Studies on structural characteristics and physiological function of a novel membrane-bound proteasome

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We have isolated the ER membrane-bound (ERb) form of proteasome that we referred to as "ERb proteasome", and studied the structural characteristics, how it binds to the ER membrane, and its possible function as a novel protease for ERAD.Isolation of subunits of ERb by HPLC and detailed analyses of isolated subunits, in comparison with those of 20S proteasome, revealed that one of two α5 subunits in 20S proteasome has been modified in ERb, which we referred to as α5' subunit. The α5' subunit had the N-terminal amino acid sequence of N-acetyl-Met-Phe-Leu-Thr-Arg-Ser-, while that of α5 subunit was Thr-Arg-Ser-. Since Met-Phe-Leu sequence corresponded to the propeptide of α5 subunit, it is highly likely that α5' subunit was produced by N-acetylation of the N-terminal Met of the precursor form of α5 subunit. To examine if α5' subunit is contributing to the membrane binding of ERb, we prepared recombinant α5-type and α5'-type mutant subunits in E.coli, and investigated the capability and specificity of phospholipid binding of the recombinant subunits as well as ERb. None of α5-type, α5'-type mutant subunit, or ERb bound to the major components of membrane such as PC, PE, PS or PI, but they specifically bound to phosphatidylinositol polyphosphates (PIP, PIP2 and PIP3), suggesting a unique characteristics of membrane binding of α5' subunit and ERb. We also examined the function of ERb and its derivative in ERAD of misfolded glycoproteins (α1-antitrypsin null Hong Kong and antithrombin Pro429Stop) using 293 cells. Pulse-chase experiments showed that ERADs of these misfolded glycoproteins were significantly enhanced when α5'-type subunit was co-transfected, suggesting the involvement of ERb in the ERAD of misfolded glycoproteins.