Subcellular localization of DRG family proteins
Nelson, Benjamin John
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GTP-binding proteins, or G-proteins, are an ancient family that evolved to carry out many crucial cell functions. The developmentally regulated G-proteins, or DRGs, are a highly conserved family of G-proteins. Two Drg genes, Drg1 and Drg2, are found in all sequenced eukaryotes and one Drg is found in archaea. The fully sequenced Arabidopsis genome contains a third family member, Drg3. DRG proteins are not found in eubacteria, but the closely related Obg proteins are present. Despite high conservation in eukaryotes, little is known about the physiological functions of DRGs. Expression data in Arabidopsis suggests that AtDrg1 and AtDrg2 mRNAs are constitutively present in most tissues while AtDrg3 message is mostly repressed. One condition that upregulates AtDrg3 mRNA is heat stress. Analysis of knockout mutants in AtDrg1 and AtDrg2 indicates they are not essential under normal growth conditions. An important aspect of any protein's function is its location within a cell and whether this location changes in response to a changing environment. A substantial amount of research on the highly related Obgs indicates that they are ribosome associated and interact with stress response factors. Previously, the ability of DRGs to bind GTP nucleotides had been shown only for two animal DRG1 proteins. Here, GTP binding properties of plant DRG1 was confirmed and, for the first time in any system, this physical property was verified for DRG2. In this study multiple biochemical assays were used to demonstrate that plant DRG1 and DRG2 associate with ribosomes. In vivo localization of GFP:DRG fusion proteins in transgenic Arabidopsis was achieved using confocal microscopy. Under normal growth conditions, both DRG1 and DRG2 localize to the cytoplasm, and some DRG1 occurs in nuclei. Under heat stress conditions, in vivo GFP localization indicated that DRG1 and DRG2 participate in "stress granule" formation. This function was confirmed in biochemical assays using pea tissue. The stress granule response is thought to preserve specific mRNAs and 48S pre-initiation complexes during stress conditions and is nearly universal in eukaryotic cells. DRG participation in this crucial stress response mechanism provides an explanation for their ubiquity and high level of evolutionary conservation.