title: The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle.
abstract: The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
authors.forename: Eri
authors.initials: E
authors.lastname: Sakata
authors.forename: Florian
authors.initials: F
authors.lastname: Stengel
authors.forename: Keisuke
authors.initials: K
authors.lastname: Fukunaga
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Saeki
authors.forename: Friedrich
authors.initials: F
authors.lastname: Förster
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Baumeister
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D017353:Gene Deletion; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D054875:Ubiquitination
chemical_list: C499663:RPN1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C109028:UBP6 protein, S cerevisiae; D000251:Adenosine Triphosphatases