Nelson, Benjamin .
Ribosome association and GTP binding properties of pea and Arabidopsis DRG proteins.
Eukaryotes contain two DRGs (DRG1 and DRG2), whereas bacteria contain related GTP binding proteins called OBGs. Amino acid identity of eukaryotic DRG proteins is 65-70%, and the level of identity of DRG1 and DRG2 paralogs from a single species is about 55%. Despite this level of conservation, little is known about DRG function. OBGs mediate responses to environmental stress, and they physically associate with ribosomes. DRGs also may regulate translation. Two goals of our work are: 1) to determine whether pea and Arabidopsis DRGs bind to ribosomes; and 2) to test whether plant DRGs can bind GTP. To address the first question, cell homogenates (mostly from pea root apices) were fractionated by differential centrifugation, by rate-zonal centrifugation, and by additional steps to purify free and membrane-bound monosomes and polysomes. DRG1 antibodies recognize a 43kd band, and DRG2 antibodies recognize 45, 43 and 30kd bands (the latter are breakdown products of 45kd). DRG1 and the 45 and 43kd DRG2 bands are found in the cytosol and associated with free monosomes. In contrast, DRG2-30kd is enriched in free and membrane bound polysomes. Nuclear fractions contain some DRG1 but little or no DRG2. Corroborating results were obtained using an Arabidopsis line transformed with FLAG-tagged rpL18, which allowed ribosomes and associated proteins to be co-purified (courtesy of J. Bailey-Serres, UC Riverside). Using a GTP-bound affinity resin, we found pea DRG1 and the 45 and 43kd forms of DRG2 can bind to GTP, whereas the 30kd form of DRG2 does not bind. Pre-incubation of tissue extracts with 10mM GTP greatly reduced binding, but GDP or ATP had no effect. Ongoing experiments are testing the effects of environmental stresses on subcellular localization of DRG proteins.
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1 - Northern Illinois University, Biological Sciences, Montgomery Hall, Dekalb, IL, 60115, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM