Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 83

5.2.1 Vps9 Family GEFs for the Rab5/21 Subfamily
Rab5 subfamily GTPases, including Rab5A-C, Rab17, Rab21 and Rab22A/B,
regulate trafficking in the early endocytic pathway. Vps9/Rabex the founder mem-
ber of the Rab5 GEF family was identified through biochemical characterization of
mammalian Rab5 and budding yeast screens for defects in vacuolar protein sorting
(Vps) (Burd et al. 1996 ; Hama et al. 1999 ; Horiuchi et al. 1997 ). A family of GEF
proteins with the Vps9 domain exists in higher eukaryotes, and humans (Carney
et al. 2006 ). The best characterised is the mammalian Vps9 orthologue Rabex
which is thought to be a major activator of Rab5 during trafficking into early
endosomes from the plasma membrane (Horiuchi et al. 1997 ). Screens in
C. elegans have shown that a second Vps9 domain GEF receptor-mediated endo-
cytosis defective 6 (RME-6) is also involved in Rab5 regulation in the clathrin-
mediated endocytic pathway, and this has been confirmed in mammalian cell lines
(Sato et al. 2005 ; Semerdjieva et al. 2008 ). Interestingly, Vps9 domain GEFs can
act on multiple although not necessarily all Rab5 subfamily members. Rabex
activates Rab5 and Rab21 with equal efficiency, k cat / K m 2.3 and 3.2 10 4 M 1 s 1 ,
but shows two orders of magnitude lesser activity towards Rab22 (Delprato
et al. 2004 ). While there is no crystal structure for Rab5 in complex with a Vps9
domain GEF, a high-resolution X-ray crystal structure of a complex of the closely
related GTPase Rab21 together with the Vps9 domain of Rabex has been solved
(Delprato and Lambright 2007 ). This structure shows that a conserved aspartate
finger residue in the Vps9 domain is inserted into the Mg 2+ and phosphate-binding
site where it contacts the P-loop lysine. The Vps9 domain engages the Rab switch
regions stabilising an open conformation for switch I. More details of the exchange
mechanism have come from the Arabidopsis thaliana Rab5 family GTPase ARA7
structure in both nucleotide-free and nucleotide-bound forms in complex with its
Vps9 domain exchange factor (Uejima et al. 2010 ). These structures indicate that
GEF interaction and insertion of the aspartate finger promote movement of the
P-loop lysine away from the beta-phosphate of GDP and towards the conserved
aspartate residue at the base of switch II. This would lead to release of the
magnesium ion. It is proposed that these changes result in deprotonation of the
beta-phosphate of GDP and destabilise GDP binding due to repulsion between this
oxygen anions and the aspartate finger (Uejima et al. 2010 ). Once magnesium and
GDP have left the binding pocket the aspartate finger of the Vps9 GEF also contacts
the P-loop lysine and stabilises the nucleotide-free form of the Rab-GEF complex.
Other evidence supporting this picture comes from analysis of the human Rab5-
Rabex GEF system. Mutation of the conserved switch II aspartate leads to a form of
Rab5 refractory to Rabex-stimulated GDP release (Langemeyer et al. 2014 ). Expul-
sion of the magnesium and GDP as a consequence of GEF binding inevitably results
in a loss of defined switch I structure and movement to a more open position. Vps9
domain GEFs may therefore promote nucleotide exchange primarily though inter-
actions with the P-loop lysine and switch II rather than promoting rearrangements
in switch I.