The long-term goal of this research is to identify the molecular machinery that regulates vesicular transport during development, especially during localization of informational macromolecules and secretion during morphogenesis. Vesicular transport is a fundamental cellular process needed for life. While many of the molecular components of vesicular transport are known, there are many gaps in our knowledge. Major controllers of vesicular transport are rab GTPases, which are regulated by rab GDP dissociation inhibitor (GDI). GDI binds avidly to rabs, but the trigger for GDI to release rabs is unknown and presumably involves interaction with different proteins. Recently, an inherited form of mental retardation in humans was shown to be caused by mutations in a gene for rab GDI, underscoring the potentially pivotal role that GDI could play in regulating vesicular transport.
The hypothesis that underlies this research is that the interaction between rab and GDI is regulated by proteins that interact with GDI. The overall goal is to identify these proteins and their role in GDI-rab interactions, using biochemical, molecular genetic and genetic techniques, using mutations in Drosophila rab GDI. This project has the following specific aims:
1. Identify proteins that bind directly to GDI, using co-immunoprecipitation with an anti-GDI antibody, binding to a GDI affinity column, and binding to GDI in a yeast two-hybrid system.
2. Identify genes encoding proteins that interact with GDI, by using gene interaction screens. One screen will select for dominant enhancers and suppressors of the Drosophila GDI mild alleles. The other screen will select for dominant enhancers of the Drosophila GDI null allele.
3. Isolation of other secretion mutants in Drosophila, using a pupal cuticle screen. Analysis of the GDI mutant phenotype indicates that secretion of the pupal cuticle is especially sensitive to defects in vesicular transport. This screen will isolate mutants with defective pupal cuticles.
