Purification of the XXXX 26 S proteasome: biochemical and molecular analyses revealed the presence of multiple isoforms.

@entity375 @entity958 @entity16626 @entity16627 @entity6395 @entity12740 @entity16628
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 @entity12740 ( @entity12740 ) 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 @entity12740 for breakdown. Here, we describe the molecular and biochemical properties of the 26 S proteasome from the plant @entity958 . Like the @entity12740 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 @entity375 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 @entity958 seedlings. These preparations are biochemically indistinguishable from those from @entity375 and mammals, including the need for ATP to maintain integrity and a strong sensitivity to the inhibitors @entity16627 , @entity16626 , @entity16628 , and @entity6395 . Mass spectrometric analysis of the complex detected the presence of almost all @entity12740 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 @entity375 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 @entity958 holoprotease raises the intriguing possibility that plants synthesize multiple 26 S proteasome types with unique properties and/or target specificities.
