Structural and genetic analysis of proteasomes

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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.