Hsm3/S5b Participates in the Assembly Pathway of the 19S Regulatory Particle of the Proteasome

Abstract

The 26S proteasome, the central enzyme of the ubiquitin-proteasome system, is comprised of the 20S catalytic core particle (CP) and the 19S regulatory particle (RP), itself composed of two subcomplexes, the base and the lid. 20S proteasome assembly is assisted by several chaperones. Integral subunits of the RP participate in its assembly, but no external factors have been identified so far. Here we characterize the yeast Hsm3 protein, which displays unique features regarding 19S assembly. Hsm3 associates with 19S subcomplexes via a carboxy-terminal domain of the Rpt1 base subunit but is missing in the final 26S proteasome. Moreover, Hsm3 is specifically required for the base subcomplex assembly. Finally, we identify the putative species-specific 19S subunit S5b as a functional homolog of the Hsm3 chaperone in mammals. These findings shed light on chaperone-assisted proteasome assembly in eukaryotes.

Intracellular Dynamics of the Ubiquitin-Proteasome-System

“…Several classical NLSs exist within the N-termini of distinct α subunits which were proposed to be either accessible rendering the CP in an import-competent conformation, or to be masked rendering the CP in an import-incompatible conformation ( Tanaka et al , 1990). Indeed, recent cryo-EM structure analysis revealed flexible and less structured α ring surfaces in Ump1-associated CP precursor complexes ( Kock et al , 2015), in compliance with our finding that importin α recognizes CP precursor complexes but not mature CP with closed α rings ( Lehmann et al , 2002). Our model upon which CP precursor complexes are imported into the nucleus was supported by the following observations ( Figure 1).…”

“…Our studies in yeast strongly suggest that newly synthesized proteasomes are imported from the cytosol into the nucleus as inactive precursor complexes and that the maturation of nuclear CP proceeds to completion post-import ( Lehmann et al , 2002). Although electron microscopy studies have shown that the NPC could expand to accommodate the longitudinal passage of the 30S proteasome, the permeability barriers towards macromolecules such as CP precursor complexes and RP assembly modules must be overcome by specific importins/karyopherins ( Pante & Kann, 2002).…”

“…Two competing models exist for RP assembly ( Funakoshi et al , 2009; Le Tallec et al , 2009; Park et al , 2009; Roelofs et al , 2009). The first posits that RP assembly occurs in modules independent of the CP with the help of four RP-dedicated chaperones, named Hsm3, Nas2, Nas6 and Rpn14 ( Funakoshi et al , 2009).…”

“…Second, in importin α mutants namely srp1-49 but not in srp1-31 , several groups found that the CP is mislocalized to the cytoplasm, providing another piece of evidence for the classical import pathway of proteasomes. Unprocessed and incompletely processed β5 subunits, crucial determinants of CP precursor complexes and pre-holo-CP, respectively, accumulate in srp1-49 mutants, while precursors of β5 subunits are hardly detectable in wild type cells ( Lehmann et al , 2002). Third, when CP maturation is delayed by UMP1 deletion, all CP reporter proteins accumulate in the nucleus, although half of the CP is not fully matured and most likely exists as pre-holo-CP ( Fehlker et al , 2003; Lehmann et al , 2008).…”

The proteasome: molecular machinery and pathophysiological roles

“…Like the CP assembly pathway, the base assembly is also a highly organized process assisted by four base-dedicated chaperone proteins, including Nas2/p27, Nas6/gankyrin = p28, Rpn14/PAAF1, and Hsm3/S5b (yeast/human: miscellaneous nomenclature) (Funakoshi et al , 2009 ;Kaneko et al , 2009 ;Le Tallec et al , 2009 ;Park et al , 2009 ;Roelofs et al , 2009 ;Saeki et al , 2009 ). Recent studies proposed renaming of Rpn14, Nas6, Nas2, and Hsm3, to RP assembling chaperones (RACs), i.e., RAC1, 2, 3, and 4, respectively (Tomko and Hochstrasser , 2011 ).…”