Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 90

structural biology and highly sensitive sequence alignment tools based on hidden
Markov modelling show that a number of Rab GEFs are united by longin/roadblock
domains (Yoshimura et al. 2010 ; Wu et al. 2011 ; Levine et al. 2013 ). TRAPP,
DENN family GEFs and the HerMon GEFs for Rab7-related GTPases all contain
longin domains. These domains may therefore act as general platforms for GTPase
regulation by promoting conformational change in the switch I and II regions,
which represents a common theme in Rab activation. The longin/roadblock plat-
form has also been found in other GTPase regulatory systems involving vesicle coat
protein complexes, the ER signal sequence receptor, and a bacterial cell polarity
MglA/MglB (Sun et al. 2007 ; Levine et al. 2013 ; Miertzschke et al. 2011 ). There-
fore these domains may represent an ancient regulatory module used to control
diverse GTPases and not only Rabs.
5.3 Rab GAPs: The TBC Domain Proteins
TBC1 (Tre-2/Cdc16/Bub2) domain GTPase activating proteins (GAPs) were orig-
inally identified in genetic screens for modulators of yeast Rab/Ypt function (Strom
et al. 1993 ; Du et al. 1998 ; Albert et al. 1999 ; Vollmer et al. 1999 ; Albert and
Gallwitz 2000 ; Eitzen et al. 2000 ). Because of this they are referred to as GAPs for
Ypts (Gyp) in yeasts. In higher eukaryotes, they are typically referred to as TBC1
domain proteins. Analysis of the evolutionary history of Rabs and Rab GAPs has
failed to create a clear picture of their relationship (Gabernet-Castello et al. 2013 ).
This type of investigation has suggested that there is an ancient complement of ten
TBC domain proteins, yet matching this to the cognate Rabs has proven difficult.
This difficulty may be in part due to the presence of other domains in addition to the
Rab GAP activity encoding TBC domain. Because of this it is important to
biochemically define the target specificity of each Rab GAP using purified proteins.
Expression of Rab GAPs has been relatively widely used as a tool to inactivate Rabs
in cellular trafficking pathways (Hsu et al. 2010 ; Yoshimura et al. 2010 ; Fuchs
et al. 2007 ; Longatti et al. 2012 ). Once a specific GAP or set of GAPs blocking a
transport pathway is known, then it is relatively simple to identify the target Rabs
using biochemical assays for GTP hydrolysis. This methodology has been used to
identify Rabs involved in endocytic and secretory trafficking, as well as cell
polarisation during cilium formation. Caution is required when interpreting such
screens, since in some cases the effect of the GAP may not always correlate with
catalytic activity (Haas et al. 2007 ; Longatti et al. 2012 ). A counterscreen with a
mutated catalytically inactive Rab GAP can be used to test for this. One complica-
tion that has arisen from such approaches is the promiscuous nature of some TBC
domain proteins in terms of their Rab target specificity.