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

Arch Biochem Biophys. 1999 Aug 1;368(1):85-97. doi: 10.1006/abbi.1999.1294.


Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees 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 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Drosophila melanogaster / enzymology*
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / growth & development
  • Electrophoresis, Gel, Two-Dimensional
  • Heterozygote
  • Immunoblotting
  • Isoelectric Point
  • Models, Biological
  • Molecular Weight
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / metabolism*
  • Mutation
  • Protease Inhibitors / pharmacology
  • Proteasome Endopeptidase Complex
  • Protein Conformation
  • Temperature


  • Multienzyme Complexes
  • Protease Inhibitors
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex