Protein Modification and Turnover
Saracco, Scott , Scalf, Mark A. , Hansson, Maria , Smith, Lloyd , Vierstra, Richard .
Proteomic Analysis of Ubiquitin-Protein Conjugates from Arabidopsis.
Post-translational modification of proteins with ubiquitin (Ub) plays a pervasive role in growth, development, and biotic/abiotic stress protection in eukaryotes. This highly conserved 76-amino acid protein becomes covalently attached via an ATP-dependent conjugation cascade through its C-terminal glycine and free lysyl amino groups in the target. Typically, ubiquitination targets proteins for degradation by the 26S proteasome but other functions are also possible. In plants, Ub plays a key role in numerous processes, including photomorphogenesis, circadian rhythms, floral homeosis, and almost all hormone signaling pathways. Despite this importance, few ubiquitination targets have been conclusively identified in plants, presumably because the ubiquitinated species are present at low levels and are short-lived. To assist in the purification of ubiquitinated proteins from intact plants, we have created Arabidopsis lines expressing 6x-His tagged versions of Ub at levels equivalent to wild-type Ub and that are functional in planta. Using a double affinity purification strategy involving a Ub-binding domain and nickel-chelate columns, we can enrich for Ub-protein conjugates from whole Arabidopsis seedlings. LC/LC-ESI-MS/MS of trypsinized products was then used to identify the ubiquitinated species. By exploiting the Ub-signature footprint, created when Ub moieties are cleaved off conjugates by trypsin, we identified the specific lysine(s) modified by Ub on a number of targets. This MS analysis has generated a library of Ub targets, some of which are now being analyzed further for their physiological importance. This work provides the first proteomic analysis of Ub-protein conjugates from an intact multicellular eukaryote.
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1 - University of Wisconsin - Madison, Genetics, 425 Henry Mall, Madison, WI, 53706, USA
2 - University of Wisconsin - Madison, Chemistry, 1101 University Ave., Madison, WI, 53706, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM