(Received for publication, July 21, 1995; and in revised form, November 7, 1995)
From the
Rab proteins are Ras-related small GTPases that are
digeranylgeranylated at carboxyl-terminal cysteines, a modification
essential for their action as molecular switches regulating
intracellular vesicular transport. Geranylgeranylation of Rabs is a
complex reaction that requires a catalytic Rab geranylgeranyl
transferase (GGTase) and a Rab escort protein (REP). REP binds
unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP
remains associated with diGG-Rab, which leads to insertion of the Rab
into a specific membrane. We used recombinant Rab1a single cysteine
mutants that accept only one GG group to study the mechanism of the
digeranylgeranylation reaction. Using the prenylation assay, gel
filtration chromatography, and density ultracentrifugation, we show
that REP, but not Rab GGTase, forms a stable complex with unprenylated,
monoGG- and diGG-Rab1a. The REPmonoGG-Rab1a complex is stable in
the presence of detergents or phospholipids, whereas the
REP
diGG-Rab1a complex partially dissociates under these
conditions. The stoichiometry of the REP
Rab complex appears to be
1:1 before prenylation. Prenylation induces a change in complex
stoichiometry, with the formation of a 2:2 or 2:1 REP
Rab complex.
A possible mechanism by which Rab proteins are digeranylgeranylated is
suggested by the current studies. We propose that each geranylgeranyl
addition is an independent reaction that leads to the production of
monoGG-Rab and diGG-Rab, respectively. The stability of the
REP
monoGG-Rab complex prevents monoGG-Rab from dissociating from
REP prior to the second geranylgeranylation reaction, ensuring
efficient digeranylgeranylation of Rab substrates.
Many eukaryotic proteins contain prenyl groups, either the
C- farnesyl or the C-
geranylgeranyl (GG), (
)attached via thioether linkage to cysteines at or near the
carboxyl terminus(1, 2, 3, 4) .
Prenyl modification is essential for function of the modified protein,
since it is required for membrane association and formation of specific
protein-protein interactions.
Rab proteins, Ras-related small
GTPases involved in the regulation of intracellular vesicular traffic
in exocytic and endocytic pathways (5, 6, 7, 8) , are among the
prenylated proteins present in
cells(9, 10, 11) . Rabs contain two cysteine
residues at or near the carboxyl terminus arranged in various motifs
such as XXCC, XCXC, or CCXX, where
C is cysteine and X is any amino acid. Both cysteine residues
present in the motif are modified by the attachment of GG groups via
thioether bonds in a complex reaction mechanism that requires two
components(12, 13) . One component is catalytic and
designated Rab GGTase (previously called Component B) or GGTase-II. It
is a tightly coupled heterodimer composed of a 60-kDa -subunit and
a 38-kDa
-subunit(14) , both related to the
- and
-subunits of the other known prenyl transferases, farnesyl
transferase and CAAX GGTase (or GGTase-I).
Rab GGTase is unique among known prenyl transferases, since it is unable to catalyze the reaction on its own, but requires the presence of an additional component, designated Rab escort protein or REP (previously designated Component A, also known as choroideremia protein)(13, 15) . REP binds to unprenylated Rab, presents it to Rab GGTase, and thereby facilitates GG transfer. After geranylgeranylation, REP remains bound to Rab in a stable complex that can be released in vitro by detergents(15) . In vivo, REP delivers geranylgeranylated Rab to its target donor membrane(16) .
Two related REPs, REP-1 and REP-2, have been
identified(17, 18) . Mutations in REP-1 give rise to
choroideremia, a retinal degeneration
disease(13, 19, 20) . In vitro,
REP-2 can assist in the prenylation of most Rab proteins as efficiently
as REP-1. A notable exception is Rab3a which displays a lower V with REP-2 than with REP-1(18) .
Another exception is Rab27, which is prenylated with 3-fold higher
affinity in the presence of REP-1 as compared with REP-2(20) . In vivo, choroideremia lymphoblasts that contain only REP-2
can efficiently prenylate all endogenous Rab substrates, except for
Rab27(20) . Rab27 is the first example of, possibly, a family
of Rabs that require preferentially either REP-1 or REP-2 for
prenylation and that might explain the retina-restricted phenotype
observed in choroideremia(20) .
Different Rabs regulate
different steps of intracellular vesicular transport. For example,
Rab1a (and Rab1b) regulate endoplasmic reticulum to Golgi transport,
while Rab5a functions in plasma membrane to endosome transport. A
current view of the cyclic function of Rab proteins in vesicular
transport is as
follows(5, 6, 7, 8, 15) .
Newly synthesized Rabs bind REP, the REPRab complex associates
with Rab GGTase and geranylgeranylation of both carboxyl-terminal
cysteines occurs. After prenylation, REP delivers diGG-Rab (presumably
in the inactive GDP-bound form) to its target donor organelle membrane.
Upon membrane association, diGG-Rab is activated by exchange of GDP for
GTP and remains associated with the transport vesicle until the
transport vesicle and the target acceptor membrane fuse. GTP is then
hydrolyzed into GDP and diGG-Rab is extracted from the acceptor
organelle membrane by Rab GDP dissociation inhibitor, which can deliver
diGG-Rab back to the donor organelle membrane and complete the cycle.
Rab GDP dissociation inhibitor and REP share structural and functional
homology, which suggests that both use similar mechanisms to associate
with diGG-Rab and deliver it to intracellular membranes. However,
details about the mechanism responsible for the partition of diGG-Rabs
to membranes is unknown at present.
Farnsworth et al.(21) have established that Rab GGTase/REP can
digeranylgeranylate Rab substrates, whether they contain a XXCC, a XCXC, or a CCXX double
cysteine motif. It has also been shown that very little, if any,
monoGG-Rab accumulates in in vitro reactions(21) .
This observation suggested that the Rab GGTase/REP enzyme system
catalyzes efficiently the digeranylgeranylation reaction either because
the K/K
of the
second GG addition is much larger than that of the first GG addition or
because dissociation of monoGG-Rab from the enzyme is slower than the
transfer of the second GG group. In this study, we used recombinant
mutated forms of Rab1a that could only accept one GG group to probe the
mechanism of the prenylation reaction, and we present evidence to
suggest that the latter hypothesis is more likely. Our results suggest
that REP forms a stable complex with monoGG-Rab1a in order to ensure
double geranylgeranylation of Rabs prior to delivery to intracellular
membranes.
Rab GGTase activity is stimulated by detergents such as
Nonidet P-40(15) . Evidence indicates that detergents stimulate
the reaction, because they act as acceptors of GG-Rab, the product of
the reaction. In their absence, REP and GG-Rab form a stable complex,
and REP is unable to undergo further rounds of catalysis. We wanted to
determine if phospholipid vesicles would act similarly to stimulate the
reaction. Fig. 1A shows an experiment where we measured the
time-dependent transfer of [H]GG to Rab1a. In the
absence of detergents, the reaction reached completion when 2 pmol of
[
H]GG were incorporated into Rab1a. In the
presence of the detergent Nonidet P-40, the reaction progressed for up
to 30 min, essentially as described before(12, 15) .
When phosphatidylcholine (PC) vesicles were used, a significant
stimulation of the reaction was observed, comparable with that obtained
with Nonidet P-40 (Fig. 1A). Under these conditions, 60
pmol of [
H]GG were incorporated into Rab1a or
30-fold stimulation over control reactions in the absence of detergents
or phospholipids.
Figure 1:
Geranylgeranylation of Rab1a wild-type
and mutants: effect of detergent and phospholipid. Each reaction
mixture contained, in a final volume of 50 µl, 5.5 µM [H]GGPP (3,000 dpm/pmol), 1 pmol of
RabGGTase, 1.4 pmol of REP-1, and either 2.5 µM Rab1a-CC (A), Rab1a-CS (B), Rab1a-SC (C), or Rab1a-SS (insets). The reactions were incubated for the indicated times
at 37 °C, in the absence (closed triangles) or presence of
phosphatidylcholine vesicles (25 µg/tube) (closed squares)
or Nonidet P-40 (1 mM) (closed circles). After
incubation, the amount of [
H]GG transferred to
Rab1a proteins was determined as described under ``Experimental
Procedures.'' Blank values determined at zero time (0.19
pmol/tube) were subtracted from each value. The insets in B and C show the same experiment plotted such that
the y axis was rescaled to 5 pmol. A control reaction mixture
containing 2.5 µM Rab1a-SS (open circles) was
included in the plot.
We used recombinant DNA techniques to generate
Rab1a mutants that contain either one or both the carboxyl-terminal
cysteines mutated to serines. The resulting proteins, Rab1a-CS, -SC,
and -SS, can accept only one or no GG groups. When we analyzed mutant
Rab1a-CS and Rab1a-SC in the same experiment, we observed that Nonidet
P-40 or PC were very weak stimulators of GG transfer (Fig. 1, B and C). As shown in the inset to Fig. 1, B and C, the incorporation of
[H]GG was only 4 pmol for Rab1a-CS and 1.8 pmol
for Rab1a-SC after 30 min, which represented 3-4-fold stimulation
over control reactions in the absence of lipids. This result suggested
that mono GG-Rab1a forms a complex with REP and/or RabGGTase that is
stable to detergent or phospholipid micelle destabilization, since the
difference in -fold stimulation between wild-type and mutant proteins
is much greater than can be accounted for by the reduced stoichiometry
of geranylgeranylation of the Rab1a mutants (one-half of the
wild-type).
If this hypothesis is correct and monoGG-Rabs are
titrating essential components of the reaction, it should be possible
to inhibit the prenylation of wild-type Rab1a with the Rab1a mutants.
We tested this hypothesis in two experiments. In the first experiment,
we used a fixed subsaturating amount of Rab1a-CC and added increasing
amounts of mutant Rab1a proteins (Fig. 2). When increasing
amounts of Rab1a-SS were added, we observed up to a 50% reduction in
the amount of [H]GG incorporated into Rab1a-CC
when the mutant protein was present at 4-fold higher concentration,
consistent with previous results(13) . When increasing amounts
of Rab1a-CS were added, we observed a more striking inhibition. There
was 65% inhibition of Rab1a-CC prenylation when both proteins were
present at equimolar concentrations (Fig. 2). This finding is
remarkable considering that the mutant protein is nevertheless a
substrate for the reaction (Fig. 1B) and that the assay
is measuring all of the [
H]GG transfered. A
similar, but somewhat less potent, effect was observed with Rab1a-SC
mutant, while a related protein that is not a Rab GGTase substrate,
Ha-Ras, did not inhibit the reaction.
Figure 2:
Inhibition of Rab1a-CC prenylation by
mutant Rab1a proteins. Each reaction mixture contained, in a final
volume of 50 µl, 1 mM Nonidet P-40, 5.5 µM [H]GGPP (3,000 dpm/pmol), 1 pmol of
RabGGTase, 1.4 pmol of REP-1, and 2.5 µM Rab1a-CC. The
reactions were incubated for 10 min at 37 °C, in the presence of
the indicated concentrations of Rab1a-CS (closed squares)
Rab1a-SC (closed triangles), Rab1a-SS (closed
circles), or Ha-Ras (open circles). After incubation, the
amount of [
H]GG transferred to Rab1a proteins was
determined as described under ``Experimental Procedures.''
Blank values determined from parallel reactions in the absence of Rab1a
proteins (0.29 pmol/tube) were subtracted from each
value.
The previous experiment
demonstrated that monoGG-Rab is more potent than unprenylated Rab in
inhibiting the prenylation of diGG-Rab, suggesting that it forms a more
stable complex with the enzymatic components of the reaction. To test
this hypothesis, we designed the following experiment (Fig. 3).
We initiated the reaction with the mutant Rab proteins in the presence
of enzyme (REP/Rab GGTase) so that stable association could occur.
After a 10-min incubation, we added an excess of Rab1a-CC, and we
measured the stimulation of the rate of the reaction upon this
addition, as an indication of the ability of the wild type Rab1a to
compete for the available enzyme in the presence of the Rab1a mutants.
As shown in Fig. 3, Rab1a-CC efficiently competes with Rab1a-SS
for [H]GG transfer (compare open and closed diamond curves), but is much more inefficient in
overcoming the inhibition imposed by Rab1a-CS (compare open and closed triangle curves). Again, we obtained an
intermediate effect with the Rab1a-SC mutant. These data suggest that
monoGG-Rab1a proteins are inhibitory for the wild-type diGG-Rab1a,
because they form a relatively stable complex with the enzymatic
components of the reaction.
Figure 3:
Geranylgeranylation of Rab1a-CC in the
presence of Rab1a mutants. Each reaction mixture, containing in a final
volume of 50 µl, 1 mM Nonidet P-40, 5.5 µM [H]GGPP (3,000 dpm/pmol), 1 pmol of
RabGGTase, and 1.4 pmol of REP-1, was incubated at 37 °C in the
presence of 2.5 µM either Rab1a-CC (squares),
Rab1a-CS (circles), Rab1a-SC (triangles), or Rab1a-SS (diamonds). At 10-min incubation, 2.5 µM Rab1a-CC
was added (closed symbols, + curves) and at the indicated
times, the amount of [
H]GG transferred to Rab1a
proteins was determined as described under ``Experimental
Procedures.''
To determine which component or
components of the reaction are inactivated by mutant Rab1a proteins, we
designed the following experiment (Fig. 4). We incubated
Rab1a-CS under standard reactions conditions, with approximately 2 pmol
each of RabGGTase and REP-1. After 5 min, Rab1a-CS incorporated 1 pmol
of [H]GG. At this point, we made fresh additions
to the reaction mixture, either RabGGTase, REP-1, or both. When REP-1
was added, either alone or in combination with RabGGTase, the
incorporation of [
H]GG into Rab1a-CS increased
rapidly and was 2-fold higher than when RabGGTase or buffer control
were added. These results suggest that REP-1 is the limiting component
of the reaction when Rab1a-CS is added, because REP-1 and Rab1a-CS form
a stable complex, as was demonstrated for REP-1 and Rab1a-CC in the
absence of detergents(15) .
Figure 4:
Geranylgeranylation of Rab1a-CS:
stimulation by REP-1. Each reaction mixture contained in a final volume
of 50 µl, 5.5 µM [H]GGPP (3,000
dpm/pmol), 1.8 pmol of RabGGTase, 2.4 pmol of REP-1, and 2.5 µM Rab1a-CS. After incubation for 5 min at 37 °C, one of the
following additions was made: none (open circles), 4.5 pmol of
RabGGTase (closed circles), 6 pmol of REP-1 (open
triangles), or 4.5 pmol of RabGGTase with 6 pmol of REP-1 (closed triangles). At the indicated times, the amount of
[
H]GG transferred to Rab1a proteins was
determined as described under ``Experimental Procedures.''
Blank values determined at zero time (0.31 pmol/tube) were subtracted
from each value.
If REP-1 is the limiting
component in the reaction, then the amount of product formed should be
proportional to the amount of REP-1 present in the reaction. In Fig. 5A, we show that the amount of REP-1 determines
the amount of [H]GG incorporated into Rab1a-CS.
Increasing amounts of REP-1, up to 10 pmol, resulted in increasing
amounts of GG-Rab1a-CS formed, even when limiting amounts of RabGGTase
(1 pmol) were present. There is a stoichiometry of approximately 0.5
pmol of [
H]GG transfered for every pmol of REP-1,
suggesting that one Rab binds a REP-1 dimer. These data also suggest
that Rab GGTase is not a stable component of the complex, since it can
catalyze the reaction when present at much lower levels than REP. In
the reverse experiment, increasing amounts of RabGGTase were unable to
generate more [
H]GG incorporation into Rab1a-CS,
when limiting amounts of REP-1 were present (Fig. 5B).
Figure 5:
Geranylgeranylation of Rab1a-CS in the
presence of limiting amounts of REP-1 or RabGGTase. Each reaction
mixture contained, in a final volume of 50 µl, 1 mM Nonidet P-40, 5.5 µM [H]GGPP
(3,000 dpm/pmol), and 2.5 µM Rab1a-CS. In A, the
reaction mixture also contained 0.9 pmol of RabGGTase with 1.2 pmol (open squares), 2.4 pmol (open circles), 4.8 pmol (open triangles), or 9.6 pmol (open diamonds) of
REP-1, and in B, the reaction mixture also contained 1.2 pmol
of REP-1 with 0.9 pmol (open squares), 1.8 pmol (open
circles), 4.8 pmol (open triangles), or 7.2 pmol (open diamonds) of RabGGTase. At the indicated times, the
amount of [
H]GG transferred to Rab1a proteins was
determined as described under ``Experimental Procedures.''
Blank values determined at zero time (0.31 pmol/tube) were subtracted
from each value.
Taken together, these results suggest that monoGG-Rab1a (Rab1a-CS)
forms a tight complex with REP-1 that is resistant to dissolution by
detergents or phospholipids. To demonstrate this binding directly, we
incubated wild-type and mutant Rab1a with GGPP in a prenylation mixture
containing RabGGTase, in the presence or absence of REP-1. Then, we
loaded the reaction mixtures on Superdex 200 gel filtration
chromatography and determined the position of elution of REP-1 and Rabs
by immunoblot following SDS-gel electrophoresis of the eluted fractions (Fig. 6). In the absence of REP-1, Rab1a-CS eluted from the gel
filtration column at fraction 10 (Fig. 6A). This
corresponds to the elution position of 30-kDa proteins and is
consistent with elution as a monomer. In the presence of REP-1, a
significant fraction of Rab1a-CS eluted earlier at fractions 6 and 7 (Fig. 6B). Fractions 6 and 7 also contained REP-1, and
they correspond to elution position of 160-kDa proteins. This finding
is consistent with the formation of a REP-1Rab1a complex, since
purified and recombinant REP-1 eluted from the same column with
apparent molecular mass of 140 kDa ( (13) and this study not
shown). Rab1a-CC and Rab1a-SS also formed complexes with REP-1, as
determined by co-elution upon gel filtration chromatography under the
same conditions described above for Rab1a-CS (Fig. 6, C and D), but Ha-Ras did not (Fig. 6E).
Figure 6:
Detection of REPRab1a complex by gel
filtration chromatography. Each reaction mixture contained, in a final
volume of 50 µl, 5 µM GGPP, 0.5 µM RabGGTase, and either 2 µM Rab1a-CS (A and B), Rab1a-CC (C), Rab1a-SS (D), or Ha-Ras (E), in the absence (A) or presence of 2 µM REP-1 (B-E). After incubation for 15 min at 37
°C, each sample was loaded onto a Superdex 200 3.2/30 column
equilibrated and run as described under ``Experimental
Procedures.'' An aliquot (30 µl) of elution fractions
2-10 was subjected to SDS-gel electrophoresis on 12.5% minigels,
the proteins transferred to nitrocellulose and detected with either
J905 anti-REP-1 antibody (0.03 µg/ml), D576 anti-Rab1a antibody
(2.5 µg/ml), or anti-Ha-Ras antibody (0.1 µg/ml), as indicated,
using the ECL system. The column was calibrated with thyroglobulin (670
kDa), aldolase (160 kDa), and ovalbumin (45 kDa), and vertical
arrows on A denote the position of elution of the
markers. Horizontal arrows denote the position of migration of
REP, Rab1a, and Ha-Ras (left side) and the indicated molecular
mass markers (right side) upon SDS-gel
electrophoresis.
To demonstrate directly that REP-1 and monoGG-Rab1a formed a complex
that was resistant to destabilization by phospholipids, we performed in vitro prenylation reactions as above in the presence of PC
vesicles and subjected them to gel filtration chromatography (Fig. 7). When loaded on Superdex 200, PC vesicles eluted in the
void of the column (fraction 2), clearly separated from the
REPRab complex (fractions 6 and 7). In the presence of REP-1,
wild-type Rab1a-CC now eluted at three different positions: in fraction
2 co-eluting with phosphatidylcholine vesicles, in fraction 6
co-migrating with REP-1, and in fraction 10 as a monomer (Fig. 7A, compare with Fig. 6C).
Strikingly, under the same conditions Rab1a-CS was not found in
fraction 2, but only in fractions 6-7 and 10, as was observed in
the absence of phosphatidylcholine vesicles (Fig. 7B,
compare with Fig. 6B). Rab1a-SS was present mostly in
fraction 10, and very little was found co-migrating with REP-1 (Fig. 7C, compare with Fig. 6D). In the
absence of REP-1, wild-type and mutant Rab1a proteins did not bind PC
vesicles and eluted in fractions 9 and 10 (not shown). These data are
consistent with the hypothesis that REP-1
monoGG-Rab1a complex is
stable to disruption by phospholipids. The observation that the
REP
Rab1a-SS complex is unstable in the presence of PC vesicles is
surprising and may reflect a less stable association of
REP
unprenylated Rab versus REP
prenylated Rab
complexes. While the REP-Rab interaction remains to be defined in more
detail, the available data suggest that there are at least two binding
sites, one involving the prenyl groups and the COOH-terminal region and
another involving one or more upsteam Rab
sequences(13, 18, 26, 27) . This
result suggests that the geranylgeranyl moiety is an important
determinant of the REP-Rab interaction.
Figure 7: Gel filtration chromatography of Rab1a proteins after prenylation reaction in the presence of phospholipids. Each reaction contained, in a final volume of 50 µl, 25 µg phosphatidylcholine vesicles, 5 µM GGPP, 0.5 µM RabGGTase, 2 µM REP-1, and either 2 µM Rab1a-CC (A), Rab1a-CS (B), or Rab1a-SS (C). After incubation for 15 min at 37 °C, each sample was loaded onto a Superdex 200 3.2/30 column equilibrated and run as described under ``Experimental Procedures.'' An aliquot (30 µl) of elution fractions 2-10 was subjected to SDS-gel electrophoresis on 12.5% minigels, the proteins transferred to nitrocellulose and detected with either J905 anti-REP-1 antibody (0.03 µg/ml) or D576 anti-Rab1a antibody (2.5 µg/ml), as indicated, using the ECL system. The column was calibrated with thyroglobulin (670 kDa), aldolase (160 kDa), and ovalbumin (45 kDa), and vertical arrows on A denote the position of elution of the markers. Horizontal arrows denote the position of migration of REP, and Rab1a (left side), and the indicated molecular mass markers (right side) upon SDS-gel electrophoresis.
In order to study the
stoichiometry of the REPRab complex, we performed glycerol
gradient ultracentrifugation. We subjected reaction mixtures containing
different combinations of REP, Rab GGTase, and wild-type and mutant
Rab1a and determined the position of elution of these proteins by
immunoblot following SDS-gel electrophoresis of the eluted fractions (Fig. 8). When subjected to density ultracentrifugation, REP
migrated to fractions 5 and 6, consistent with a 60-kDa protein (Fig. 8A), Rab GGTase migrated to fraction 7,
consistent with a 80-kDa protein (Fig. 8B) and Rab1a
wild-type and mutants peaked at fraction 3, consistent with 30-kDa
proteins (not shown). When Rab GGTase and Rab1a-CS were incubated
together and applied on the glycerol gradient, no changes in migration
were observed, suggesting that these proteins did not form a complex (Fig. 8C). However when Rab1a-CS was incubated with
REP, its migration did shift significantly to fractions 5 and 6,
co-migrating with REP (Fig. 8D). Similar results were
obtained with Rab1a-CC and Rab1a-SS (not shown). We conclude that REP
behaves as a monomeric protein upon density ultracentrifugation and
that the migration of the REP
Rab complex is most consistent with
a 1:1 stoichiometry.
Figure 8:
Glycerol Gradient Ultracentrifugation of
REP, Rab GGTase and Rab1a. Reaction mixtures contained 50 mM sodium Hepes (pH 7.2), 5 mM MgCl, 1 mM dithiothreitol, 10 µM unlabeled GGPP in a final
volume of 50 µl, in the presence of 4 µM REP-1 (A), 4 µM RabGGTase (B), 4 µM Rab1a-CS and 4 µM RabGGTase (C), or 4
µM Rab1a-CS and 4 µM REP-1 (D).
After incubation for 15 min at 37 °C, reaction mixtures were loaded
onto 4 ml 7.5-30% glycerol gradients in 20 mM Tris-HCl
(pH 7.5) and 1 mM dithiothreitol and spun as described under
``Experimental Procedures.'' An aliquot of each fraction (30
µl) was subjected to SDS-gel electrophoresis on 12.5% minigels and
the proteins transferred to nitrocellulose and detected with either
J905 anti-REP-1 antibody (0.03 µg/ml), H492 anti-Rab GGTase
antiserum (1:5,000 dilution), or D576 anti-Rab1a antibody (8.6
µg/ml), as indicated, using the ECL system. Each gradient was
calibrated with internal standards, catalase (230 kDa), aldolase (160
kDa), and ovalbumin (45 kDa), and vertical arrows on each
panel denote the position of elution of the markers. Horizontal
arrows denote the position of migration of REP, Rab GGTase
-
and
-subunits, and Rab1a (left side) and the indicated
molecular mass markers (right side) upon SDS-gel
electrophoresis.
To analyze the stoichiometry of the complex
after prenylation, we included Rab GGTase in the reaction mixture and
subjected the reaction mixtures to density ultracentrifugation. Under
the same conditions described above, the migration of monoGG-Rab or
diGG-Rab shifted and peaked in fraction 8 (Fig. 9, A and B). It is noticeable that REP migration is also
significantly shifted toward later fractions. Under the same
conditions, Rab1a-SS migrated to fractions 5 and 6 (Fig. 9C), the migration observed for unprenylated
Rab1a in the presence of REP-1. We conclude that prenylation induces a
change in the stoichiometry of the REPRab complex, likely to a
dimeric complex.
Figure 9:
Stoichiometry of REPRab1a complex by
glycerol gradient ultracentrifugation. Reaction mixtures contained 50
mM sodium Hepes (pH 7.2), 5 mM MgCl
, 1
mM dithiothreitol, 10.5 µM [1-
H] GGPP, 4 µM REP-1, and 4
µM RabGGTase in a final volume of 50 µl, in the
presence of 4 µM Rab1a-CS (A), 4 µM Rab1a-CC (B), or 4 µM Rab1a-SS (C).
After incubation for 15 min at 37 °C, reaction mixtures were loaded
onto 4 ml 7.5-30% glycerol gradients in 20 mM Tris-HCl
(pH 7.5) and 1 mM dithiothreitol and spun as described under
``Experimental Procedures.'' An aliquot of each fraction (30
µl) was subjected to SDS-gel electrophoresis on 12.5% minigels and
the proteins transferred to nitrocellulose and detected with either
J905 anti-REP-1 antibody (0.03 µg/ml), H492 anti-Rab GGTase
antiserum (1: 5,000 dilution), or D576 anti-Rab1a antibody (8.6
µg/ml), as indicated, using the ECL system. The same filters were
exposed to a PhosphorImager plate for 18 h to visualize the
[
H]GG-Rab1a protein. Each gradient was calibrated
with internal standards, catalase (230 kDa), aldolase (160 kDa), and
ovalbumin (45 kDa), and vertical arrows on each panel denote
the position of elution of the markers. Horizontal arrows denote the position of migration of REP, Rab GGTase
- and
-subunits, Rab1a and [
H]GG-Rab1a (left
side), and the indicated molecular mass markers (right
side) upon SDS-gel electrophoresis.
A possible mechanism by which Rab proteins are
digeranylgeranylated is suggested by the current studies. We propose
that each geranylgeranyl addition is an independent reaction that leads
to the production of monoGG-Rab and diGG-Rab, respectively. However,
the monoGG-Rab product does not accumulate, because it forms a complex
with REP that is resistant to disruption by detergents and
phospholipids, whereas the REPRab or the REP
diGG-Rab
complex is not. The stability of the REP
monoGG-Rab complex
prevents monoGG-Rab from dissociating from REP prior to the second
geranylgeranylation reaction, ensuring efficient digeranylgeranylation
of Rab substrates.
In the present work, we confirm and extend
studies previously published. First, we provide a possible mechanism
for the inefficient prenylation of mutant Rabs that can only accept one
GG group, as reported in vitro(10, 22) or in vivo(28, 29) . We show that the
prenylation of Rab1a mutants is strictly dependent on, and
stoichiometric with, the levels of REP present in the reaction.
Furthermore, we demonstrate that REP and Rab associate in a stable
complex, confirming previous observations(15) . Rab GGTase
appears not to be a stable component of the monoGGREP complex.
Rab GGTase is able to catalyze GG transfer even when present in much
lower amounts than REP. Also, the position of co-elution of Rab1a and
REP on gel filtration chromatography or glycerol gradient
ultracentrifugation is inconsistent with the presence of Rab GGTase as
part of the complex. Therefore, we suggest that each prenylation
reaction is an independent event that may involve
dissociation-reassociation of Rab GGTase. Second, we provide a possible
mechanism for the absence of accumulation of monoGG-Rab1a in
vitro, as reported by Farnsworth et al.(21) . As
discussed above, we show the formation of a stable REP
monoGG-Rab
complex that may prevent the dissociation of monoGG-Rab from REP until
the second GG addition occurs.
Our results suggest that there is not an absolute order of addition of GG groups to the two adjacent cysteine residues in Rab1a, since either Rab1a mutant (CS or SC) can accept a prenyl group. However, the amino-terminal cysteine is somewhat preferred, since prenylation of that cysteine is more efficient than the carboxyl-terminal one. Also, Rab1a-CS is a more potent inhibitor of wild-type Rab1a prenylation (that is, REP recycling), than Rab1a-SC. Given the inherent flexibility of Rab GGTase, which is able to prenylate adjacent cysteines with or without a spacer amino acid in between the cysteine residues, it is possible that the digeranylgeranylation of Rab1a-CC in vivo is actually ordered. Further experiments will be needed to clarify this issue.
We
obtained essentially the same results in identical biochemical
experiments where we used wild-type and single cysteine mutants of
Rab3a, a substrate that contains a XCXC motif rather
that XXCC present in Rab1a. ()This suggests that
the mechanism of digeranylgeranylation is similar for all Rabs and
involves a lipid-resistant transitional complex. However, several Rabs,
including Rab8 and Rab13, contain only one cysteine residue within a
carboxyl-terminal CAAX motif, where A is an aliphatic
residue. Inasmuch as GG transfer to these Rabs could theoretically be
catalyzed by either Rab GGTase or CAAX GGTase, it remains to
be established which enzyme is actually responsible for the reaction
under steady-state in vivo conditions.
The gel filtration
experiments presented here with the recombinant protein ( Fig. 6and Fig. 7) and previously published with the
purified protein (13) showed that REP elutes as an 140-kDa
protein and suggested that it exists in solution as a dimer. However,
the behavior of a protein upon gel filtration is proportional to its
Stokes radius rather than its molecular weight, and for nonglobular
proteins, those two parameters are quite different. We attempted to
dissociate the putative dimer by denaturation, but we were unable to
shift REP migration on gel filtration chromatography from 140 to 70 kDa
(not shown). It is noteworthy that REP migrates anomalously upon
SDS-gel electrophoresis (to 95 kDa rather than its predicted molecular
mass of 73 kDa) for unknown reasons. For these reasons, we used a more
reliable method, namely density ultracentrifugation, to study the
stoichiometry of the REPRab complex. Glycerol gradient
ultracentrifugation suggested that REP is a monomer. The discrepancy in
apparent molecular mass of REP-1 by gel filtration chromatography and
density ultracentrifugation suggests that REP-1 is an elongated
molecule with a large Stokes radius. The stoichiometry of the
REP
Rab complex prior to prenylation appears to be 1:1 ( Fig. 8and Fig. 9). However, upon prenylation we observed
a significant shift in the migration of both REP-1 and Rab1a,
consistent with the formation of a 2:2 or a 2:1 complex. We cannot
distinguish between these two possibilities with the present
experiments. We also cannot rule out that the shift is due to the
binding of Rab GGTase to the REP
Rab complex, but this possibility
is unlikely for the reasons detailed above. The significance of this
shift in complex stoichiometry is unclear but may be important for the
next step of the reaction, the REP-mediated delivery of diGG-Rab to
intracellular membranes.
The issues raised by this work may be
addressed with future studies detailing the kinetics of the prenylation
reaction, biophysical studies on REP and the REPRab complex, and
the role of REP in the delivery of prenylated Rabs to intracellular
membranes.