The regulatory complex of Drosophila melanogaster 26S proteasomes: Subunitcomposition and localization of a deubiquitylating enzyme

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterizat ion, purified 26S proteasomes were resolved by two-dimensional (2D) gel ele ctrophoresis and subunits composing the regulatory complex (RC) were identi fied by amino acid sequencing and immunoblotting, before corresponding cDNA s were sequenced. 17 subunits from Drosophila RCs were found to have homolo gues in the yeast and human RCs. An additional subunit, p37A, not yet descr ibed in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibi tor complexes allowed for the first time to localize one of the RC's specif ic functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were dete rmined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete.