PHOSPHORYLATION OF ATPASE SUBUNITS OF THE 26S PROTEASOME

The 26S proteasome complex plays a major role in the non-lysosomal deg radation of intracellular proteins. Purified 26S proteasomes give a pa ttern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimen sional polyacrylamide gel electrophoresis of proteasomes immunoprecipi tated from [P-32]phosphate-labelled human embryo lung L-132 cells reve aled the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of t he core 20S proteasome. Comparison with the positions of the regulator y polypeptides revealed a minor phosphorylated form to be S7 (MSS1). A ntibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at t he position of major phosphorylated polypeptides suggesting that sever al of the ATPase subunits may be phosphorylated. The phosphorylation o f S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and t hen S4 was immunoprecipitated with subunit-specific antibodies. Antibo dies against the non-ATPase subunit S10, which has been suggested by o thers to be phosphorylated, did not coincide with the position of a ph osphorylated polypeptide. Some differences were observed in the 2D-PAG E pattern of proteasomes immunoprecipitated from cultured cells compar ed to purified rat liver 26S proteasomes suggesting possible differenc es in subunit compositions of 26S proteasomes, (C) 1998 Federation of European Biochemical Societies.