(Received for publication, July 19, 1995; and in revised form, September 18, 1995)
From the
In vitro synthesis and post-translational prenylation
of Rab5 is accomplished using reticulocyte lysate supplemented with
prenyl precursors (Sanford, J. C., Pan, Y., and Wessling-Resnick,
M.(1993) J. Biol. Chem. 268, 23773-23776). When Rab5 is
translated in the presence of biotin-lysine-tRNA, it incorporates
biotin-lysine into its peptide backbone and is efficiently prenylated;
since this modification is dependent on guanine nucleotide binding,
biotin-Rab5's functional integrity must be maintained. Prenylated
biotin-Rab5 associates with a 45-kDa reticulocyte GDP dissociation
inhibitor (GDI), sedimenting as a 70-kDa particle on 5-20%
sucrose density gradients. The GDI-Rab5 complex can be captured using
streptavidin-linked agarose beads. Only Rab5 peptides that are
substrates for prenylation are found to cosediment with the lysate GDI
on sucrose gradients. Post-translational association of Rab5 and GDI is
a novel finding, since previous reports suggested Rab5 remains
associated with Rab escort protein (REP) after prenylation (Alexandrov,
K., Horiuchi, H., Steele-Mortimer, O., Seabra, M. C., and Zerial,
M.(1994) EMBO J. 13, 5262-5273). Since
post-translational prenylation is catalytically mediated by REP, our
study suggests that a complex between Rab5 and this factor is transient
in nature. Thus, newly synthesized and prenylated Rab5 is most likely
escorted to its target membrane by a GDI acceptor molecule. Biotin-Rab5
provides a novel tool for future efforts to capture and characterize
additional accessory factors required for Rab protein function in
vesicle transport.
Intracellular vesicle transport involves a series of regulated
budding and fusion events that are dependent on members of the Rab
family of GTP-binding
proteins(1, 2, 3, 4) . Rab5 is a
particularly well characterized member of this family. Rab5 is
associated with early endosomes and the plasma membrane(5) ,
and the internalization of transferrin (6) and uptake of
horseradish peroxidase (7) have been shown to be enhanced by
overexpression of this factor. In contrast, endocytosis is perturbed
when the GTP-binding mutants Rab5 and Rab5
are overexpressed(6, 7, 8) . Cell-free
systems also have demonstrated a role for Rab5 in endosome-endosome
fusion(2, 7, 9) . Although the precise
function of Rab proteins in membrane transport remains undefined, it
has been proposed that these GTPases act as regulators of the assembly
and disassembly of protein complexes involved in the docking and fusion
of vesicles(10) .
Important structural elements required for
Rab function are prenyl groups that are post-translationally attached
to the protein's C-terminal cysteines(11, 12) .
Rab5 mutants lacking these key cysteine residues fail to stimulate
endocytosis in vivo(7) and do not support vesicle
fusion activity in cell-free assays(2) . Non-prenylated Rab5
that contains the C-terminal cysteines also fails to stimulate
endosome-endosome fusion in vitro(9) . Rab proteins,
including Rab5, are post-translationally prenylated by geranylgeranyl
transferase II(13, 14) . This enzyme is composed of
two components: an dimer with homology to farnesyl
transferase (15) and geranylgeranyl transferase I(16) ,
and Rab escort protein (REP)
or component A(13) .
REP was originally identified to have significant homology with the
gene product responsible for the retinal degenerative disorder,
chorioderemia(17) . REP forms a complex with Rab proteins to
enable functional prenylation by the catalytic
subunits of
geranylgeranyl transferase II(18) . More recently, REP has been
assigned a role in the delivery of newly synthesized Rab5 to membranes.
Alexandrov et al. (19) have demonstrated that Rab5 can
be modified in a reconstitution assay using purified prenylation
reaction components and that Rab5 remains associated with REP after
modification to be delivered to membranes in the absence of other
cytosolic factors. These observations help to explain why the in
vitro prenylation reaction is limited in the absence of
detergents, which help to dissociate the Rab-REP complex; delivery and
release of Rab proteins to membranes subsequent to prenyl transfer
would help to recycle REP and thus to recover catalytic activity of
geranylgeranyl transferase II. Otherwise, this enzyme appears to act in
a stoichiometric fashion in vitro(18) .
The apparent ability of REP to target Rab proteins to membranes resembles a functional characteristic of another Rab binding factor, GDP dissociation inhibitor (GDI). GDI was first identified and purified by its ability to inhibit GDP release(20) . The binding of Rab proteins to GDI is dependent on their post-translational prenylation(21, 22) . GDI also has been shown to selectively recruit Rab proteins to their target membranes(23, 24) ; release of the Rab from GDI at the membrane surface is accompanied by GDP/GTP exchange(24, 25) . Finally, GDI has a well characterized role in the retrieval of Rabs from membranes and is responsible for maintaining a soluble pool of the GTP-binding proteins (20, 26, 27) .
We have devised a novel method to capture protein complexes in association with biosynthetically biotinylated Rab5. Using this approach, we find that newly synthesized and prenylated Rab5 associates with a cytosolic GDI. In contrast to the findings of Alexandrov et al.(19) , stable complexes between REP and newly synthesized Rab5 are not observed in the cell-free reticulocyte lysate system. Since the assembly of the Rab5-GDI complex is dependent on protein prenylation and occurs in the absence of membranes, we conclude that immediately after geranylgeranylation, Rab5 must rapidly dissociate from REP to bind GDI in the reticulocyte lysate. These novel findings indicate that biosynthetic biotinylation can be a useful tool to explore protein-protein interactions of members of the Rab family and their accessory factors, including yet-to-be-identified guanine nucleotide exchange factors and GTPase-activating proteins (GAPs).
Figure 1:
Biotin-Rab5 is prenylated. Translation
of Rab5 was carried out in the presence of
[S]methionine (50,000 cpm/pmol) and
biotin-lysine charged tRNA (Promega) for 30 min at 30 °C. For
post-translational processing, reaction mixtures were adjusted to 40%
reticulocyte lysate with 12 mM Tris-Cl, pH 8.0, 0.6 mM DTT, 3.0 mM MgCl
, and 100 µM
mevalonate was added. Incubation was continued at 37 °C for 16 h.
After synthesis and processing, Rab5 peptides (250 fmol) were
electrophoresed on urea-acrylamide gradient gels and transferred to
nitrocellulose. The immunoblot was then incubated with
streptavidin-linked alkaline phosphatase (Avidin-AP, left
panel). The biotin-Rab5/streptavidin-linked alkaline phosphatase
complex was detected using NBT and BCIP as described under
``Experimental Procedures.'' The immunoblot was also exposed
to film (1 day of exposure), and the autoradiograph is shown (right
panel). The mass of electrophoresed molecular size markers is
shown on the left in kDa.
Our previous work has demonstrated that newly
synthesized Rab5 becomes post-translationally modified with two
geranylgeranyl groups and that the prenylated isoform can be detected
by its characteristic mobility shift on urea-acrylamide gradient
gels(36) . Accordingly, further incubation of S-labeled biotin-Rab5
at 37 °C with the
isoprenoid precursor mevalonate added to the lysate results in
conversion of the peptide to a greater mobility isoform (Fig. 1). Since geranylgeranylation of Rab5 is dependent on its
guanine nucleotide binding capacity(29) , the fact that
biotin-Rab5 is fully processed into this isoform indicates the
functional integrity of the GTP-binding protein is not perturbed by the
incorporation of biotinylated lysine residues.
Figure 2:
Biotin-Rab5 sediments in a 70-kDa complex.
Rab5 was synthesized, biotinylated, and prenylated as described in Fig. 1. The reticulocyte lysate containing biotin-Rab5 was then
layered on a continuous 5-20% sucrose gradient and centrifuged at
165,000 g for 17 h at 4 °C. One hundred and
fifty-µl fractions were collected from the bottom of the gradient
and were incubated with streptavidin-linked agarose for 16 h at 4
°C to collect biotin-Rab5. Protein captured on streptavidin-agarose
was boiled in Laemmli buffer and electrophoresed on a urea-acrylamide
gradient SDS gel. The arrow at right indicates
unprocessed Rab5, and the arrowhead at right designates the prenylated isoform. The positions of molecular size
markers in the gradient are indicated by arrows at the top of the figure.
Figure 3: Detection of REP in reticulocyte lysate. Reticulocyte lysate and K562 cell cytosol (600 µg/lane) were electrophoresed 8% SDS-PAGE gels. Proteins were transferred to nitrocellulose and incubated with anti-REP1 (1:400 dilution) in 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 5% bovine serum albumin, and 0.02% Tween 20 for 1 h. Immunocomplexes were detected with goat anti-rabbit IgG-linked alkaline phosphatase as described under ``Experimental Procedures.''
Figure 4: Detection of proteins immunologically related to GDI1 and GDI2. Bovine brain cytosol and reticulocyte lysate (120 µg) were electrophoresed on 12% SDS-PAGE gels, and protein was electrophoretically transferred to nitrocellulose. The Western blots were then probed with antibodies against bovine Rab3A GDI, a GDI1-like species (1:1,000 dilution) (left panel), and a synthetic GDI2 peptide (1:15,000 dilution) (right panel). The latter antibody cross-reacts with GDI1- and GDI2-like isoforms. After extensive washing, the immunoblots were incubated with goat anti-rabbit IgG-linked alkaline phosphatase, followed by incubation with NBT/BCIP as described for Fig. 1. The mass of electrophoresed molecular size standards is shown on the left in kDa.
Figure 5:
GDI
cosediments with Rab5. Rab5 was translated, prenylated,
and fractionated on a 5-20% sucrose gradient as described for Fig. 2, except that the synthetic peptide was not biotinylated.
Thirty-µl aliquots of sucrose gradient fractions were
electrophoresed on urea-acrylamide gradient gels and immunoblotted to
detect the 45-kDa GDI2-like species (upper panel). The lower panel is the autoradiograph of
S-labeled
Rab5 in these same gradient fractions. The position of molecular size
standards in the sucrose gradient is indicated at the top of
the figure.
To verify that Rab5 and the 45-kDa GDI isoform exist in
the predicted 1:1 complex in the reticulocyte lysate, gradient
fractions containing biosynthetically biotinylated and prenylated
Rab5
were incubated with streptavidin-linked agarose
beads. Proteins captured by the beads were electrophoresed and
transferred to nitrocellulose, and the immunoblot was then probed with
anti-GDI2 antibodies. Autoradiography confirms the isolation of
S-labeled biotin-Rab5 on the streptavidin-linked beads as
demonstrated by the results of Fig. 6(left panel).
Furthermore, biotin-Rab5 bound to the streptavidin-agarose was indeed
associated with the 45-kDa GDI2-like species (right panel).
These results indicate that complex formation does not interfere with
the binding of the biotinylated product to the beads. Conversely,
biosynthetic biotinylation apparently does not disrupt Rab5-GDI
interactions. Incubation of immunoblots of the same sample with
anti-REP1 antibodies failed to detect this protein in complex with
biotin-Rab5 (data not shown). These observations are consistent with
the apparent M
of prenylated Rab5 on sucrose
gradients and confirm that the sedimentation pattern observed is due to
a 1:1 complex of the newly synthesized product with the GDI2-like
isoform.
Figure 6:
Identification of a
biotin-Rab5[GDI complex. Sucrose gradient fractions
containing biotin-Rab5 (400 µl) were incubated with 75 µl of
streptavidin-linked agarose. Proteins captured on the beads were
electrophoresed and transferred to nitrocellulose as described for Fig. 2. The Western blot was then probed with anti-GDI2 peptide
antibodies (right panel). Autoradiography confirms that
S-labeled biotin-Rab5 was isolated (left panel).
Control experiments with the streptavidin-linked agarose fail to
capture the 45-kDa GDI isoform alone.
Figure 7:
Sedimentation of co- and
post-translationally processed Rab5. Rab5 was synthesized as described
under ``Experimental Procedures.'' After a 20-min translation
reaction, 900 fmol of peptide were removed from the reaction and
diluted in 50 mM HEPES, pH 7.5, 1 mM
MgCl, 1 mM DTT, and 5 µM GDP on ice.
Post-translational prenylation of the remaining Rab5 was initiated by
supplementing the reaction mixture with 100 µM geranylgeranyl pyrophosphate and adjusting the reaction conditions
to 12 mM Tris-Cl, pH 8.0, 3 mM MgCl
, 0.6
mM DTT, 100 µg/ml RNase A, and 40% (v/v) reticulocyte
lysate. At the post-translational times indicated (1 h and 24 h),
1200-fmol samples were removed and diluted as described above. Samples
were then fractionated on 5-20% sucrose gradients and analyzed as
described for Fig. 2.
Our results clearly
contradict the idea that, subsequent to prenylation, newly synthesized
Rab5 remains associated with REP. However, Alexandrov et al. (19) were also able to demonstrate that REP can mediate the
transfer of Rab5 to membranes. Therefore, to rule out the possibility
that contaminating membranes provide for the release of Rab5 from REP
followed by its immediate retrieval by GDI, the reticulocyte lysate was
precleared at 350,000 g for 15 min prior to
prenylation reactions. Results obtained with precleared lysate were
found to be identical to those shown in Fig. 2and Fig. 5(data not shown).
To further investigate potential
Rab5-REP complex formation during synthesis and processing, a series of
Rab5 mutants was studied. We have previously demonstrated that a
GTPase-defective mutant, Rab5 is poorly processed in
vitro(29) . For the purposes of this study, we further
investigated the post-translational processing of Rab5
,
a point mutant that constitutively binds GDP(8) . Fig. 8compares the time course of prenylation determined for
Rab5
, Rab5
, and Rab5
. For
these experiments, translated peptides were adjusted to 10 nM in the in vitro prenylation reaction and 100
µM mevalonate was added. Aliquots of each reaction were
removed, quenched in Laemmli buffer, and electrophoresed on
urea-acrylamide gradient gels (panel A). The amount of
modified product was determined by densitometric scanning of the
unprocessed (upper band) and prenylated (lower band) isoforms. As shown
in panel B, Rab5
is modified with the same rate
and efficiency as wild type, unlike Rab5
. This
observation confirms that the GDP-bound state of Rab5 is indeed the
preferred conformation for geranylgeranyl transferase function. Thus,
Rab5
is predicted to interact preferentially with REP.
Nonetheless, sucrose density gradient analysis (Fig. 9)
demonstrates that Rab5
sediments in the same 70-kDa
complex observed for wild type. The hydrolysis-defective mutant
Rab5
exhibits the same sedimentation behavior. Finally,
a C-terminal truncation mutant, Rab5
, which lacks
the cysteine acceptor sites for prenylation, was also translated and
fractionated on a sucrose density gradient. Although this peptide is an
inhibitor of Rab5
prenylation in
vitro(29) , most likely due to competitive interactions
with REP, only a M
25,000 isoform is observed
in the gradient sedimentation analysis (Fig. 9).
Rab5
does not associate with the GDI2-like
isoform, since this interaction would require prenylation of C-terminal
cysteines(21) . We conclude that stable Rab5-REP complexes
cannot be identified by sucrose density ultracentrifugation and that
the association between Rab5 and REP may be transient in nature. All of
our results are consistent with the rapid transfer of newly synthesized
and prenylated Rab5 from the REP/geranylgeranyl transferase II complex
to the 45-kDa GDI. An alternate explanation of our data is that the
GDI2 isoform may actually provide the Rab escort function during and
after the post-translational modification reaction; however, this
scenario is unlikely since non-prenylated Rab5 fails to interact with
the 45-kDa protein (Fig. 2, Fig. 5, and Fig. 9)
and the closely related bovine Rab3A GDI cannot substitute for REP in in vitro assays of geranylgeranyl transferase II
function(19) .
Figure 8:
Prenylation of Rab5,
Rab5
, and Rab5
. Rab5 peptides were
synthesized and adjusted to a final concentration of 10 nM in
12 mM Tris-Cl, pH 8.0, 0.6 mM DTT, 3.0 mM
MgCl
, 100 µg/ml RNase A with 40% (v/v) reticulocyte.
Prenylation was then initiated by addition of 100 µM mevalonate, and incubation was continued at 37 °C. At the
indicated times, 3-µl samples (30 fmol) were diluted into 77 µl
of Laemmli buffer to be electrophoresed on urea-acrylamide gradient SDS
gels. Panel A presents autoradiographs of the
S-labeled peptides demonstrating the time-dependent
conversion to a greater mobility isoform, which is indicative of
geranylgeranylation(29) . These radiolabeled bands were
densitometrically scanned, and the percentage of peptide modified is
shown as a function of time in panel B: Rab5
(
), Rab5
(
), and Rab5
(
).
Figure 9:
Sucrose density gradient analysis of Rab5
mutants. Rab5, Rab5
, Rab5
,
and Rab5
were synthesized and the concentrations
were adjusted to 10 nM before post-translational prenylation
as described for Fig. 1. Reticulocyte lysate containing 500 fmol
of each mutant was fractionated by sucrose density gradient
ultracentrifugation. Gradient fractions were electrophoresed on
urea-acrylamide gradient gels and subsequently processed for
fluorography as shown. Sucrose density gradients were calibrated with
the molecular size standards indicated at the top of the
figure.
Biosynthetic biotinylation of Rab5 can be accomplished by its in vitro synthesis in the presence of biotin-lysine tRNA. Since incorporation of biotin-lysine does not interfere with the post-translational modification of Rab5 with geranylgeranyl groups, the guanine nucleotide binding properties of biotin-Rab5 appear to be unimpaired(29) . Thus, biotin-Rab5 can serve as a novel reagent to capture and identify factors that regulate this GTP-binding protein's activity. Our results demonstrate that the only GDI isoform present in reticulocyte lysate, a 45-kDa GDI2-like species, forms a complex with nascent prenylated biotin-Rab5. This complex sediments as a 70-kDa particle and can be captured on streptavidin-linked agarose beads through biotin adduct(s) on Rab5.
Cytosolically disposed Rab proteins are known to complex with
GDIs(23, 28) . Rab3A GDI was first purified from
bovine brain based on its ability to decrease the rate of GDP
release(20) . A closely related 55-kDa homolog, GDI1, and a
second isoform of 45-kDa, GDI2, were subsequently identified by
molecular cloning of mouse GDIs(28) . Nishimura et al. (37) have also cloned two GDI isoforms from rat brain, called
GDI and GDI
. The latter shares amino acid sequence with a
47-kDa protein purified from CHO cells(38) ; thus, two classes
of GDI isoforms are known to exist: mouse GDI2, rat GDI
, and the
47-kDa CHO GDI represent lower molecular mass isoforms, while bovine
Rab3A GDI, mouse GDI1, and rat GDI
display higher mobility on SDS
gels and closer sequence similarity(38) . This is clearly a
multigene family, and at least five distinct mouse GDI genes are
predicted based on Southern analysis(39) . Our results show
that Rab5 associates with a GDI2-like species present in rabbit
reticulocyte lysate. Since the 45-kDa protein is the only GDI isoform
that can be detected, this result is not surprising. Shisheva et
al.(28) have demonstrated that both GDI1 and GDI2
extract membrane-bound Rab5 with the same efficiency. Furthermore, the
results of Yang et al.(38) indicate that Rab proteins
partition to form complexes with the most abundant GDI isoform
available. The latter observation suggests there is little functional
difference between the various GDIs, an idea that is strongly supported
by biochemical studies (28, 37) .
One unexpected
finding made in our study is the observation that newly synthesized
Rab5 associates with the reticulocyte GDI. The inability to detect a
predominant Rab5-REP complex is particularly perplexing, since Zerial
and co-workers have previously documented that Rab5 remains associated
with REP upon prenylation(19) . In the latter study, the
complex between REP and Rab5 was identified by gel filtration
chromatography over Superose 12; accordingly, we also have fractionated
the products of our in vitro synthesis and prenylation
reaction over Superose 12 but do not observe the hallmark 120-kDa
complex to distinguish REP-Rab5 interactions. (
)A singular
difference between our study and the report by Alexandrov et al.(19) is that while the reticulocyte lysate contains the
necessary complement of factors required for post-translational
modification, the latter group employed purified components, and
therefore GDI was not available during the course of Rab5 prenylation.
Subsequent to its prenylation, Alexandrov et al.(19) demonstrated the apparent release of Rab5 from REP to
membranes. However, this process potentially could have been mediated
by a membrane-associated form of GDI, consistent with its established
role in Rab protein targeting(23) . Although REP is thought to
associate with both prenylated and non-prenylated Rab
proteins(18) , our findings agree with evidence characterizing
the ability of GDI to recognize only prenylated Rabs(22) ,
particularly since non-modified Rab5 and Rab5
do
not assemble into the multimeric 70-kDa complex.
If REP were to
mediate the association of a newly synthesized Rab protein with
membranes, one would predict that a round of GTP hydrolysis would be
required prior to its retrieval and binding by GDI. This idea stems
from observations that reveal the concomitant exchange of GDP for GTP
upon membrane binding of Rab proteins and the selective recruitment of
GDP-bound Rabs by GDI from
membranes(24, 25, 26, 27) .
Therefore, two additional lines of evidence argue that GDI serves as a
soluble escort for newly synthesized Rab proteins: 1) we find that the
complex between synthetic Rab5 and the reticulocyte GDI forms in the
complete absence of membranes; and 2) we observe that Rab5 behaves identical to the wild-type protein vis à vis its post-translational processing and
association with GDI. The latter mutant constitutively binds GDP (8) ; therefore, our results suggest that GTP hydrolysis is not
required for association of nascent Rab proteins with GDI. Moreover,
since the kinetics of Rab5
prenylation are identical to
those observed for the wild-type protein, GTPase function may not be
required for post-translational prenylation. This issue is raised
because in contrast to Rab5
and Rab5
, the
GTPase-defective mutant Rab5
, is inefficiently
processed. Based on the results of the present study, this effect is
most likely due to the predominant GTP-binding state of this mutant.
Although we cannot completely rule out the possible presence of a
Rab5-GAP activity capable of stimulating GTP hydrolysis by
Rab5
, this conclusion is consistent with our previous
data, which indicated that the GDP-bound form of Rab5 is the preferred
substrate conformation for the Rab geranylgeranyl transferase and
possibly REP interactions(29) . These observations suggest that
Rab5's GTPase activity does not play a major role in its
post-translational modification and insertion into membranes.
Finally, our study clearly demonstrates the utility of biosynthetic biotinylation as an approach to capturing Rab protein accessory factors. Biotin-Rab5 provides a useful tool to isolate key factors involved in vesicle transport. Candidate elements include guanine nucleotide exchange factors and GAPs, which are thought to interact specifically with individual Rab family members(40, 41) . Future experiments will focus on endosomal membrane proteins that may be identified associated with biotin-Rab5 and captured on avidin-linked beads, much like the soluble complex with reticulocyte lysate GDI isolated and characterized in this study.