Purification of the Arabidopsis 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms.
The 26 S proteasome is a multisubunit protease complex responsible for degrading a wide range of intracellular 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.
Yang
Peizhen
P
Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706-1574, USA.
Fu
Hongyong
H
Walker
Joseph
J
Papa
Charles M
CM
Smalle
Jan
J
Ju
Yu-Ming
YM
Vierstra
Richard D
RD
D000251:Adenosine Triphosphatases / Q000737:chemistry; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003546:Cysteine Endopeptidases; D030541:Databases, Genetic; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D015151:Immunoblotting; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D046988:Proteasome Endopeptidase Complex; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases