The 26S proteasome, a molecular machine responsible for regulated protein degradation, consists of a proteolytic core particle (20S CP) associated with 19S regulatory particles (19S RPs) subdivided into base and lid subcomplexes. The assembly of 19S RP base subcomplex is mediated by multiple dedicated chaperones. Among these, Hsm3 is important for normal growth and directly targets the carboxyl-terminal (C-terminal) domain of Rpt1 of the Rpt1-Rpt2-Rpn1 assembly intermediate. Here, we report crystal structures of the yeast Hsm3 chaperone free and bound to the C-terminal domain of Rpt1. Unexpectedly, the structure of the complex suggests that within the Hsm3-Rpt1-Rpt2 module, Hsm3 also contacts Rpt2. We show that in both yeast and mammals, Hsm3 actually directly binds the AAA domain of Rpt2. The Hsm3 C-terminal region involved in this interaction is required in vivo for base assembly, although it is dispensable for binding Rpt1. Although Rpt1 and Rpt2 exhibit weak affinity for each other, Hsm3 unexpectedly acts as an essential matchmaker for the Rpt1-Rpt2-Rpn1 assembly by bridging both Rpt1 and Rpt2. In addition, we provide structural and biochemical evidence on how Hsm3/S5b may regulate the 19S RP association to the 20S CP proteasome. Our data point out the diverse functions of assembly chaperones.AAA ATPase | Arm/HEAT repeats | two-hybrid assay | native gel T he ubiquitin-proteasome system is a major proteolytic system in the cytosol and nucleus of all eukaryotic cells that regulates various essential cellular processes by degrading proteins, which, in most cases, are conjugated to ubiquitin (1-3). The 26S proteasome, the most downstream element of this pathway, is responsible for protein degradation. It comprises the catalytic core particle (20S CP) capped by one or two regulatory particles (19S RPs or PA700 in mammals), forming RP 1 CP and RP 2 CP complexes, respectively (4). The 20S CP encloses the protease active sites (5), whereas the 19S RP functions in substrate recognition, deubiquitination, unfolding, and translocation into the 20S CP and provides the ATP and ubiquitin dependence on the 20S CP (reviewed in 2).The 19S RP can be subdivided into two subcomplexes, namely, the base and the lid. The subunit architecture of the 19S RP has been further improved very recently (6, 7). The base contains six homologous ATPase subunits of the AAA family (referred to as Rpt1-6 in yeast) plus non-ATPase subunits: Rpn1, Rpn2, and Rpn13 (reviewed in 8). By analogy to proteasome-activating ATPase complexes in prokaryotes and archaeas, the 19S RP ATPases are presumed to assemble into a six-membered ring that directly abuts the 20S CP (reviewed in 8, 9). The Rpt1-Rpt5-Rpt4-Rpt3-Rpt6-Rpt2 arrangement within the ring, first proposed based on phylogenetic hypotheses (10), was recently experimentally confirmed (11). Rpt subunits consist of a coiled coil (CC) segment and an OB-fold domain, followed by an AAA ATPase domain (12, 13). The AAA ATPase domain comprises the C domain, which is a four-helix bundle. Protruding from the C...
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