Subunit compositions and catalytic properties of proteasomes from developmental temperature-sensitive mutants of Drosophila melanogaster

Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits β6/C5 (DTS5) and β2/Z (DTS7). At nonpermissive temperature (29°C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25°C). Two- dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to α6/C2 (five species), α2/C3 (three species), α7/C8 (three species), α5/zeta, and β1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (3-7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (2 and 3), decreases in Y (25 kDa; 2-fold) and 31-kDa (9; 2-fold) subunits, and increases in 52-kDa (1; 1.9-fold) and 24-kDa (13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits 3-7 and were decreases in 9 (2.5-fold) and 14 (3-fold) and a loss of 2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (16) in DTS/+ preparations, with about a 14-fold greater amount of 16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.