Proteomic Analysis of Affinity-Purified Proteasomes Identifies a Suite of Assembly Chaperones in Arabidopsis

ABSTRACT: The 26S proteasome is an essential protease for the selective elimination of dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this multi-catalytic, ATP-dependent proteolytic machine in plants, we exploited Arabidopsis particles tagged at either the core protease (CP) or regulatory particle (RP) sub-complexes for rapid affinity purification followed by deep sequence analysis via mass spectrometry. Studies on proteasomes purified from whole seedlings via either a CP or RP subunit with or without ATP, which is needed to maintain the holo-proteasome complex, identified all known core subunits but failed to detect subunit isoform preferences, suggesting that Arabidopsis does not construct unique proteasomes sub-types. We also identified a suite of proteasome-interacting proteins, including likely orthologs of the yeast and mammalian chaperones Pba1, Pba2, Pba3, and Pba4 that assist in CP assembly; Ump1 that helps connect CP half-barrels; Nas2, Nas6, and Hsm3 that assist in RP assembly; and Ecm29 that promotes CP-RP association. Analysis of proteasomes enriched from seedlings exposed to the proteasome inhibitor MG132 revealed the accumulation of novel assembly intermediates, which reflect partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Unlike in yeast, genetic analyses of Arabidopsis UMP1 showed that this chaperone is essential, with mutants missing the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single mutants impacting ump1a also accumulate a collection of proteasome assembly intermediates, consistent with its importance for CP construction. With this list of chaperones, it should now be possible to study the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more core subunits.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

SUBMITTER: David C Gemperline  

LAB HEAD: Richard D Vierstra

PROVIDER: PXD014610 | Pride | 2019-09-25

REPOSITORIES: Pride