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J. Biol. Chem., Vol. 279, Issue 8, 6401-6413, February 20, 2004

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Purification of the Arabidopsis 26 S Proteasome

BIOCHEMICAL AND MOLECULAR ANALYSES REVEALED THE PRESENCE OF MULTIPLE ISOFORMS^*

Peizhen Yang, Hongyong Fu¶||, Joseph Walker, Charles M. Papa¶, Jan Smalle, Yu-Ming Ju¶, and Richard D. Vierstra**

From the Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706-1574 and the ^¶Institute of Botany, Academia Sinica, 128, Sec 2, Academy Road, Taipei, Taiwan 11529

The 26 S proteasome is a multisubunit protease complex responsible for degrading a wide range of intracelular proteins in eukaryotes, especially those modified with polyubiquitin chains. It is composed of a self-compartmentalized core protease (CP) that houses the peptidase active sites appended on either or both ends by a regulatory particle (RP) that identifies appropriate substrates and translocates them into the lumen of the CP for breakdown. Here, we describe the molecular and biochemical properties of the 26 S proteasome from the plant Arabidopsis thaliana. Like the CP and the ATPase ring of the RP, the RP non-ATPase subunits are often encoded by two transcriptionally active genes with some pairs displaying sufficient sequence divergence to suggest functional differences. Most RPN subunits could functionally replace their yeast counterparts, implying that they have retained their positions and activities within the complex. A method was developed to purify the 26 S proteasome intact from whole Arabidopsis seedlings. These preparations are biochemically indistinguishable from those from yeast and mammals, including the need for ATP to maintain integrity and a strong sensitivity to the inhibitors MG115, MG132, lactacystin, and epoxomicin. Mass spectrometric analysis of the complex detected the presence of almost all CP and RP subunits. In many cases, both products of paralogous genes were detected, demonstrating that each isoform assembles into the mature particle. As with the yeast and animal 26 S proteasomes, attenuation of individual RP genes induces a coordinated up-regulation of many of the other 26 S proteasome genes, suggesting that plants contain a negative feedback mechanism to regulate the 26 S proteasome levels. The incorporation of paralogous subunits into the Arabidopsis holoprotease raises the intriguing possibility that plants synthesize multiple 26 S proteasome types with unique properties and/or target specificities.

Received for publication, October 31, 2003

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY230828 (RPN1a), AY230829 (RPN1b), AY230830, AY239610 (RPN2a, alleles 1 and 2), AY242527 (RPN2b), AY230831 (RPN3a), AY230832 (RPN5a), AY230833 (RPN5b), AY230834-37 (RPN6, alleles 1-4), AY230838 (RPN7), AY230839-40 (RPN8a, alleles 1 and 2), AY258412 (RPN8b), AY230841 (RPN9a), AY230842 (RPN9b), AY230843-45 (RPN11, two allele 1 and one allele 2), AY230846-47 (RPN12a, allele 1-2), AY258413 (RPN12b).

^* This work was supported by United States Department of Energy Grant DE-FG02-88ER13968, National Science Foundation Arabidopsis 2010 Program Grant MCB-0115870, and Research Division of the University of Wisconsin College of Agriculture and Life Sciences Grant Hatch 142-E443 (to R. D. V.) and by Republic of China National Science Council Grants 90-2311-B-001-067, 91-2311-B-001-129, and 91-2317-B-001-018 (to H. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains an additional figure and table.

These two authors contributed equally to this work.

|| To whom correspondence should be addressed: Institute of Botany, Academia Sinica, 128, Sec 2, Academy Rd., Nankang, Taipei, Taiwan 11529, Republic of China. Tel.: 11-886-2-27823050 (ext. 422); Fax: 11-886-2-27827954; E-mail: hongyong{at}gate.sinica.edu.tw. ^** To whom correspondences should be addressed: Dept. of Genetics, 445 Henry Mall, University of Wisconsin, Madison, WI 53706-1574. Tel.: 608-262-8215; Fax: 608-262-2976; E-mail: vierstra{at}wisc.edu.

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