Protein Modification and Turnover
Yang, XiaoYuan , Chen, WenPing , Gray, William M. , Cohen, Jerry D. .
Development of stable isotope labeling methods for measuring protein turnover rates in plants.
Plants respond to changing environmental and intracellular signals rapidly, and such regulatory responses are critical to their normal growth and development. The goal of our work is to develop methods to measure dynamic protein turnover rates in plants using whole plant isotopic labeling strategies. Such methods should provide robust information on the actual turnover rates under specific conditions and at different developmental stages.
In our initial studies we determined the labeling efficiency of amino acid pools in Arabidopsis. Seedlings were labeled with 0, 20, 30, or 40atom% D2O or 99+atom% 15N salts for different durations on agar plates. Free amino acids were isolated and changes in mass isotopomer distribution (MID) of selected amino acids were monitored by GC-MS. We found that using 30% D2O the amino acids MID could reach the asymptotic value in 5 days and the turnover rates of Ala, Asp and Glu were very fast (< 3 hours and for Ala ~0.6 h). Leu and Phe had longer half lives, in some cases >10 h. Using 15N, however, the apparent half lives were much longer than that measured using D2O, likely due to 15N-recycling. In D2O labeled seedlings the abundance of Ala increased markedly above that of the control seedlings and the increase was greater as the percentage of D2O was increased. Current work centers on development of methods 1) to optimize the the rapid exchange of 15N from the plant nitrogen pools, 2) determine the utility of both 15N and D2O for labeling proteins with different in vivo rates of turnover, 3) to further understand the consequences of high level stable isotope enrichment by physiological and microarray analyses.
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1 - University of Minnesota, Department of Plant Biology
2 - University of Minnesota, Department of Horticultural Science
3 - University of Minnesota, Department of Horticultural Science, 1970 Folwell Avenue 305 Alderman Hall, St.paul, MN, 55108, USA
mass isotopomer distribution.
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM