Structural and genetic analysis of proteasomes

26 S蛋白酶体是一种真核生物的ATP依赖性蛋白酶，是一种分子量为2000 kDa的大型多亚基复合物，由40多种不同的组分组成。它是一个类似于伞的颗粒，由一个圆柱形的催化机器（称为20 S蛋白酶体）和两个末端模块（称为PA 700或19 S复合物）组成，具有可能的调节功能，以相反的方向连接到中心部分的两端，形成具有酶活性的蛋白酶体。PA 700含有20多个分子量为25-110 kDa的异质亚基，可分为两个亚组：一个亚组至少有6种结构相似且在进化中高度保守的ATP酶，另一个亚组有15种以上结构与ATP酶家族成员无关的异质亚基。在本研究期间，我们通过蛋白质化学和分子生物学技术确定了人26 S蛋白酶体的10个新的调节亚基的一级结构，其中包括两个新的ATP酶亚基。根据这些结果，结合我们以前的报道，我们成功地分离出了编码26 S蛋白酶体复合体的大部分组分的cDNA，从分子水平上提供了这个多亚基复合体的结构信息，并对酿酒酵母进行了遗传分析，阐明了单个亚基的细胞功能，发现蛋白酶体对酵母细胞的增殖是必需的。与大多数催化型20 S蛋白酶体和6个调节型ATP酶亚基的基本作用不同，一些非ATP酶亚基对细胞活力是不可缺少的，这表明它们可能具有冗余功能。本研究获得的结构和遗传学结果为阐明26 S蛋白酶体的结构与功能关系提供了重要信息。

The 26S proteasome, a eukaryotic ATP-dependent protease, is a large multisubunit complex with a molecular weight of 2000 kDa, consisting of over 40 distinct components. It is a cumbbell-like particle, consisting of a cylindrical catalytic machine (termed the 20S proteasome) and two terminal modules (named PA700 or 19S complex), having possible regulatory functions, that is attached to both ends of the central portion in opposite orientations to form the enzymatically active proteasome. PA700 contains more than 20 heterogeneous subunits of 25-110 kDa, which can be classified into two subgroups : a subgroup of at least six ATPases that are structurally similar and highly conserved in evolution, and a subgroup of over 15 heterogeneous subunits that are structurally unrelated to the members of the ATPase family. During this research period, we have determined primary structures of ten new regulatory subunits containing two novel ATPase subunits of the human 26S proteasome by protein-chemical and molecular-biological techniques. By these results and together with our previous reports, we have succeeded to isolate cDNAs encoding most of all components of the 26S proteasome complec, providing structural information of this multisubunit complex at molecular level.Moreover we have carried out genetic analysis in Saccaromyces cerevisiae to clarify the cellular functions of individual subunit and found that proteasomes are essential for proliferation of the yeast cells. Intruguingly, some of non-ATPase subunits were indespensable for cell viability, unlike essential roles of most catalytic 20S proteasomal and 6 regulatory ATPase subunits, suggesting that they may have redundant functions. Structural and genetic results obtained by present study could provide important information to clarify the structure-function relationships of the 26S proteasome.