The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

ABSTRACT

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

ACKNOWLEDGMENTS

We thank Olivier Coux, Alfred Goldberg, and Inge Wefes for assistance in refining the proteasome purification procedure; Keiji Tanaka and Akio Toh-e for providing unpublished data; Chris Larsen for constructing the His₆-Rpt4 plasmid; Chris Larsen and Seth Sadis for useful discussions and comments; an anonymous reviewer for helpful comments on the text; Michel Ghislain and Carl Mann for the rpt1 knockout construct and polyclonal antibodies to Rpt1 and Rpt6; Richard Diaz, Olivier Coux, and Fred Goldberg for ubiquitin-lysozyme conjugates; Steve van Nocker and Richard Vierstra for the polyclonal antibody to Rpn10; Akio Toh-e for antibodies to Rpn3, Rpn4, and Rpn12; Michelle Mischke and John Chant for anti-Cdc10 antibodies; and Andrei Lupas and Takashi Toda for helpful discussions.

This work was supported by NIH grant GM43601 (to D.F.) and by fellowships from the NIH and the Massachusetts Division of the American Cancer Society (to D.M.R.) and the Damon Runyon Walter Winchell Cancer foundation (to M.H.G.).