| Abstract Detail
Protein Modification and Turnover Book, Adam [1], Holmes, James [2], Lee, Sang Sook [2], Hansson, Maria [2], Vierstra, Richard [2]. Compositional Analysis of the Arabidopsis 26S Proteasome. Plants rely on protein degradation for a variety of essential functions, including the maintenance of free amino acid pools, the removal of damaged proteins, and the control of regulatory protein abundance. The major machine for this breakdown is the 26S proteasome, an ATP-dependent, 2-MDa proteolytic complex. The 26S proteasome is regulated by various mechanisms, including the use of alternative subunit isoforms, capping with different regulatory complexes, association with accessory proteins, and/or various post-translational modifications such as phosphorylation and the addition of N-acetyl glucosamine. Several potential accessory proteins have been identified in Arabidopsis thaliana, including the PA200 regulatory complex, the scaffolding protein ECM29, the ubiquitin-specific protease UBP6, and the HECT E3s UPL6 and 7. Preliminary studies using a phospho-protein specific stain on purified 26S proteasomes has identified several potentially phosphorylated subunits. To help further define this heterogeneity we have begun to characterize the Arabidopsis complex by biochemical and mass spectrometric (MS) methods, using either proteasomes purified conventionally or enriched via a rapid affinity approach. The affinity approach exploits tagged versions of various proteasome subunits that can be used to rapidly enrich for the complex. The tagged subunits are expressed in mutant backgrounds missing the corresponding wild-type subunit to encourage high incorporation of the complementing subunit. We will discuss the success of this approach and our initial MS/MS analysis of conventionally purified 26S proteasomes for protein composition and various post-translational modifications.
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1 - University of Wisconsin - Madison, Genetics, 425 Henry Mall, Madison, WI, 53706, USA
2 - University of Wisconsin - Madison, Genetics
Keywords:
26S Proteasome
Affinity Purification.
Presentation Type: Plant Biology Abstract
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P37017
Abstract ID:1873 |
