Arabidopsis DRI2 (DRG interacting protein-2): Analysis of protein-protein interactions, expression patterns and mutant phenotypes

GTP binding proteins (G proteins) are vital to many cellular processes including signal transduction, translation, cellular trafficking, cytoskeleton construction, and nuclear translocation. G proteins act as molecular switches in these processes. There are several types of effector proteins that modify the activity of the G protein. Other interacting proteins are likely to act as upstream or downstream effectors in signal transduction pathways.; The Developmentally Regulated GTP binding protein (DRG) family is highly conserved. Eukaryotes possess two orthologous groups called DRG1 and DRG2. Amino acid identity within an orthologous group is extremely high. For example, DRG1s from plants, mammals, and yeast are 65-70% identical. Amino acid identity for 2 paralogues from a given species is about 57%. Although the specific function of the DRGs remains unknown, the important functions played by other G proteins and their conservation suggest that they play a vital role in cell physiology.; Proteins that interact are often involved in the same pathway and biological function. Global yeast interaction experiments reveal that both DRG1 and DRG2 interact with the same protein, YDR152w. The apparent orthologue of this gene in Arabidopsis is called DRG Interacting protein 2 (DRI2). Analysis of expression patterns, mutagenesis, and protein interaction partners of AtDRI2 may provide clues to the function of an important family of G proteins, the DRGs. Expression patterns of Dri2 were examined by a promoter::gusA fusion construct, Northern blot, and Western blot. Expression pattern analysis revealed similar patterns to those of the Drgs. Western blots on mutant Drg2 and Dri2 lines revealed that when one protein is knocked out, the other protein is no longer present. This could suggest that DRG2 and DRI2 interact with each other within Arabidopsis and could be reciprocally responsible for each others stability. The BacterioMatch Two-Hybrid System and affinity precipitation experiments confirmed that DRI2 and the DRGs interact. The BacterioMatch Two-Hybrid System also was used to screen a cDNA library for other potential interacting proteins within the Arabidopsis genome. Proteins identified as putative interacting proteins with DRG1, DRG2, and DRI2 revealed potential components of a signal transduction pathway, a ubiquitin-proteasome pathway, and the ribosome complex.