Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 80

Careful analysis of Rab evolution indicates that the last common eukaryotic ancestor
possessed 12 Rabs that we know to be involved in ER-Golgi trafficking, endocytosis,
endocytic recycling, lysosome-related organelle formation, lipid storage and cilium
function (Elias et al. 2012 ; Klopper et al. 2012 ). The size of the Rab family differs
between species, but generally increases in metazoan lineages and is reduced in some
single cell eukaryotes such as yeasts. Human cells express a subset of 66 Rabs, while
budding yeast express 11 Rab/Ypts. The increased number of Rabs in higher eukary-
otes is thought to reflect the greater number of cellular compartments, and increased
complexity of cell polarisation in metazoans compared to single cell eukaryotes. Like
other Ras superfamily proteins, Rabs switch between two states: an inactive
GDP-bound state and an active GTP-bound state (Wittinghofer and Vetter 2011 ).
This cycle of activation and inactivation is under the control of cellular regulators
promoting GDP-GTP exchange during Rab activation and GTP hydrolysis during
Rab inactivation (Barr and Lambright 2010 ;Barr 2013 ). Accordingly, each Rab has a
cognate GDP-GTP exchange factor (GEF) promoting release of GDP and subsequent
binding of GTP, and a GTPase activating protein (GAP) stimulating the slow intrinsic
GTP hydrolysis. This cycle of activation and inactivation is linked to a cycle of
membrane binding and release. Rabs are prenylated at the C-terminus and this
modification is required for targeting to membrane surfaces (Khosravi-Far
et al. 1991 ; Peter et al. 1992 ). When in the GDP-bound inactive form Rabs rapidly
partition between a cytoplasmic pool bound to a protein termed GDI (guanine
nucleotide dissociation inhibitor) and a membrane-associated pool that is accessible
to GEFs (Soldati et al. 1993 ;Wuetal. 2010 ). GDI interacts with the GDP-bound
form of the Rab nucleotide-binding domain, and also shields the hydrophobic
prenylated tail of the Rab from the aqueous environment of the cytoplasm (Rak
et al. 2003 ). Rabs accumulate at the membrane surface where their cognate GEF is
located following GDP-GTP exchange, because GTP-bound Rabs have greatly
reduced affinity for GDI and therefore do not partition into the cytoplasm (Soldati
et al. 1993 ; Rak et al. 2003 ;Blumeretal. 2013 ). In this chapter I will focus on the
mechanisms by which GEF and GAP regulatory factors act on their target Rabs.
5.2 Rab GEF Families and Mechanism
Rab GEFs initiate a kinetic proofreading system for vesicle and target organelle
membrane surfaces by promoting accumulation of active Rabs only at the required
sites (Barr 2013 ). Rab GEFs and their target Rabs are therefore key determinants of
membrane trafficking. As for many other components of the membrane trafficking
machinery the first candidate Rab GEFs were initially identified by means of
genetic screens in budding yeast. Later biochemical characterisation then matched
the GEFs to their targets Rab GTPases, and cell biological studies have confirmed
these functional relationships (Mizuno-Yamasaki et al. 2012 ; Hutagalung and
Novick 2011 ). As a result the identity of GEFs for the major Rabs controlling
secretory and endocytic trafficking is known (Barr and Lambright 2010 ).