| Small G proteins of the Ras superfamily were reported as major regulators of various cellular functions and are known to play a role in numerous human pathologies, such as cancer. They are currently classified according to their amino acid sequence into five subfamilies (Ras, Rho, Rab, Arf and Ran). In order to systematically study their functions and redundancy, we have selected C. elegans as an experimental model. This organism was chosen since it provides a reliable simple model for the study of small G proteins: In addition to providing experimental advantages over other higher eukaryotes, C. elegans expresses well conserved small G proteins, in limited number. We used this model to perform an integrative analysis of the biochemical activities of small G proteins and to study the role of these enzymes in the maintenance of the endoplasmic reticulum homeostasis.;Another biochemical characteristic studied in this project was the role of small G proteins in the regulation of the endoplasmic reticulum (ER) homeostasis. We have used a GFP-based reporter assay in living worms to test the effect of small G proteins silencing on the induction of the Unfolded Protein Response (UPR). We have established the importance of CRP-1 for the transcriptional activation of genes required for the UPR activation and have showed that, through its interaction with the AAA ATPase CDC-48 (VCP/p97/CDC-48) and the kinase ATM-1, CRP-1 is essential for the worm to develop normally under stressful conditions.;We have first constructed a library of vectors containing 53 C. elegans GTPase genes cloned in various expression vectors. This strategy appeared very efficient since the success rate of our cloning approach was above 89%. Using two high throughput technologies, we have then developed assays to measure enzymatic activities (GTP affinity, exchange and hydrolysis activities) of small G proteins. Integration and analysis of these enzymatic results revealed that the amino acid sequence clustering only partially represents the enzymatic characteristics of small G proteins. This suggests that a biochemical classification would be more representative of small G protein function similarities than their current amino acid sequence-based classification. To develop on this idea, we have generated and collected data related to RNAi phenotypes, cellular and tissue localizations and mRNA co-expression to integrate various functional properties and proposed a more complete clustering of the Rho subfamily of proteins. |
