到26 S蛋白酶体位置的所有Rpt和Rpn蛋白的免疫共沉淀。这些结果表明T.在细胞裂解时，其明显解离成19 S和20 S复合物。RNA干扰选择性地阻断蛋白酶体20 S核心和Rpt亚基的表达导致泛素化蛋白的显著积累，伴随着细胞生长的停止。酵母RPT 2基因在T.布鲁氏菌Rpt 2缺陷细胞不能挽救致死表型，从而证实了两种Rpt 2之间的不相容性。霸王布氏11 S调节子（PA 26）缺陷型RNA干扰细胞生长正常，提示T.布鲁塞。
The 26 S proteasome, a complex between the 20 S proteasome and 19 S regulatory units, catalyzes ATP-dependent degradation of unfolded and ubiquitinated proteins in eukaryotes. We have identified previously 20 S and activated 20 S proteasomes in Trypanosoma brucei, but not 26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate, a process that requires ATP but is inhibited by lactacystin, and the lactacystin-sensitive turnover of ubiquitinated proteins in the intact cells. T. brucei cDNAs encoding the six proteasome ATPase homologues (Rpt) were cloned and expressed. Five of the six T. brucei Rpt cDNAs, except for Rpt2, were capable of functionally complementing the corresponding rpt deletion mutants of Saccharomyces cerevisiae. Immunoblots showed the presence in T. brucei lysate of the Rpt proteins, which co-fractionated with the yeast 19 S proteasome complex by gel filtration and localized in the 19 S fraction of a glycerol gradient. All the Rpt and putative 19 S non-ATPase (Rpn) proteins were co-immunoprecipitated from T. brucei lysate by individual anti-Rpt antibodies. Treatment of T. brucei cells with a chemical cross-linker resulted in co-immunoprecipitation of 20 S proteasome with all the Rpt and Rpn proteins that sedimented in a glycerol gradient to the position of 26 S proteasome. These data demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon cell lysis. RNA interference to block selectively the expression of proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal phenotype, thus confirming the incompatibility between the two Rpt2s. The T. brucei 11 S regulator (PA26)-deficient RNA interference cells grew normally, suggesting the dispensability of activated 20 S proteasome in T. brucei.