Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes

Abstract

Eukaryotic 20S proteasomes are composed of two alpha-rings and two beta-rings, which form an alphabetabetaalpha stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1-Dmp2, in budding yeast. Dmp1-Dmp2 directly bound to the alpha5 subunit to facilitate alpha-ring formation. In Deltadmp1 cells, alpha-rings lacking alpha4 and decreased formation of 20S proteasomes were observed. Dmp1-Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3-a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1-Dmp2-alpha5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the beta-rings are assembled.

Identification of minimum Rpn4‐responsive elements in genes related to proteasome functions

“…To confirm that the increased proteasome subunits were properly assembled, cell lysates were separated by glycerol gradient centrifugation, followed by measurement of proteasome activity and Western blot. The proteasome activity was increased in both the 20S CP fractions (fractions [16][17][18][19][20] and the 26S proteasome fractions (fractions 24-30) by Rpn4 overexpression, compared to control cells (Fig. 1B).…”

“…Yeast cells harboring galactose-inducible plasmids were incubated in SGal medium lacking uracil for 2 h. Protein extraction, glycerol gradient centrifugation, and assays for peptidase activity were performed as described previously [20].…”

The proteasome: molecular machinery and pathophysiological roles

“…Currently, experimental techniques have been developed to defi ne the molecular mechanisms underlying the assembly of the CP by identifying a set of proteasome-dedicated assembling chaperones. We identifi ed four proteasome-dedicated chaperones that assist α ring formation named proteasome assembly chaperone (PAC) 1 -4 in mammals and found that these chaperones form a pair of functional heterodimers, PAC1-PAC2 and PAC3-PAC4 (Hirano et al , 2005(Hirano et al , , 2006Yashiroda et al , 2008 ). While the PAC1-PAC2 and PAC3-PAC4 complexes have different roles at different steps, they cooperate with each other in the assembly of α rings.…”

Structural Basis for Specific Recognition of Rpt1p, an ATPase Subunit of 26 S Proteasome, by Proteasome-dedicated Chaperone Hsm3p

“…We previously reported the crystal structures of Pba3-Pba4 complexed with the ␣5 subunit of the CP (15) and Rpn14 (20). Although these studies provided a structural basis for mechanisms underlying proteasome assembly, detailed mechanisms regarding how the RP is formed remain mostly unclear.…”

“…For the 20 S CP assembly, five distinct chaperones are identified as follows: Ump1 (9, 10); PAC1-PAC2 complex (11) (called Pba1-Pba2 complex (12) in yeast) and PAC3-PAC4 complex (13) (Pba3-Pba4 complex in yeast) (14,15). These chaperones help the initiation and progression of the assembly process by transiently associating with proteasome precursors.…”