Functional characterisation of Arabidopsis DRGs : Clues from the DRG2 interactor PDL1

Plume, Andrew Michael (2002). Functional characterisation of Arabidopsis DRGs : Clues from the DRG2 interactor PDL1 PhD Thesis, School of Integrative Biology, The University of Queensland.

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Author Plume, Andrew Michael
Thesis Title Functional characterisation of Arabidopsis DRGs : Clues from the DRG2 interactor PDL1
School, Centre or Institute School of Integrative Biology
Institution The University of Queensland
Publication date 2002
Thesis type PhD Thesis
Supervisor Assoc. Prof. Jimmy Botella
Subjects L
780105 Biological sciences
270300 Microbiology
Abstract/Summary The GTP hydrolases (GTPases) are a ubiquitous superfamily of proteins with diverse cellular roles. Members of the recently-discovered DRG subfamily have been implicated in human disease and may play a role in cell division, differentiation or death. While recent work in our laboratory has focused on the expression and subcellular localisation of DRG1 and DRG2 in the model plant Arabidopsis thaliana (Etheridge et al., 1999; Etheridge, 2002), our understanding of the role of these proteins in planta remains unclear. A yeast two-hybrid (Y2H) library screen using Arabidopsis DRG2 as bait identified 16 interacting proteins, 75% of which are predicted to reside in cellular compartments other than the cytoplasm. Since DRG2 has previously been localised to cytoplasmic vesicles, this observation suggests that DRG2 could be involved in the trafficking of these proteins to their correct cellular locations. A single DRG2 interactor, DRGIP4, was selected for detailed study and its interaction with DRG2 was confirmed in vivo and in vitro. Based on sequence similarity to Pseudomonas PCD/DCoH, DRGIP4 was renamed PDL1 (PCD/DCoH-like protein 1). Members of the widespread PCD/DCoH family are bifunctional proteins which possess catalytic and transcriptional coactivation functions in different cellular compartments and in different oligomeric states. PDL1 is encoded by a single-copy gene in Arabidopsis and shares remarkable secondary and tertiary structural conservation with members of the PCD/DCoH family and is also capable of homomeric associations. PDL1 contains an N-terminal chloroplast transit peptide (cTP) which is functional in planta and is important for interaction with DRG2, suggesting that these proteins interact in the cytoplasm prior to the import of PDL1 into the chloroplast. PDL1 lacks most of the residues important for PCD/DCoH enzymatic activity but may retain a transcriptional coactivation function in the chloroplast. PDL1 homologs in other plants also contain an N-terminal cTP and a PCD/DCoH domain. pdl1 mRNA is expressed at moderate to high levels in the aerial tissues but not in the roots. Accumulation of pdl1 transcripts is light-inducible, and the pdl1 promoter contains several cis-elements which may be responsible for light-responsive transcription. Expression of the intron-GUS reporter gene under the control of the pdl1 promoter generally correlates with mRNA accumulation but reveals tight spatial control of gene expression. Overexpression of pdl1 in transgenic plants does not result in an obvious phenotype. However, downregulation of pdl1 in transgenic plants results in a pale green leaf phenotype associated with a reduction in photosynthetic pigment content and chloroplast numbers per cell. Leaf internal architecture and chloroplast ultrastructure are unaffected in these plants. This phenotype is similar to the arc (accumulation and replication of chloroplasts) mutants and mutants in the chloroplast division and protein import machinery. PDL1 may therefore be involved in the process or regulation of chloroplast division in Arabidopsis. The pdl1 downregulation phenotype is also associated with pleiotropic effects on plant growth. A second PCD/DCoH-like protein, PDL2, was identified in the Arabidopsis genome sequence. Like PDL1, PDL2 shares limited primary sequence similarity to members of the PCD/DCoH family but retains characteristic secondary and tertiary structural features. PDL2 contains an N-terminal mitochondrial transit peptide (mTP) and is expressed at low levels in all plant tissues. PDL2 homologs in other plants also contain an N-terminal mTP and a PCD/DCoH domain. The significance of PDL2 for the function of PDL1 and DRG2 is not yet clear. Overexpression of drg1 in transgenic plants does not result in an obvious phenotype, while downregulation of drg1 in transgenic plants affects general aspects of plant growth which may be unrelated to DRG1 function. To investigate the role of DRGs in a different model system, homologs of drg1 and drg2 were isolated from Saccharomyces cerevisiae (baker’s yeast) and Schizosaccharomyces pombe (fission yeast). Knockout of either or both genes in transgenic yeast is non-lethal and results in sensitivity to agents which disrupt intra- and inter-molecular protein interactions. This phenotype is consistent with a role in protein trafficking. Overexpression of drg1 or drg2 in S. cerevisiae does not result in an obvious phenotype, while overexpression of either gene in S. pombe results in a slow growth phenotype. DRG1 and DRG2 are localised in the cytoplasm in S. pombe. The results presented here suggest that DRGs may be involved in the trafficking of proteins to different subcellular compartments. This research has provided a foundation for the detailed functional characterisation of plant and yeast DRGs and of the novel PDL family in Arabidopsis.

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Created: Fri, 21 Nov 2008, 16:23:55 EST