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Published October 2000 | Published
Journal Article Open

Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes


Ubiquitin-dependent proteolysis is catalyzed by the 26S proteasome, a dynamic complex of 32 different proteins whose mode of assembly and mechanism of action are poorly understood, in part due to the difficulties encountered in purifying the intact complex. Here we describe a one-step affinity method for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core particles from budding yeast cells. Affinity-purified 26S proteasomes hydrolyze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hydrolyzable analog ATP-gamma -S unexpectedly revealed that a large number of proteins, including subunits of the skp1-cullin-F-box protein ligase (SCF) and anaphase-promoting complex (APC) ubiquitin ligases, copurify with the 19S cap. To identify these proteasome-interacting proteins, we used a recently developed method that enables the direct analysis of the composition of large protein complexes (DALPC) by mass spectrometry. Using DALPC, we identified more than 24 putative proteasome-interacting proteins, including Ylr421c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalian 19S cap (PA700 complex). Additional PIPs include the heat shock proteins Hsp70 and Hsp82, the deubiquitinating enzyme Ubp6, and proteins involved in transcriptional control, mitosis, tubulin assembly, RNA metabolism, and signal transduction. Our data demonstrate that nucleotide hydrolysis modulates the association of many proteins with the 26S proteasome, and validate DALPC as a powerful tool for rapidly identifying stoichiometric and substoichiometric components of large protein assemblies.

Additional Information

© 2000 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0). Submitted April 28, 2000; Revised July 14, 2000; Accepted July 24, 2000 We thank Craig Correll for antibodies to Cdc53 and Skp1, Kiran Madura for the rub1Delta yeast strain, Wade Harper for Sic1 baculovirus, Carl Mann for antibodies to Rpt1 and Rpt6, John Monaco for antibody to LMP7, Alex Varshavsky laboratory members and Cecile Pickart for tetraubiquitin chains, the Varshavsky laboratory for the ubiquitin fusion reporter plasmid, Zack Pitluck and David Gonda for antibody to Cdc34, Jae Hong Seol for the TEV2myc9-tagging cassette, and Joseph Walker and Richard Vierstra for antibody to Rpn10. We thank members of the laboratory, especially Wenying Shou, for their helpful comments. This work was supported by National Institutes of Health grant GM-52466 to R.J.D. R.J.D. was supported by grant DO-649/1-1 from the Deutsche Forschungsgemeinschaft. J.Y. was supported by the National Center for Research Resources. National Institutes of Health Yeast Biotechnology Resource Center grant RR-11823-03.

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