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降解的主要途径。泛素-蛋白酶体系统调节细胞周期、抗原提呈和植物自身相容性，我们分离了蛋白酶体和蛋白复合物相互作用的蛋白酶体亚基，并对其结构和功能进行了分析。水稻蛋白酶体的四个ATP酶亚基由重复基因编码。我们纯化了水稻蛋白酶体的19 S复合体，并分析了各基因产物在水稻不同组织中的表达。各产物的组织特异性表达表明存在参与组织特异性蛋白质代谢的多种形式的19 S复合物。分析了酵母20 S蛋白酶体的翻译后修饰。确定了α 7亚基的磷酸化位点。RPN 4最初被鉴定为26 S蛋白酶体的一个亚基，是蛋白酶体多个亚基的转录调节因子。我们用TAP法从酵母细胞中分离出RPN 4复合物。Bud 32被报道与蛋白酶体的许多亚基相互作用。我们从酵母单倍体细胞中分离出Bud 32复合物。该复合物由四种蛋白质组成：Kael、Cgi 121、Bud 32和Gon 7。该复合物最近被报道为KEOPS（激酶，推定的内肽酶和其他小尺寸蛋白质）/EKC（内肽酶样激酶染色质相关），其调节端粒维持或几个基因的转录。这些蛋白质在细胞核和胞质溶胶中观察到。Bud 32的二倍体缺失突变体显示随机出芽，而不是在正常细胞中观察到的双极出芽模式。缺失突变体在复合体其他成员上的出芽模式不是随机的。从二倍体细胞中分离的Bud 32复合物含有类似的蛋白质。观察到一些额外的次要组分。这些结果表明，Bud 32本身调节二倍体酵母细胞的出芽模式。

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.