Structural and functional analysis on protein complex of ubiquitin-proteasome system

泛素-蛋白酶体系统降解蛋白质是细胞内蛋白质分解代谢的主要途径。泛素-蛋白酶体系统调节细胞周期、抗原提呈和植物自交亲和性。我们分离了蛋白酶体和蛋白复合体相互作用的蛋白酶体亚基，并对其结构和功能进行了分析。水稻蛋白酶体的4个ATPase亚基由重复基因编码。我们纯化了水稻蛋白酶体的19S复合体，并分析了每个基因产物在不同水稻组织中的表达。每种产物的组织特异性表达表明参与组织特异性蛋白质代谢的多形式19S复合体的存在。对酵母20S蛋白酶体的翻译后修饰进行了分析。测定了α7亚基的磷酸化位点。RPN4最初被确定为26S蛋白酶体的一个亚单位，是蛋白酶体几个亚单位的转录调节因子。我们用TAP法从酵母细胞中分离出RPN4复合体。据报道，Bud32与蛋白酶体的许多亚单位相互作用。我们从酵母单倍体细胞中分离到了Bud32复合体。该复合体由Kael、Cgi121、Bud32和Gon7四种蛋白组成。该复合体最近被报道为Keops(用于激酶、假定的内肽酶和其他小分子蛋白)/EKC(内肽酶样激酶染色质相关)，它调节几个基因的端粒维持或转录。这些蛋白在胞核和胞浆中均可观察到。Bud32的二倍体缺失突变体表现为随机萌发，而不是正常细胞中观察到的两极萌发模式。该复合体其他成员的缺失突变体的萌发模式不是随机的。从二倍体细胞中分离到的Bud32复合体含有相似的蛋白质。观察到了一些额外的次要成分。这些结果表明，Bud32本身调节二倍体酵母细胞的萌发模式。

Protein degradation by ubiquitin-proteasome system is a major path of protein catabolism in cells. The ubiquitin-proteasome system regulates cell cycle, antigen presentation and plant self compatibility.We have isolated proteasome and protein complex interacting proteasome subunits, and analyzed structure and functions. Four rice ATPase subunits of the proteasome were encoded by duplicated genes. We purified rice 19S complex of the proteasome and analyzed the expression of each gene products in various rice tissues. Tissue specific expression of each product suggests the preesence of multiform 19S complex engaged in the tissue-specific protein metabolism. The posttranslational modification of yeast 20S proteasome was analyzed. The phosphorylation sites of alpha7 subunit was determined. RPN4, originally identified as a subunit of 26S proteasome, is a transcriptional regulator for several subunits of proteasome. We isolated RPN4 complex from yeast cells by TAP method. Bud32 is reported to interact many subunits of proteasome. We isolated Bud32 complex from yeast haploid cells. The complex consisted of four proteins, Kael, Cgi121, Bud32 and Gon7. This complex was reported recently as KEOPS (for kinase, putative endopeptidase and other proteins of small size)/EKC (Endopeptidase-like Kinase Chromatin-associated), which regulates telomere maintenance or transcription of several genes. These proteins observed in nucleus and also in cytosol. The duploid deletion mutant of Bud32 shows random budding instead of bipolar budding pattern observed in normal cells. Budding pattern of deletion mutants on other members of the complex was not random. Bud32 complex isolated from duploid cells contained similar proteins. Some additional minor components were observed. These results suggest that Bud32 itself regulates budding pattern of duploid yeast cells.