Abstract
The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly.
Highlights
The chaperones Ump[1] and Pba1–Pba[2] promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta[7]
In order to enrich the cellular 15S precursor population and to enable selective affinity purification, we used a yeast strain lacking the C-terminal extension of b7 (b7DCTE)[18] and expressing an N-terminally FLAG-6 Â His (FH)-tagged version of Ump[1]
The b7DCTE mutation does not affect the structure of the 15S intermediate because the b7 subunit is absent from this complex[18]; lack of the CTE causes an accumulation of the 15S precursor complex (PC) because it inhibits 20S core particle (CP) formation by complex dimerization[20]
Summary
The chaperones Ump[1] and Pba1–Pba[2] promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta[7]. The heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening In this way, the Pba1–Pba[2] chaperone is recycled for a new round of proteasome assembly. The two proteins form a stable heterodimer, which was reconstituted and crystallized with mature 20S CP from yeast[25] In this structure, the C-terminal HbYX motif of Pba[1] interacts with the intersubunit pocket between a5 and a6, which can bind to the HbYX motif of Blm[10] or of the 19S RP subunit Rpt[5] (refs 7,26,27). The deletion of Ump[1] can rescue lethality caused by deletions of the pro-peptides of either b5 or b6, pointing to a possible physical interaction between Ump[1] and these pro-peptides[19,16]
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