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Published online 11 July 2000. doi:10.1083/jcb.150.1.119
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© The Rockefeller University Press, 0021-9525/2000/7/119/ $5.00
The Journal of Cell Biology, Volume 150, Number 1, July 10, 2000 119-130

Original Article

The Regulatory Complex of Drosophila melanogaster 26S Proteasomes: Subunit Composition and Localization of a Deubiquitylating Enzyme

Harald Hölzla, Barbara Kapelaria, Josef Kellermanna, Erika Seemüllera, Máté Sümegib, Andor Udvardyb, Ohad Medaliac, Joseph Sperlingc, Shirley A. Müllerd, Andreas Engeld, and Wolfgang Baumeistera
a Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany
b Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary
c Department of Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
d Maurice E. Müller Institute, Biocenter, University of Basel, CH-4056 Basel, Switzerland

Correspondence to: Wolfgang Baumeister, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. Tel:+49-89-85782642 Fax:+49-89-85782641 E-mail:baumeist{at}biochem.mpg.de.

Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity.

The masses of 26S proteasomes with either one or two attached RCs were determined 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.

Key Words: protein degradation, ubiquitin, ubiquitin hydrolase, ATP-dependent proteolysis , electron microscopy


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