Redundancy Between Proteasome Chaperones Nas2 and Hsm3 During Proteasome Assembly

Abstract

Eukaryotic proteasomes consist of a 20S cylindrical core particle (CP) capped with a 19S regulatory particle (RP) on one or both sides. The regulatory particle can be subdivided into base and lid complexes. The base contains a ring of six homologous AAA‐ATPase subunits; Rpt1 to Rpt6. The efficient assembly of the base requires several proteasome‐specific chaperones. Two of these, Nas2 and Hsm3, show redundancy and bind to neighboring Rpt subunits, Rpt5 and Rpt1 respectively. However, Nas2 and Hsm3 are never found on the same sub complex and it is unclear why they are redundant. Here, we show that the reported rescue of Hsm3 deletion strains by over expression of Rpt1 depends on the presence of Nas2. Interestingly, in an Hsm3ΔNas2Δ strain no increased levels of Rpt1 were detected upon overexpression of Rpt1. This suggest that, at least in part, the Nas2 and Hsm3 redundancy depends on the ability of either protein to stabilize Rpt1 in the cell. Consistent with a potential role of Nas2 in Rpt1 stabilization, we observed a direct interaction between Nas2 and Rpt1 in vitro. This binding site mapped to the N‐terminal region of Rpt1. To understand the importance of the domains in Nas2, we studied the effects of over expression of N‐terminal domain or the PDZ domain of Nas2 in hsm3Δnas2Δ strain and observed differences in their ability to rescue the temperature sensitivity of these cells. Biochemical and structural studies have suggested the binding of Nas2 and Hsm3 to proteasome sub complexes is mutually exclusive and suggests a role of Hsm3 in Nas2 expulsion. Consistent with this, we observed the presence of Nas2 on larger assembly intermediates in strains deleted for HSM3. In all, our data suggest Nas2 and Hsm3 show redundancy at the step of Rpt1 incorporation into proteasome complexes and indicate pliability in the assembly pathway.Support or Funding InformationNIH‐NIMGS (1R15GM112142‐01A1)