Regulation of endocytosis and secretion by Rab GTPase activating proteins

Vesicle traffic in eukaryotic cells is a tightly organized process
involving a multitude of regulatory proteins. Key regulators of
this traffic are small GTPases called Rabs. With about 60 members
in the human genome, they constitute the largest subgroup in the
superfamily of Ras like monomeric GTPases. They recruit effector
proteins to specific membranes and thus define the identity of
organelles. Rabs switch between an active, GTP bound state and an
inactive GDP bound state. Key regulators of this conversion are
RabGAPs, which accelerate the hydrolysis of bound GTP. All RabGAPs
are characterized by the presence of a TBC domain. In the human
genome 40 RabGAPs were identified, most of which had not been
studied so far. To assign them to their specific Rab proteins, a
novel reverse yeast two-hybrid screening method was developed. This
identified a GAP for Rab5 termed RabGAP-5. RabGAP-5 stimulated the
GTPase activity of Rab5. Its expression inactivated Rab5 and
redistributed the Rab5 effector EEA1 from early endosomes to the
cytoplasm. RabGAP-5 also blocked the Rab5 dependent uptake of EGF
and transferrin from the plasma membrane. When RabGAP-5 was
depleted, the size of endosomes was increased, indicating elevated
Rab5-GTP levels. Endocytosed EGF was unable to exit the endosome,
indicating that trafficking through endosomes was also blocked. To
identify GAPs and Rabs implicated in the regulation of early
secretory events simultaneously, a second novel screening method
was established. It involved the analysis of phenotypes caused by
the inactivation of endogenous target Rabs via the overexpression
of RabGAPs. Changes in Golgi morphology, ERGIC organisation and the
proceeding of secretion were only observed with one candidate
RabGAP, the highly conserved protein TBC1D20. TBC1D20 showed
activity towards Rab1 and Rab2 in vitro, and acted primarily on
Rab1 in vivo. In contrast to all other RabGAPs it has a
transmembrane domain, which localises it to the ER. TBC1D20
interacts with RTN-1 on ER membranes. This interaction modulates
the activity of TBC1D20. These data indicate a novel function for
Rab1 in regulating ER exit, and thus extend the classical view of
RabGAPs as regulators of active Rab lifetime.