From the
Rab GDP dissociation inhibitors (GDIs)
Rab
proteins represent a family of at least 30 different Ras-like GTPases
that function in the processes by which membrane vesicles identify
and/or fuse with their targets (see (1, 2, 3, 4) for review). Almost
every membrane-bound organelle in the secretory and endocytic pathways
bears a distinct set of Rabs on its surface. Rabs are doubly
geranylgeranylated at or near their carboxyl termini, which leads to
their membrane association. The specificity of Rab localization is
provided by structural determinants unique to each family
member(5, 6, 7, 8) that appear to
be recognized by distinct sets of proteins on organelle
surfaces(9, 10) .
Like Ras, Rab proteins
interconvert between two conformations. Active Rabs have GTP bound; GTP
hydrolysis converts the protein into its inactive, GDP-bound
conformation. Thus, transport vesicles bear Rab proteins with bound
GTP; concomitant with or after membrane fusion, Rabs are converted into
their GDP-bound states. In this manner, target membranes acquire
vesicle-derived Rab proteins in their GDP-bound conformations.
In
pulse-chase experiments, Novick and co-workers (11) showed that
Rab proteins cycle from the plasma membrane back to secretory granules
via a cytosolic intermediate. Indeed, at steady state, the bulk of a
given Rab is membrane-associated; however, between 10 and 50% can be
detected in the cytosol. Given that Rabs are stably prenylated, it was
not initially clear how membrane dissociation might be accomplished. In
1990, Takai and co-workers (12) reported the discovery and
purification of a protein from bovine brain cytosol that blocked the
dissociation of [
Araki et al.(14) discovered that bovine
brain GDI could displace Rab3A from membranes in its GDP- but not
GTP-bound conformation. Membrane displacement resulted in the formation
of soluble, equimolar complexes of Rab3A bound to GDI. As first
predicted by Matsui et al.(13) , it is now clear that
bovine brain GDI can interact with a variety of Rab proteins including
yeast Sec4 and can also release essentially all Rab proteins from
membranes, in the presence of GDP but not
GTP(15, 16, 17, 18, 19, 20, 21, 22) .
The ability of GDI to distinguish GTP- and GDP-bound conformations of
Rabs provides GDI with the capacity to retrieve ``spent'' Rab
proteins from target membranes into the cytosol, allowing their return
to the correct organelle for subsequent rounds of intracellular
transport.
Northern blot analyses indicated that GDI was expressed in a
variety of rat tissues(13) . Two RNAs were detected in rat
brain (3.1 and 2.3 kilobases); the 2.3-kilobase RNA was also detected
in lung, thymus, heart, liver, spleen, kidney, and small intestine.
The existence of two Rab-GDI mRNAs indicated that there may be more
than one GDI isozyme. Nishimura etal. (23) have now shown that rat brain expresses two GDI isoform
RNAs (
Whereas mouse GDI-1 and GDI-2 are 90 and 72%
identical in deduced amino acid sequence to bovine brain GDI-
Despite the existence of
multiple isoforms of GDI, functional differences have not yet been
detected. GDI-
Using partial proteolysis, Araki et al.(27) generated carboxyl-terminally truncated Rab3A protein
and showed that the carboxyl terminus was essential for Rab3A
interaction with GDI and with membranes. Carboxyl-terminal prenylation
was critical for GDI interaction, because GDI did not alter the rate of
GDP dissociation from bacterially expressed Rab3A nor did these
proteins form a complex (27) . However, carboxyl methylation of
the prenylated protein was not required(28, 29) . An
intact carboxyl terminus and geranylgeranylation was also required for
Rab9-GDI interaction(18) .
Monogeranylated Rab proteins also
complex with GDI(18, 19, 29) , although
perhaps to a lesser extent than diprenylated Rab proteins (cf. (29) ). Palmitoylation of one cysteine, followed by
farnesylation or geranylgeranylation of the second cysteine, led to a
similar decrease in the ability of Rab6 to interact with
GDI(29) .
In addition to prenylation, GDI interaction seems
to involve structural determinants of Rab proteins (cf. (29) ). GDI interacts only with Rab proteins and not with other
doubly geranylgeranylated constituents(17) . GDI also binds
preferentially to Rabs in their GDP-bound conformations. This suggests
that GDI may recognize structural determinants thought to undergo a
nucleotide-dependent conformational change, such as the effector
domain. The importance of Rab structural determinants is underscored by
the unexpected observation that Sec4p, the yeast secretory vesicle Rab,
interacts with yeast GDI even in the absence of
prenylation(20) .
If GDI serves to recycle Rab proteins after each round of
intracellular transport, mutations in GDI would be expected to
interfere with a variety of intracellular transport events. Garrett et al.(26) have now shown that depletion of yeast GDI
using a regulated promoter led to multiple defects in protein transport
and that the GDI1 gene is in fact allelic with an already
identified pleiotropic secretory mutant gene, sec19. Moreover,
depletion of GDI led to the concomitant depletion of the cytoplasmic
pool of Sec4p. These data strongly support a Rab retrieval role for GDI
in living cells. When Rab proteins accumulate at their fusion targets
in their GDP conformations, subsequent transport events cannot take
place.
The ability of GDI to retrieve Rab proteins from membranes
and the existence of a cytosolic pool of Rab proteins bound to GDI
suggested that GDI functions to present them to the transport machinery
located on distinct organelles. This hypothesis was confirmed by the
observation that purified GDI-
Rab proteins that are competent for
membrane recruitment in vitro are functional in these systems
in that they have been shown to stimulate endosome fusion (10) or receptor transport(9) . Direct proof that GDI
presents functional Rab proteins to specific organelle membranes comes
from immunodepletion experiments in which removal of GDI and Rab
proteins bound to GDI led to complete loss of the ability of cytosol to
stimulate intracellular transport of proteins between late endosomes
and the trans-Golgi network in vitro(30) . Rab1B-GDI
complexes were also required for the transport of proteins from the
endoplasmic reticulum and through the Golgi stack(31) .
GDI
increases the efficiency with which Rab proteins can be utilized by the
transport machinery by increasing the selectivity with which they are
delivered to their correct membrane targets(30) . This was
shown in experiments designed to test whether GDI served only a
solubilizing function for prenylated Rab proteins. Delipidated serum
albumin could solubilize prenylated Rab9 and deliver it to membranes in
a functional form. However, unlike Rab9 delivered by GDI, Rab9
delivered by serum albumin complexes associated nonspecifically with
membrane fractions. By binding very tightly to Rab9 (K
Fig. 1summarizes our current view of GDI function.
Unoccupied GDI retrieves GDP-bearing Rab proteins from target membranes
after a vesicular transport event (Step 1). Prenylated Rab-GDI
complexes are then recognized by target membrane-specific proteins.
Recent experiments have shown that Rab proteins are recruited with high
selectivity (9, 10) by a process that displays an
apparent K
Figure 1:
A model for GDI function. Step 1, unoccupied, cytosolic GDI displaces prenylated, GDP-bearing
Rab proteins from target membranes. Step 2, GDI presents
prenylated Rabs to the donor membranes from which nascent transport
vesicles will form. Such membranes would include the portions of the
endoplasmic reticulum from which vesicles form, the Golgi complex,
endosomes, and the plasma membrane. According to this model, Rab-GDI
complexes are recognized by a Rab-specific GDI displacement factor (GDF). Rab-GDP would then become membrane-associated
concomitant with GDI release into the cytosol (Step 3).
Rab-GDP would then be converted to Rab-GTP by the action of a
membrane-bound, Rab nucleotide exchanger. The proteins that mediate
organelle-specific association of Rabs are not yet
known.
Rab proteins are prenylated by Rab geranylgeranyl transferase (34) . A required accessory factor for this enzyme, termed Rab
escort protein (REP), is similar in sequence to GDI-
Despite the fact that
REP shares so many properties with GDI, yeast REP cannot substitute for
GDI in yeast cells in which the GDI gene has been disrupted.
The role of REP is to bind unprenylated
Rabs and ensure their prenylation. If REP bound very tightly to
prenylated Rabs, release of these proteins from REP would be
rate-limiting for prenylation. Perhaps by differing in their affinities
for prenylated Rabs, REP is designed to facilitate prenylation while
GDI can facilitate accurate Rab targeting and recycling.
Another characteristic of GDI-
In
summary, GDI is an elegantly designed recycling and presentation factor
for prenylated Rab proteins. The emerging diversity of GDIs and the
significance of their (apparent) post-translational modification (33, 38) are not yet understood. Another major
remaining challenge will now be to identify proteins with which GDI
interacts.
INTRODUCTION
Multiple Rab-GDI Isoforms
Structural Requirements for Rab Protein-GDI Interaction
GDI Is Required for Rab Protein Recycling in Living Cells
GDI Presents Functional Rab Proteins to the Transport
Machinery in Vitro
A Model for GDI Function
Rab Escort Protein-1 Shares Properties with GDI
GDI-
Inhibits Intracellular Transport in Vitro
FOOTNOTES
REFERENCES
(
)are relatively abundant cytosolic proteins that bind
prenylated Rab-GTPases with high specificity. GDIs have the capacity to
deliver Rab proteins to their specific membrane-bound compartments and
to retrieve Rabs from their fusion targets after they have completed a
catalytic cycle. This Minireview will summarize the current available
information regarding this interesting class of protein escorts.
H]GDP from Rab3A. The protein
was termed GDP dissociation inhibitor (GDI), and the corresponding cDNA (13) predicted a M
of 50,565, consistent
with the electrophoretic mobility of the purified protein (
54,000
kDa).
- and
-forms), whereas other rat tissues produce
predominantly the
-mRNA. The two rat proteins are
86%
identical in amino acid sequence; the rat
-form is 99% identical
to bovine brain GDI, which is now termed GDI-
. Shisheva etal. (24) reported the primary structures of two
mouse GDI isozymes, which they have termed GDI-1 and -2. These workers
reported that GDI-1 RNA was the predominant form in mouse kidney
whereas GDI-2 was the major form in lung. Brain, skeletal muscle, and
pancreas contained both. Although a few years ago Ueda etal. (16) presented data suggesting that a GDI
isolated from rat liver cytosol appeared not to interact with Rab11,
Rab 11 has now been shown to interact with both GDI-
and
-
(23) .
,
respectively(24) , rat GDI-
and -
are 99 and 86%
identical to bovine GDI-
, respectively(23) . The nature of
the differences in sequence among the bovine, mouse, and rat isozymes
would be consistent with the existence of more than two GDI isoforms in
mammalian cells. Perhaps the mouse GDI-2 gene encodes a third
form, and the true mouse
gene, which might be predicted to be
closer in sequence to the bovine
gene, has yet to be cloned.
Indeed, sequences of additional GDI isoforms will soon be
available.
(
)
and -
show comparable capacities to slow GDP
dissociation from Rab3A and Rab11 (23) , and both release a
variety of Rabs from cellular membranes(22, 24) . The
proteins differ in their relative abundance in different cell
types(22, 25) , and GDI-
shows a more particulate
distribution than GDI-
(25) . The significance of these
differences is not yet clear and will require further study. The fact
that yeast contain a single GDI gene that is essential for viability (26) suggests that GDI isozymes may overlap significantly in
terms of their functional capabilities.
does indeed have the capacity to
deliver cytosolic Rab proteins to the appropriate organelle, using
permeabilized cells (10) or enriched membrane
fractions(9) . Prenylated Rab5 and Rab9 proteins were
reconstituted with purified GDI-
, and membrane transfer was then
monitored. Membrane targeting was accompanied by the displacement of
GDI, followed by the exchange of bound GDP for GTP. Since membrane
association preceded the nucleotide exchange
process(9, 10) , it was proposed that a GDI
displacement factor catalyzes the recruitment of Rab proteins onto
specific organelle membranes.
23 nM(32) ), GDI
appears to suppress the promiscuous interaction of Rab proteins with
inappropriate target membranes. In this manner, GDI increases the
efficiency of Rab utilization.
of <50 nM. While
it appears that the membrane interaction site for Rab-GDI complexes is
Rab-specific, it is not yet clear whether the protein (or proteins)
that comprise this site interact directly with GDI, in addition to
that portion of the Rab protein that would be presented by GDI (Fig. 1, Step 2). In the last step, GDI is displaced
(Step 3) and returned to the cytosol; the Rab associates with
membranes first in its GDP-bound conformation prior to what is presumed
to be a catalyzed nucleotide exchange event. Zerial and coworkers (39) have very recently described an activity in preparations
of clathrin-coated vesicles that may represent a GDF for Rab5.
The
cellular concentration of GDI- in CHO cells has been estimated to
be
170 nM(32) . In addition, CHO cell lines
contain more GDI-
than GDI-
(22, 25) .
Furthermore, the level of GDI-
per mg of cytosol protein is much
higher in bovine brain (12) and Madin-Darby canine kidney cells (33) than in CHO cells. The presence of individual Rab proteins
at nanomolar concentrations in the cytosol and the high affinity of GDI
for prenylated Rabs suggest that there is adequate unoccupied GDI to
direct Rab recycling.
(35) .
Current data support a model in which REP presents Rabs to
prenyltransferase and then could transfer them to another target
molecule(35) . An obvious candidate recipient molecule would be
GDI. However, it was recently shown that prenylated Rab-REP complexes
were able to deliver prenylated Rabs to endosome membranes in
permeabilized cells(36) . The characteristics of REP-mediated
Rab delivery paralleled those previously described for GDI-mediated Rab
delivery in the same cell type(10) . Moreover, REP-1 depressed
the rate of GDP release from Rab5 and could retrieve Rab5 from
membranes with potency comparable with that of GDI(36) . These
experiments suggest that newly prenylated Rabs might be delivered
directly to membranes by REP, rather than GDI.
(
)Thus, for some subtle and yet to be discovered
reason, REP and GDI must play distinct roles in vivo. In this
regard, it is important to note that GDIs are more abundant in the
cytosol than REP(19, 32, 36) . In addition,
Rab-REP complexes seem to be of lower affinity than Rab-GDI
complexes.
(
)The weaker interaction of REP with
prenylated Rabs may lead to some mistargeting of Rab proteins in the
same manner as Rab-bovine serum albumin complexes lead to
mistargeting(30) , which might subsequently be corrected by the
availability of excess GDI.
Inhibits Intracellular Transport in Vitro
is that it inhibits the
transport of proteins between membrane-bound compartments when added to in vitro reactions that reconstitute such
events(21, 30, 31) . GDI-
inhibits the
transport of proteins from the endoplasmic reticulum to the
Golgi(31) , between Golgi cisternae(21, 31) ,
and from late endosomes to the trans-Golgi network(30) .
Indeed, this property was used to reveal a requirement for a Rab
protein in endoplasmic reticulum-to-Golgi transport and in intra-Golgi
transport(21, 30) . In the case of the transport of
proteins between endosomes and the trans-Golgi network, cytosolic
Rab9-GDI complexes represent a significant fraction of the total
activity of the cytosol in terms of the ability of cytosol to stimulate
that transport process(30, 37) . In order for Rab
proteins to function, they must first be delivered to the correct
membrane-bound compartment. GDI is a potent inhibitor of Rab protein
membrane delivery(9, 10) . Perhaps by blocking the
delivery of Rabs that are essential, cytosol-derived transport factors, in vitro transport reactions are suppressed. Another model
would be that GDI inhibits transport by scavenging Rabs from membranes.
However, according to this scenario, it is not clear why such complexes
could not be reutilized (and therefore be active, rather than
inhibitory) during an in vitro transport reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.