Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in Arabidopsis .

Autor:	Gemperline DC; Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706., Marshall RS; Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706.; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130., Lee KH; Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706., Zhao Q; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130., Hu W; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130., McLoughlin F; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130., Scalf M; Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706., Smith LM; Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706., Vierstra RD; Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706 rdvierstra@wustl.edu.; Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2019 Nov 15; Vol. 294 (46), pp. 17570-17592. Date of Electronic Publication: 2019 Sep 27.
DOI:	10.1074/jbc.RA119.010219
Abstrakt:	The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types. We also detected 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. Proteasomes from seedlings exposed to the proteasome inhibitor MG132 accumulated assembly intermediates, reflecting partially built proteasome sub-complexes associated with assembly chaperones, and the CP capped with the PA200/Blm10 regulator. Genetic analyses of Arabidopsis UMP1 revealed that, unlike in yeast, this chaperone is essential, with mutants lacking the major UMP1a and UMP1b isoforms displaying a strong gametophytic defect. Single ump1 mutants were hypersensitive to conditions that induce proteotoxic, salt and osmotic stress, and also accumulated several proteasome assembly intermediates, consistent with its importance for CP construction. Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits. (© 2019 Gemperline et al.)
Databáze:	MEDLINE
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