(Received for publication, June 6, 1995)
From the
Rab GTPases are localized to the surfaces of distinct
membrane-bound organelles and function in transport vesicle docking
and/or fusion. Prenylated Rab9, bound to GDP dissociation
inhibitor-, can be recruited selectively onto a membrane fraction
enriched in late endosomes; this process is accompanied by nucleotide
exchange. We used this system to address whether each Rab uses a
distinct machinery to associate with its cognate organelle. Purified,
prenylated Rab1B, Rab7, and Rab9 proteins were each reconstituted as
stoichiometric complexes with purified GDP dissociation
inhibitor-
, and their recruitment onto endosome- or ER-enriched
membrane fractions was quantified. The two late endosomal proteins,
Rab9 and Rab7, were each recruited onto endosome membranes with
approximate apparent K
values of 9 and 22
nM, respectively. However, while control Rab9
GDP
dissociation inhibitor-
complexes inhibited the initial rate of myc-tagged Rab9 recruitment with an apparent K
of
9 nM, Rab7 complexes
inhibited this process much less effectively (apparent K
112 nM). Similarly,
complexes of the endoplasmic reticulum-localized Rab1B protein were
even less potent than Rab7 complexes (apparent K
405 nM). Rab9 complexes
inhibited Rab7 recruitment with the same low efficacy as Rab7 complexes
inhibited Rab9 recruitment. These experiments distinguish,
biochemically, the recruitment of different Rab proteins onto a single
class of organelle. Since Rab7 and Rab9 are both localized at least in
large part, to late endosomes, this suggests that a single organelle
may bear multiple Rab recruitment machines.
Rab proteins are small GTPases that participate in the processes
by which transport vesicles identify and fuse with their cognate target
membranes (for review, see Zerial and Stenmark(1993), Nuoffer and
Balch(1994), and Pfeffer(1994)). Over 30 different Rab proteins have
been identified. Distinct sets of Rab proteins are found on the ER, ()Golgi, intermediate compartment between the ER and the
Golgi, on early endosomes and the plasma membrane, and on late
endosomes. Some are redundant isoforms that carry out a common function (cf. Singer-Krüger et al.(1994));
most others are unique and essential for a particular step of
intracellular transport.
Although Rab proteins are readily
identifiable as a subfamily of Ras-like GTPases, the proteins are
nevertheless highly diverse. For example, Rab9 is only 30%
identical to Rabs 1A, 2, 3A, 3B, 4, and 5 (Chavrier et al.,
1990a). Rab proteins display the greatest extent of sequence diversity
at their carboxyl termini. In domain-swap experiments, the
hypervariable domain was shown to contain important targeting
information and could relocalize a Rab protein to a different location
(Chavrier et al., 1991; Brennwald and Novick, 1993). Other
regions of the molecule are also important for Rab protein localization
and function, in particular, the regions that correspond to
-helix
3-loop 7 of Ras (Dunn et al., 1993; Brennwald and Novick,
1993; Stenmark et al., 1994).
Given the organelle-specificity of Rab proteins, it seemed likely that organelle-specific receptors would mediate Rab recruitment. However, the recruitment machinery is difficult to saturate, both in vivo (Bucci et al., 1992; Lombardi et al., 1993) and in vitro (Soldati et al., 1994; Ullrich et al., 1994), and thus other scenarios have been proposed. One alternative is that Rab recruitment occurs in two steps. Recognition could be accomplished by a catalytic process in which the recruited Rab does not remain bound to the initial entry site; this initial membrane binding event could be followed by subsequent transfer to a saturable receptor site.
Recent studies in which Rab recruitment has been reconstituted using purified components (Soldati et al., 1994) or permeabilized cells (Ullrich et al., 1994) have provided new information regarding the mechanism of Rab protein recruitment. Prenyl Rab proteins, complexed with GDP dissociation inhibitor (GDI), first associate with the appropriate membranes in their GDP-bound conformations prior to a nucleotide exchange event (Soldati et al., 1994; Ullrich et al., 1994). Rab recruitment displays a saturable initial rate, consistent with a catalytic process. GDI displacement precedes nucleotide exchange, and may represent the catalytic event that underlies Rab recruitment.
We sought to test whether two different Rab proteins that reside on a single class of organelle use a common machinery to achieve their organelle-specific localizations. We focused on Rab7 and Rab9, two proteins of the late endosome (Chavrier et al., 1990b; Lombardi et al., 1993). While Rab9 functions in the transport of proteins between late endosomes and the trans-Golgi network (Lombardi et al., 1993; Riederer et al., 1994), work on the yeast homolog of Rab7, Ypt7p, suggests that this protein functions in late endosome fusion (Wichmann et al., 1992).
In this study we
show that like Rab9, prenyl Rab7 can be recruited onto late endosomes
when added to a membrane fraction in complex with GDI-. Prenyl
Rab1B shows a comparable capacity for recruitment onto ER-enriched
membranes. Competitive inhibition experiments suggest that each of
these proteins utilizes a distinct but related machinery to accomplish
selective membrane targeting.
Rab7 and Rab9 are both localized to late endosomes. Rab7 was first shown to be a late endosome constituent based upon immunoelectron microscopic colocalization with the 300-kDa, cation-independent mannose 6-phosphate receptor (Chavrier et al. (1990b), see also Gorvel et al.(1991)). Rab9 also shows dramatic co-localization with this late endosome marker, as determined by confocal immunofluorescence microscopy (Lombardi et al., 1993). Both Rab7 and Rab9 are present at very low abundance, and thus a direct co-localization was not straightforward. To circumvent this problem, we examined the distributions of these proteins in Chinese hamster ovary cells stably expressing 50-fold higher than normal levels of Rab9 that were subsequently transiently transfected with a Rab7 expression construct. As shown in Fig. 1, the proteins showed significant co-localization in these cells. The bulk of the anti-Rab7 staining overlapped with the distribution of Rab9 in perinuclear structures and other punctate compartments throughout the cytoplasm; Rab7 was also detected in structures, which stained less strongly for Rab9. In summary, Rab7 and Rab9 overlap significantly in their distribution in Chinese hamster ovary cells; some endosomes stained more strongly for Rab9 than Rab7, and vice versa.
Figure 1: The majority of Rab9 and Rab7 colocalize on late endosomes. Chinese hamster ovary cells expressing canine Rab9 (Riederer et al., 1994) were transiently transfected with a plasmid encoding Rab7 under the control of the SV40 early promoter. Cells were grown in plastic dishes, fixed 24-48 h postelectroporation, and then processed for indirect immunofluorescence using a mouse monoclonal antibody to Rab9 (Soldati et al., 1993) and affinity purified rabbit anti-Rab7 antibodies, followed by goat anti-mouse IgGs (coupled to Texas Red) and goat anti-rabbit IgGs (coupled to fluorescein isothiocyanate). Left column, anti-Rab9 staining; right column, anti-Rab7 staining.
To compare the recruitment
properties of Rab9 with those of other Rab proteins, we expressed Rab7,
Rab9, myc-tagged Rab9 and His-tagged Rab1B proteins
individually using recombinant Baculoviruses and purified each of the
proteins from the CHAPS-solubilized membranes of Baculovirus-infected
insect cells (Soldati et al., 1995). Purified Rab proteins
were mixed with equimolar amounts of GDI-, and complexes formed
spontaneously upon dialysis to remove detergent (Soldati et
al., 1994, 1995). Complex formation was verified in each case by
gel filtration on Sephacryl S-100. As shown in Fig. 2, under
these conditions, the Rab
GDI complexes contained stoichiometric
amounts of GDI and the respective Rab proteins.
Figure 2:
Coomassie Blue-stained SDS-polyacrylamide
gel electrophoresis of RabGDI complexes. Complexes were
reconstituted using geranylgeranylated Rab9, Rab7 and Rab1B proteins (rabs) purified from insect cell membranes after infection
with the respective recombinant baculoviruses and GDI-
(GDI) purified from bovine brain cytosol. Mobilities of size
markers are indicated; their masses are shown in
kDa.
Prenyl Rab9
associates with GDI- with an apparent K
of
23 nM (Shapiro and Pfeffer, 1995); thus reconstituted
complexes formed under these conditions are stable. Moreover, when
prenyl Rab9 is present in molar excess relative to GDI-
during
reconstitution, uncomplexed molecules aggregate and thus precipitate.
Such aggregates are readily detected by ultracentrifugation or gel
filtration chromatography. All of the Rab
GDI complexes used in
this study remained soluble and failed to sediment upon
ultracentrifugation.
As targets for membrane recruitment, we utilized membranes enriched in late endosomes, as judged by their content of the 300 kDa, cation-independent mannose 6-phosphate receptor, or ER, based on the enrichment of protein disulfide isomerase (Table 1). The late endosome fraction also contains Golgi membranes, as determined by the distribution of GlcNAc transferase I (Corthésy-Theulaz et al., 1992). The endosome-enriched membranes were enriched 16-fold in Rab9 protein relative to the ER fraction. Rab7 was twice as abundant as Rab9 protein in the endosome-fraction but was only 3.8-fold enriched relative to ER. Differences in enrichment indicate nonidentical protein distributions. Such differences could be due, for example, to the presence of Rab9 on both endosomes and the trans-Golgi network (Lombardi et al., 1993), which co-fractionates with endosomes, while Rab7 may reside uniquely on late endosomes. Alternatively, Rab7 may be present on an overlapping, but slightly distinct population of late endosomes (see Fig. 1).
Rab1B was enriched 13-fold in the endosome-(Golgi)-enriched fraction relative to ER membranes. Rab1B has been reported to be an excellent marker of the ER-Golgi intermediate compartment (Griffiths et al., 1994), which apparently fractionates with Golgi and endosomes upon buoyant density centrifugation. Nevertheless, the ER fraction contained 20-fold more Rab1B than Rab9.
Figure 3:
A, the
initial rate of Rab7 recruitment onto a late endosome-enriched membrane
fraction is saturable. Points represent the average of at least
duplicate determinations (S.E. 10%). Rab7 recruitment onto late
endosomes was linear for
15 min; the initial rate was obtained
from standard 250-µl reactions carried out for 5-10 min with
the indicated amounts of Rab7
GDI complexes. The apparent K
(22 nM) was determined by
Lineweaver-Burk analysis (B).
As shown in Table 2, Rab7 membrane recruitment was a selective process. Under conditions in which Rab9 was recruited 6.8-fold more efficiently onto endosome-enriched membranes than onto lysed red blood cell membranes, Rab7 was recruited with a very similar degree of specificity (6.6-fold). Both Rab proteins were recruited to a small, but significant extent onto membranes of an ER-enriched fraction; this may be due to the presence of contaminating late endosomes, since low levels of 300 kDa mannose 6-phosphate receptors were detected in that fraction (Table 1). All of these experiments were carried out using equal amounts of membrane protein; this also represented comparable levels of phospholipid (Table 1). Red blood cell ghosts yielded background levels of recruitment in all cases (Table 2).
We also examined the
recruitment of prenyl Rab1B onto the membrane fractions used in this
study. Complexes of prenyl Rab1B bound to GDI- delivered Rab1B
efficiently onto ER membranes; 0.76 pmol were recruited onto 3 µg
of membrane in 40 min in reactions containing 4 pmol of Rab1B protein.
Despite the fact that Rab1B was enriched 13-fold in the
endosome-(Golgi)-enriched fraction relative to ER membranes, the
protein was not recruited with a correspondingly higher efficiency onto
these membranes; recruitment levels were close to those obtained for
the ER-enriched fraction (0.72 pmol/3 µg/40 min). These results are
consistent with the notion that Rab proteins are recruited onto a
specific membrane but may accumulate elsewhere as part of their
functional cycle. In this case, recruitment of Rab1B may be primarily
onto ER-derived membranes; accumulation may represent Rab1B bound to
early Golgi compartments and/or the ER-Golgi intermediate compartment.
This is at first surprising, because the Rabs are present at very
different levels. The most reasonable explanation is that the
recruitment machinery, which appears to act catalytically, is not
limiting for recruitment under the experimental conditions employed.
It is important to note that each of the Rab proteins were recruited
with similar efficiencies (Table 2). In addition, each of the Rab
protein preparations displayed similar capacities for GTP binding and
hydrolysis (not shown). Thus, differences observed in the experiments
described below cannot be explained by differences in the activities of
the purified RabGDI complexes.
As shown
in Fig. 4A, addition of as little as a 4-fold excess of
Rab9GDI complexes led to a significant inhibition of both the
initial rate and extent of myc-tagged Rab9 recruitment. By all
criteria tested, the recruitment of myc-tagged Rab9 was
indistinguishable from that of wild-type Rab9 protein. A roughly
10-fold excess of Rab9
GDI complexes led to almost complete
inhibition of myc-Rab9 recruitment (Fig. 4B).
Virtually identical results were obtained when myc-Rab9 was
tested as an inhibitor of Rab9 recruitment (not shown).
Figure 4:
Rab9GDI complexes inhibit the rate
and extent of myc-Rab9 recruitment onto late endosomes. A, kinetics of membrane association of myc-Rab9 was
monitored in standard reactions containing 4 pmol of myc-Rab9
GDI complexes in the absence (
) or
presence of either 16 pmol (
) or 32 pmol (
) of
Rab9
GDI complexes. B, the initial rates of myc-Rab9 recruitment were extrapolated from the linear phase
(up to 15 min) of reactions such as those presented in A,
carried out in the presence of the indicated molar excesses of
competitor Rab9
GDI complexes. C, the data presented in B) were replotted as the inverse of the initial rate versus the concentration of inhibitor. The plot yielded a
nonlinear curve consistent with the action of two distinct competitor
entities. Decomposition of the biphasic curve into two linear portions
permited an estimate of the apparent K
of
the two competitors (28 nM and 9
nM).
We next
attempted to analyze this data using kinetic methods. Lineweaver-Burk
analysis of competition experiments carried out at different myc-Rab9GDI concentrations suggested that Rab9
GDI
complexes behaved as competitive inhibitors (data not shown). Such
analyses were complicated, however, by signal-to-noise considerations.
It is important to note that Rab recruitment is difficult to measure
with high precision (<10% error) because the assay requires
immunoblotting and densitometric quantitation of all samples. In
addition, rather than assaying a single purified enzyme in solution,
the assay utilizes prenylated Rab
GDI complexes mixed with
enriched, but nevertheless, heterogeneous membrane fractions. Despite
these limitations, crude analyses yielded very useful information, as
will be described below.
A semireciprocal replot of the data
presented in Fig. 4B (Fig. 4C) yielded
a biphasic curve that could be decomposed into two lines that would
correspond to apparent K values of
9 and
28 nM. The nonlinear results indicated that Rab9
GDI
complex preparations contained two inhibitors of different potencies.
It is already well established that GDI-
is a strong inhibitor of
Rab recruitment (Soldati et al., 1994; Ullrich et
al., 1994). The ability of GDI to inhibit Rab9 recruitment was
therefore carefully quantified (Fig. 5). In this case, analysis
of the inhibitory potential of GDI as a function of the reciprocal of
the initial rate yielded a single line corresponding to an apparent K
of
26 nM. This value was very close to
one of the values obtained when Rab9
GDI complexes were analyzed
(28 nM).
Figure 5:
GDI is a strong competitor of Rab protein
recruitment. A, the initial rates of membrane recruitment of
Rab9 (), Rab7 (
), and Rab1B (
) proteins were
determined as indicated in Fig. 4B in the presence of
the indicated amounts of free GDI. B, when the data presented
in A were replotted as in Fig. 4C, the data
yielded a single slope corresponding to the action of a single
competitor with an apparent K
of 26
nM.
The equilibrium binding constant for the
association of Rab9 with GDI- is less than 23 nM (Shapiro
and Pfeffer, 1995). Thus, at concentrations of Rab9
GDI
significantly less than 20 nM, the complexes are likely to be
unstable. In addition, when Rab9
GDI complexes are added to
inhibit myc-Rab9 recruitment, every mole of Rab9 and myc-tagged Rab9 that becomes membrane-associated generates an
equal mole of free GDI. This free GDI then works together with
remaining Rab9
GDI complexes to inhibit subsequent rounds of myc-Rab9 recruitment. At low Rab9 concentrations, the
predominant inhibitor would be free GDI; at higher Rab9 concentrations,
the predominant inhibitor would be complexes of Rab9
GDI.
Given
these complexities, the simplest explanation of our data is that
Rab9GDI complexes inhibit myc-Rab9 recruitment with an
apparent K
9 nM, essentially
identical to the apparent K
for Rab9 recruitment
(9 nM) under these conditions. The close similarity of these
values (given the experimental error of
10%) validated this system
to investigate possible competition between Rab7 and Rab9 proteins.
Moreover, these experiments suggest that complexes of Rab9
GDI are
more potent inhibitors of Rab9 recruitment than free GDI alone.
Figure 6:
Rab7GDI and Rab1B
GDI weakly
inhibit Rab9 recruitment and influence the initial rate but not the
extent of the reaction. Rab7
GDI (A-C) and
Rab1B
GDI (D-F) as competitors of Rab9
recruitment are shown. A, the time-dependent membrane
association of Rab9 was monitored in standard reactions containing 4
pmol of Rab9
GDI complexes in the absence (
) or presence of
either 4 pmol (
) or 20 pmol (
) of Rab7
GDI complexes. B, the initial rate of Rab9 recruitment presented as a
function of Rab7
GDI complex concentration (
). The initial
rate of Rab7 recruitment was influenced by the presence of an excess of
Rab9
GDI complexes (
) in a very similar way. C, a
replot of the data presented in B. Decomposition of the
biphasic curve into two linear portions permitted an estimate of two
apparent K
values: 35 and 112
nM. D, the kinetics of membrane association of Rab9
was as in A in the absence (
) or presence of either 16
pmol (
) or 32 pmol (
) of competitor Rab1B
GDI
complexes. E, the initial rate of Rab9 recruitment presented
as a function of Rab1B
GDI complex concentration (
). The
initial rate of Rab1B recruitment was influenced by the presence of an
excess of Rab9
GDI complexes (
) in a very similar way. F, replot of the data presented in E. Decomposition
of the biphasic curve into two linear portions permitted an estimate of
two apparent K
values: 41 and 405
nM.
The inhibitory contribution of the Rab7GDI
complexes was about 12-fold lower than the apparent K
for Rab9
GDI complexes. These data suggest strongly that
Rab7 is recruited by a machinery that is distinct from that used by
Rab9.
Also shown are the results of experiments in which
Rab9GDI complexes were tested as inhibitors of Rab7 recruitment (Fig. 6B, open symbols). Rab9 inhibited Rab7
recruitment to essentially the same extent as Rab7 inhibited Rab9
recruitment.
An important control for these experiments was Rab1B, which is clearly distinct in its localization from Rab7 and Rab9 proteins. Since Rab1B is localized to ER-Golgi compartments, it would not be expected to inhibit the recruitment of Rab7 or Rab9. However, if Rab1B became membrane-associated during the incubation, or if the complexes were unstable, free GDI would be generated, which could inhibit Rab7 or Rab9 recruitment.
Fig. 6, D-F, shows that Rab1BGDI complexes were
indeed weak inhibitors of Rab9 recruitment. Rab9
GDI complexes
inhibited Rab1B recruitment to a very comparable extent (Fig. 6E, open symbols) as Rab1B inhibited
Rab9 recruitment (solid symbols). As expected, a replot of the
reciprocal of the initial rate as a function of Rab1B
GDI complex
concentration yielded a nonlinear set of data that could be represented
by two slopes corresponding to apparent K
values
of 41 and 405 nM. Again, it seems likely that the 41 nM component corresponded to the released GDI. This would indicate
that Rab1B
GDI complexes, as such, were 45-fold less potent than
Rab9 complexes in inhibiting Rab9 recruitment. Together, these data
suggest strongly that Rab proteins utilize distinct but homologous
machineries to direct their selective membrane recruitment.
Fig. 7compares the recruitment of prenyl Rab9 from a
conventional GDI complex with that of Rab9 when cross-linked to GDI.
Although less efficient than wild-type Rab9 protein, chemically
cross-linked Rab9GDI complexes (Rab9XGDI) displayed
significant membrane association. In addition, membrane association of
Rab9XGDI was saturable (not shown). In contrast, GDI-
alone showed
much lower, but nevertheless, detectable levels of membrane
association. This experiment shows that Rab9 can be recognized by
membrane components while associated with the much larger GDI
molecule. This is consistent with a model in which GDI presents the
Rab9 protein to the recruitment machinery and contributes selectivity
to this process (Dirac-Svejstrup et al., 1994).
Figure 7:
Cross-linked RabGDI complexes bind
to membranes and are competitive inhibitors of Rab protein recruitment. A, membrane association of Rab9 (
), cross-linked
Rab9
GDI complexes (rab9XGDI) or free GDI-
(
).
Each set of reactions contained 4 pmol of substrate. B,
initial rates of Rab9 recruitment were extrapolated from time course
experiments as shown in Fig. 4A for reactions
containing the indicated amounts of cross-linked complexes of Rab1B and
GDI (
), Rab7 and GDI (
), and Rab9 and GDI (
). Once
replotted as in Fig. 4C, the data from B yielded linear plots from which we derived apparent K
values for Rab1B (4.4 µM),
Rab7 (871 nM), and Rab9 (345 nM). C, the
initial rates of Rab9 (medium gray bars), Rab7 (light gray
bars), and Rab1B (dark gray bars) recruitment were
obtained from reactions performed in the absence (none) or presence of
a 15-fold molar excess of the cross-linked complexes of Rab9 and GDI (rab9XGDI), Rab7 and GDI (rab7XGDI), or Rab1B and GDI (rab1BXGDI).
We next
examined the relative inhibitory potentials of cross-linked complexes
of Rab1B, Rab7, and Rab9 with GDI. As would be expected, Rab1B
complexes showed very little inhibition when GDI could not be liberated (Fig. 7B). The approximate apparent K determined from these data was 4.4 µM. Rab9
cross-linked to GDI (Rab9XGDI) was the strongest inhibitor, with an
apparent K
of
345 nM. This
represents an approximately 20-fold loss in the capacity of Rab9 to
inhibit as a cross-linked species. Nevertheless, Rab7 cross-linked to
GDI was significantly less potent an inhibitor of Rab9 recruitment than
were similarly cross-linked Rab9 complexes, despite the fact that Rab7
inhibitory potential was reduced only about 9-fold in the cross-linked
complexes.
The cross-linking conditions used in these experiments were necessarily harsh because they were designed to yield preparations lacking any uncross-linked constituents. It seems reasonable to assume that the decreased potencies of the cross-linked complexes were due to the attachment of BS3 at important surface sites.
While cross-linked Rab1BXGDI complexes essentially failed to inhibit Rab9 or Rab7 recruitment, they were relatively strong inhibitors of Rab1B recruitment (Fig. 7C, right). Rab7XGDI and Rab9XGDI most potently inhibited the recruitment of their cognate Rab constituent. Rab7XGDI and Rab9XGDI also inhibited the recruitment of the other late endosomal Rab protein (Fig. 7C). Nevertheless, Rab7XGDI more potently inhibited Rab7 recruitment than Rab9 recruitment; similarly, Rab9XGDI more potently inhibited Rab9 recruitment than Rab7 recruitment.
In summary, these data confirm
the conclusion that GDI- can act as a general inhibitor when
released during a parallel recruitment process. When this complication
is eliminated, Rab7 cross-linked to GDI is a weaker inhibitor of Rab9
recruitment than Rab9 cross-linked to GDI.
In this study, we have compared the membrane recruitment of
two late endosomal Rab proteins. Despite the fact that both proteins
overlap in terms of their subcellular localization, the proteins were
readily distinguished in terms of their capacities to inhibit Rab9
recruitment. Rab7 and Rab9 were each recruited with high selectivity
onto endosome membranes with apparent K values of
10-20 nM. Rab9 competed for the recruitment of myc-tagged Rab9 with an apparent K
of 9
nM, but Rab7 was much less potent in inhibiting Rab9
recruitment, displaying an apparent K
of
112
nM. These data suggest strongly that Rab proteins utilize
distinct machineries for their membrane recruitment. Because Rab7 can
inhibit Rab9 recruitment to some extent, and Rab9 can inihibit Rab7
recruitment to an equivalent extent, it appears that the recruitment
devices are distinct but homologous. This is not surprising, given the
similarity of Rab7 and Rab9 proteins (57%), and the similarity in
function of the two Rab recruitment machineries.
Do these experiments imply that a single organelle contains multiple and distinct Rab recruitment devices? The available data are not yet sufficient to permit this conclusion. Although Rab7 and Rab9 overlap significantly in their distribution with the mannose 6-phosphate receptor, the proteins may initially be recruited onto distinct subsets of late endosomes which then fuse. Resolution of this issue will require the identification of the proteins responsible for Rab protein recruitment and determination of their specific distributions.
While
Rab1BGDI and Rab7
GDI complexes inhibited to varying
degrees, the initial rate of Rab9 recruitment, neither complex
inhibited the overall extent of this process. Similarly, Rab9
GDI
inhibited the initial rate but not the extent of Rab7 recruitment. In
contrast, Rab9
GDI did inhibit the overall extent of myc-tagged Rab9 recruitment, and His-tagged Rab1B
GDI
complexes also inhibited the overall extent of Rab1B recruitment (data
not shown). Thus, these data distinguish an entry site from a
downstream binding site for Rab proteins on organelle surfaces. Access
to the entry site would be inhibited by GDI and heterologous
Rab
GDI complexes; interaction with a downstream target would not.
The data suggest further that each Rab interacts with specific and distinct downstream target molecules. Such downstream proteins
could be V-SNAREs or other specific constituents of the transport
machinery.
Our experiments indicate that the Rab protein entry site
interacts directly with RabGDI complexes. This conclusion is
supported by the rapid and saturable binding of cross-linked
Rab9
GDI complexes to membranes and the ability of those complexes
to inhibit subsequent Rab protein recruitment (Fig. 7). Late
endosome-enriched membranes (3 µg) bound 0.2 pmol of cross-linked
Rab9
GDI. This is likely to represent a minimum estimate of the
number of entry sites since cross-linked Rab9
GDI complexes were
less potent than the native complexes in inhibiting Rab9 recruitment.
If the entry site is comprised of a protein of 25-50 kDa, that
protein would be present at
0.1% of the total protein in the
endosome-enriched fraction. This level is consistent with the low
abundance of late endosomal Rab proteins in the cell.
The ability of myc-Rab9, but not Rab1B or Rab7, to inhibit the overall extent
of Rab9 binding suggests that the capacity of endosomes for Rab9
recruitment is likely to reflect the abundance of a downstream,
Rab-specific binding site. At saturation, 1 pmol of Rab9 is bound per 3
µg of endosome membranes. This would imply that a Rab9 receptor, of
assumed mass of 25-50 kDa, might represent as much as
1% of total endosome protein.
A GDF could represent the entry site and could also be a part of (or tightly associated with) a nucleotide exchange protein on the membrane surface. This nucleotide exchange protein may or may not be Rab-specific; alternatively, it may be of intermediate specificity, acting on a subset of Rab proteins. To date, genetic approaches have led to the identification of a membrane-associated protein capable of enhancing the intrinsic rate of nucleotide exchange by Sec4p (Moya et al., 1993). This protein, termed Dss4p, was most active on Sec4p but also showed significant activity on ypt1p (yeast Rab1), which is 47.5% identical to Sec4p. A related mammalian protein, Mss4, shows preference for Sec4p but can act on other Rab proteins (Burton et al., 1993). If Dss4 and Mss4 act on multiple Rab proteins in vivo, they could not alone be responsible for the organelle-specific localization of Rab proteins.
Prenylation of
Sec4p is not required for Dss4 action, and Dss4 appears not to utilize
Sec4pGDI complexes as substrates. (
)This suggests that
an additional component may also be required to first displace GDI
prior to Dss4 action. A GDF may represent the Rab-specific component of
the Rab recruitment machinery that might be responsible for the
sequestration of Rabs onto specific membrane compartments.
After membrane association and nucleotide exchange, Rabs then appear to interact with a downstream target that also recognizes Rab-specific structural determinants. Thus, Rab delivery is controlled by the cooperation of perhaps as many as four factors: GDI, GDF, a nucleotide exchanger, and a downstream membrane receptor. The diversity and abundance of Rab proteins and the apparent complexity of their accurate targeting are likely to reflect the importance of this class of transport factors.
The presence of Rab proteins on membranes representing both the beginning and end of their functional cycles implies that the distributions of Rab proteins in membrane fractions reported here must be interpreted with care. It is interesting that in proportion to the amount of Rab1B present in the membrane, the denser, ER-enriched membranes were 10 times more active in Rab1B recruitment than the Golgi-endosome-enriched fraction. It is not clear whether Rab1B accumulates at the destination site of transport vesicles on which it functions or whether it is concentrated on the less dense ER-Golgi intermediate compartment, but the recruitment machinery is present on both light and denser membrane-bound compartments. Again, localization of the recruitment machinery itself will resolve these questions.
In summary, Rab recruitment is a selective process that utilizes Rab-specific components. Rab recruitment is mediated by a protein that recognizes Rab proteins bound to GDI. Recruitment is accompanied by GDI release and subsequent exchange of bound GDP for GTP. The next challenge will be to identify the proteins responsible for the organelle- and Rab-specific targeting of this class of Ras-like GTPases.