title: Base-CP proteasome can serve as a platform for stepwise lid formation.
abstract: 26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11-m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11-Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
authors.forename: Zanlin
authors.initials: Z
authors.lastname: Yu
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
authors.forename: Nurit
authors.initials: N
authors.lastname: Livnat-Levanon
authors.affiliation: Department of Biochemistry & Molecular Biology, Monash University, Clayton, VIC 3800, Australia.
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
authors.forename: Wissam
authors.initials: W
authors.lastname: Mansour
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Nakasone
authors.affiliation: Department of Biology and Environment, University of Haifa at Oranim, Tivon 36006, Israel.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Castaneda
authors.affiliation: Department of Biology and Environment, University of Haifa at Oranim, Tivon 36006, Israel.
authors.forename: Emma K
authors.initials: EK
authors.lastname: Dixon
authors.affiliation: Department of Biology and Environment, University of Haifa at Oranim, Tivon 36006, Israel.
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
authors.forename: Noa
authors.initials: N
authors.lastname: Reis
authors.affiliation: Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, U.S.A.
authors.forename: Elah
authors.initials: E
authors.lastname: Pick
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel glickman@tx.technion.ac.il.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Glickman
mesh_terms: D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D008958:Models, Molecular; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C115451:RPN11 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C472775:Rpn6 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex; C573321:Rpn8 protein, S cerevisiae; C000597966:Rpn9 protein, S cerevisiae; C061553:ATP dependent 26S protease
keywords: 19S regulatory particle; 20S core particle; 26S proteasome; MPN; PCI; base; lid; rpn11-m1