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 dynam ic 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 metho d for purifying intact 26S proteasomes, 19S regulatory caps, and 20S core p articles from budding yeast cells. Affinity-purified 26S proteasomes hydrol yze both model peptides and the ubiquitinated Cdk inhibitor Sic1. Affinity purifications performed in the absence of ATP or presence of the poorly hyd rolyzable analog ATP-gamma-S unexpectedly revealed that a large number of p roteins, including subunits of the skp1-cullin-F-box protein Ligase (SCF) a nd anaphase-promoting complex (APC) ubiquitin Ligases, copurify with the 19 S cap. To identify these proteasome-interacting proteins, we used a recentl y developed method that enables the direct analysis of the composition of l arge protein complexes (DALPC) by mass spectrometry. Using DALPC, we identi fied more than 24 putative proteasome-interacting proteins, including Ylr42 1c (Daq1), which we demonstrate to be a new subunit of the budding yeast 19 S cap, and Ygr232w (Nas6), which is homologous to a subunit of the mammalia n 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 si gnal transduction. Our data demonstrate that nucleotide hydrolysis modulate s the association of many proteins with the 26S proteasome, and validate DA LPC as a powerful tool for rapidly identifying stoichiometric and substoich iometric components of large protein assemblies.