Prenylation of RabGTPases, Their Delivery to Membranes, and Rab Recycling - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 6

again to allow delivery to the correct target membrane, unless there is a generic
RabGAP at the ER and Golgi that acts on them, for which there is no evidence.
After prenylation of Rab proteins in the complex between REP, Rab, and
RabGGTase, a further incoming lipid substrate molecule (GGPP) is able to disso-
ciate the RabGGTase and leave the Rab protein as a soluble complex with REP
(Thoma et al. 2001b )(Fig. 1.1 ). This high-affinity complex [ K d in the nM range (Wu
et al. 2007 )] has to be disrupted to allow Rabs to be attached to membranes. A similar
situation arises with recycling of Rabs by GDI, which has similar properties to REP in
terms of binding and solubilizing prenylated Rab proteins. In both cases, there is a
stable interaction that has to be overcome to allow insertion of the prenyl groups into
the membrane.
It has been suggested that the attachment of Rabs to membranes is catalyzed by a
GDF (GDI displacement factor) that is able to disrupt the stable GDI:Rab com-
plexes (Dirac-Svejstrup et al. 1997 ). A molecule that appears to have the desired
properties is Pra-1 (Yip3 in yeast), an intrinsic membrane protein reportedly
localized in the late Golgi and early endosomes with activity towards Rab9 and
other endosome-associated Rabs (Rab5, Rab7) (Sivars et al. 2003 ). Although there
is no convincing evidence for their mode of action, other members of the Yip family
are Rab interacting proteins thought to be candidate GDFs. However, recent
evidence has been presented indicating that the activities of these proteins are not
required for correct localization of yeast Rabs (Cabrera and Ungermann 2013 ).
The lack of definitive evidence on the existence and properties of GDFs for Rabs
was a reason for the enthusiastic reception of publications suggesting that one of the
over 250 proteins injected by Legionella pneumophila into infected cells is a bona
fide GDF as well as being a GEF towards host cell Rab1 (Ingmundson et al. 2007 ;
Machner and Isberg 2007 ). While the GEF properties of DrrA/SidM have been
amply confirmed and extensively characterized, it was also shown that the apparent
GDF activity of this protein was actually a product of its GEF activity (Schoebel
et al. 2009 ), and in fact all Rab GEFs will have this apparent activity towards their
cognate Rabs. The source of this effect is the preference of GDI for Rab:GDP rather
than for Rab:GTP. It was shown that replacement of GDP by GTP in prenylated
Rabs leads to a loss of affinity of about three orders of magnitude towards both GDI
and REP (Wu et al. 2010 ). Because of this, Rab:GDP that dissociates from its
complex with GDI or REP spontaneously will undergo nucleotide exchange in the
presence of a cognate GEF, thus preventing rebinding to GDI and possibly allowing
membrane insertion. These considerations lead to the notion that the presence of a
GEF at a specific membrane location might lead to trapping of its activated cognate
Rab at the same location (Schoebel et al. 2009 ; Wu et al. 2010 ).
Since molecules with genuine GDF activity towards Rab:GDI complexes have
so far proven elusive, with the exception of Yip3, the question of their necessity for
the process arises. Since Rab:GDI and in particular Rab:REP complexes dissociate
slowly, it appears that acceleration over the rate seen in vitro is likely to be required
in vivo. One possibility that has not yet been considered is the role of membranes in
this process, except in the very general sense that interaction with a membrane will
make a thermodynamic contribution to the process by providing a trap, whose