2008 Eighth Annual Beckman Scholars Symposium
Beckman Scholars Presentations
Saturday, July 29, 2006

Rab GTPases play critical roles in vesicular transport in all cells. There are multiple Rab genes and each Rab gene product acts as a zip code or address label for a particular compartment in the cell. Rab GDP dissociation inhibitor (GDI) plays an essential role in returning Rabs from acceptor membranes to donor membranes. Single GDIs interact with multiple Rabs, and thus, GDI may act as a universal regulator of this Rab-recycling process. During a yeast two-hybrid Drosophila cDNA library screen within the Cheney lab using wild-type GDI as the bait, a novel protein coded for by the CG5469 gene was found to interact with GDI. This GDI Interactor 3, Gint3, may act as a GDI Displacement Factor (GDF), causing the release of specific Rabs from GDI at specific membranes. Through mutational analysis of the GDI-Gint3 interaction using various GDI mutants, we can identify the necessary factors for this interaction to occur and begin to understand the role of Gint3 in the Rab-GDI cycle. Our results show that GDI and Gint3 interact in a Liquid ß-Galactosidase Quantitative Assay measuring activation of the lacZ promoter in the MATCHMAKER GAL4 Yeast Two-Hybrid System III relative to a p53-T-antigen positive control interaction. However, the L319, AK307, R240A, H1, and J3C mutant GDI proteins do not interact with Gint3 when standardized against a p53-T-antigen positive control and compared to various negative controls. The results of the interaction of GDI and the GDI mutant proteins with Gint3 suggest a model for the interaction whereby Gint3 interacts with GDI bound to the Rab or through the GDI-bound Rab at the lipid-binding pocket of Domain II of GDI. In order for this interaction to occur, GDI must be in its “on” conformation with a functional lipid-binding pocket able to extract the Rab from the membrane. Most likely, Gint3 acts as a GDF or GDF effector protein in helping GDI release a specific Rab at a specific donor membrane.