Dimerization of small GTPase Rab5

Rab proteins are small GTPases, localized to distinct cellular compartments , regulating specific steps of intracellular membrane trafficking. One memb er of the Rab family, Rab5, consists of three isoforms. Rab5a, Rab5b, and R ab5c, which have been shown to play an important role in early events of en docytosis. Using the yeast two-hybrid system, we have identified several cy tosolic proteins that interact with the Rab5b Q79L (decreasing intrinsic an d GTPase-activating protein-stimulated GTPase activities). Surprisingly, mo st positive clones were Rab5b or Rab5c, indicating that Rab5 could dimerize among extra-isoforms in the yeast two-hybrid system. In vitro and in vivo chemical cross-linking assays demonstrated that lipid-unmodified wild-type Rab5b purified from Escherichia coli, wild-type Rab5b, or a dominant active form Rab5b Q79L expressed in human 293T cells dimerized. Furthermore, the same assays using a Rab5b R81A substitution mutant showed that the Arg(81) in the Switch II region [the second GTP/GDP binding motif (residues 74-93)] was essential for Rab5b dimerization. These results suggest that Rab5 isof orms can be dimerized depending upon the GTP-bound conformation., but indep endent upon a lipid modification.