Purification and functional properties of a Rab8-specific GEF (Rabin3) in action remodeling and polarized transport.

Considering the large number of Rab proteins, only a few Rab-specific exchange factors have been found and characterized. Rab8 is involved in mediating polarized membrane traffic through reorganization of actin and microtubules. It is possible to use the yeast two-hybrid technique to find potential Rab activators. A human protein (Rabin8) and its rat equivalent (Rabin3) were found to bind Rab8 and function as nucleotide exchange factors for Rab8 but not for Rab3A and Rab5. Endogenous and ectopically expressed Rabin8 frequently colocalize with cortical actin. This association is increased by cytochalasin D and phorbol esters that also induced the translocation of both Rabin8 and Rab8 to lamellipodia-like structures. We also show that a GFP-fused Rabin8 behaves identically in this respect. Furthermore, coexpression of Rabin8 with the dominant negative mutant of Rab8 leads to translocation of Rabin8 onto vesicular structures enriched in cell protrusions, indicating that both Rab8 and Rabin8 are involved in mediating polarized membrane transport. This chapter presents a detailed description of the methods and protocols developed to find and characterize a Rab8-specific activator.