Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP binding proteins found in archaea, plants, fungi and animals. Their function is poorly understood but they are implicated in cell division, proliferation, and growth, as well as several human medical conditions. The research reported here has utilised a variety of approaches including structural biology, biochemistry, expression profiling, and mutant analysis in order to investigate the cellular function of DRG proteins in plants. Recombinant, biologically active atDRG1 and atDRG2 protein from Arabidopsis thaliana was purified using in vitro refolding and was used in both structural studies and biochemical analysis. Crystallographic studies were carried out for both atDRG1 and atDRG2 across 3840 unique, independent crystallisation conditions for each protein. Heterogeneous nucleation was also used in a separate crystallography screen in order to induce nucleation and subsequent crystal growth however no diffraction quality protein crystal were produced in this study. The nucleotide binding and hydrolysis properties of recombinant atDRG1 and atDRG2 were measured in vitro, representing the first biochemical characterisation of DRG proteins. Both atDRG1 and atDRG2 were found to bind GDP and GTP in vitro without the assistance of exogenous exchange or activation factors. The Kcat for GTP hydrolysis by atDRG1 and atDRG2 was found to be 7.44 x 10-4 min-1 and 1.18 x 10-3 min-1 respectively which is consistent with proteins related to the DRG subfamily. An Arabidopsis thaliana atDRG2a knockout mutant was identified and characterised in this study as well representing the first DRG knockout mutant in a multicellular organism. We found that complete knockout of atDRG2a is not lethal in Arabidopsis and that the nearly identical atDRG2b protein is not upregulated in response to an absence of atDRG2a in the cell. The mutant did not display an obvious phenotype compared to wild-type. The expression profiles of the three Arabidopsis thaliana drg genes, drg1, drg2a, and drg2b, were characterised using drg promoter:GUS Arabidopsis transgenics and revealed several interesting features. Under normal conditions, drg1 and drg2a transcripts are present in all cells whilst drg2b transcripts are undetectable. When heat stress is applied, drg2b and drg1 are specifically up regulated and drg2a is not. During seed imbibition, drg2a and drg1 are specifically upregulated whilst drg2b is not. The expression pattern of the drg family closely mirrors that of chaperone/heat shock proteins and this would agree with previous research that suggests that DRG2a may perform a chaperone role. The ability of DRGs to bind nucleotides without assistance, their slow rate of GTP hydrolysis, heat stress activation, abundance in seeds, cytosolic localization, and domain conservation, all agree with the models proposed for spoOB associated G-protein (Obg) function, whereby Obgs stabilise or refold ribosomes or other proteins in response to stress. It is possible that DRGs perform a similar and complementary function to Obgs, specifically during heat stress, despite the low level of sequence conservation between Obgs and DRGs.
| Identifer | oai:union.ndltd.org:ADTP/253963 |
| Creators | Anthony O'Connell |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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