From the Department of Biochemistry and Molecular
Biology, Medical College of Ohio, Toledo, Ohio 43614-5804 and
§ Department of Pharmacology, Case Western Reserve
University, Cleveland, Ohio 44106
Received for publication, February 16, 2001
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ABSTRACT |
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The targeting of various Rab proteins to
different subcellular compartments appears to be determined by variable
amino acid sequences located upstream from geranylgeranylated cysteine
residues in the C-terminal tail. All nascent Rab proteins are
prenylated by geranylgeranyltransferase II, which recognizes the Rab
substrate only when it is bound to Rab escort protein (REP). After
prenylation, REP remains associated with the modified Rab until it is
delivered to the appropriate subcellular membrane. It remains unclear
whether docking of the Rab with the correct membrane is solely a
function of features contained within the prenylated Rab itself (with
REP serving as a "passive" carrier) or whether REP actively
participates in the targeting process. To address this issue, we took
advantage of a mutation in the The Rab family of proteins consists of more than 40 Ras-related
GTPases, each involved in specific steps of vesicular transport (1-3).
Rab1A and Rab1B are two of the most extensively studied members of the
Rab family. Both proteins are found in membranes of the
ER,1 Golgi apparatus and
intermediate vesicles between these compartments (4, 5). As suggested
by their localization, these GTPases function in the anterograde
trafficking of proteins from the ER to the Golgi compartment (4, 6,
7).
According to accepted models (3, 8, 9), Rab proteins cycle on and off
donor and acceptor membranes in a guanine
nucleotide-dependent fashion. In the GDP-bound form, which
is presumed to represent the inactive state, the Rab proteins are
typically associated with an accessory protein, guanine nucleotide
dissociation inhibitor (GDI), in the cytosol. Upon delivery to the
membrane, interaction with an exchange factor promotes substitution of
GTP for GDP, placing the Rab protein in the active conformation (10).
The activated Rab protein then facilitates the targeting of a budding transport vesicle to the correct acceptor membrane by mediating vesicle-N-ethylmaleimide-sensitive factor attachment protein
receptor (v-SNARE) interactions with the corresponding
target-SNARE (t-SNARE) (11, 12). At the acceptor membrane, the
intrinsic GTPase activity of the Rab protein may be stimulated by
interaction with a GTPase-activating protein, returning it to the
GDP-bound conformation. In this form, the Rab protein can be removed
from the membrane by GDI, completing the cycle.
All Rab proteins are modified post-translationally by 1 or 2 20-carbon
geranylgeranyl groups that are linked covalently to cysteine residues
at their C termini. This isoprenoid modification (prenylation) is
necessary for two facets of Rab function. First, it is required for
association of the GTPase with the membrane (13, 14). Second, it
promotes optimal interaction of the GDP-bound form of the Rab protein
with GDI (15, 16). Prenylation of Rab proteins is catalyzed by
geranylgeranyltransferase type II (GGTase II) (17, 18). GGTase II will
prenylate the target cysteine residues only if the nascent Rab
substrate is first bound to a carrier protein termed Rab escort protein
(REP) (19, 20). REP initially binds to the Rab protein while it is in
the GDP-bound state (21). After the prenyl groups are added to the C
terminus of the Rab protein, GGTase II is released, whereas REP remains bound to the prenylated protein and delivers it to the membrane. Chavrier et al. (22) used chimeric proteins to show that the hypervariable domain near the C terminus of each Rab protein contains information required for targeting to a specific subcellular membrane compartment. However, the precise role of the chaperone
(i.e. REP) in this process has yet to be defined. One
possibility is that REP acts cooperatively with the Rab protein to
promote association with a specific docking complex at the target
membrane. An alternative possibility is that REP is a passive carrier
that does not interact directly with putative Rab-docking complexes at
the acceptor membrane.
In the present study we explore the role of REP in the membrane
targeting of Rab1B, taking advantage of our previous observations that
amino acid substitutions within the Rab1B Generation of Rab1B Constructs--
cDNAs encoding
MycRab1B( Prenylation of Rab Proteins in Vitro--
Expression of
recombinant MycRab1B proteins was induced in E. coli
BL21(DE3)pLysS, and bacterial lysates were prepared as described
previously (26). Total protein was determined by the method of Bradford
(27), and the relative amount of each MycRab1B protein in each
bacterial lysate was determined by immunoblot analysis using a
monoclonal antibody (9E10) against the Myc epitope, as described
previously (24). The abilities of different Rab1B constructs to serve
as substrates for GGTase II were compared by adding aliquots of
bacterial lysate containing equal amounts of Rab protein to a 50-µl
reaction mixture consisting of 50 mM HEPES, pH 7.4, 1.0 mM dithiothreitol, 5.0 mM MgCl2,
0.2 mM Nonidet P-40, 1 µCi of [3H]GGPP, 20 ng of recombinant REP, 20 ng of recombinant GGTase II. Prenylation of
the bacterially expressed Rab proteins by GGTase I was determined in a
50- µl reaction mixture consisting of 50 mM Tris, pH 7.7, 20 mM KCl, 5.0 mM MgCl2, 25 µM ZnCl2, 1.0 mM dithiothreitol,
0.5 mM Zwittergent 3-14, 1 µCi of
[3H]geranylgeranyl pyrophosphate, and 10 ng of purified
GGTase I. The reactions were stopped after 90 min at 37 °C by the
addition of 1× SDS sample buffer (28). Three quarters of each assay
sample was subjected to SDS-PAGE and fluorography to determine
incorporation of [3H]geranylgeranyl moiety into the
recombinant Rab protein. The remainder of each sample was subjected to
immunoblot analysis to confirm equal protein loading in the assays.
Subcellular Fractionation of Rab Proteins Expressed in HEK293
Cells--
Cells expressing various MycRab1B constructs were harvested
from 100-mm dishes 24 h after transfection. The cells were
collected by centrifugation at 500 × g for 5 min, and
the pellet was homogenized in 80 mM PIPES, pH 6.8, 5.0 mM EGTA, 1.0 mM MgCl2, 0.05%
Triton X-100, and Complete Mini-EDTA-Free protease inhibitors
(Roche Molecular Biochemicals). The resulting cell lysate was
centrifuged at 100,000 × g for 30 min at 4 °C in a
Beckman TLA100.2 rotor. The supernatant and particulate fractions were
analyzed by SDS-PAGE and Western blotting. A rabbit polyclonal antibody
against the Myc epitope (Upstate Biotechnology, Inc., Lake Placid, NY)
was used to detect the epitope-tagged Rab proteins, with goat
anti-rabbit-horseradish peroxidase as the secondary antibody.
Quantification of the signal produced by Pierce SuperSignal reagents
was performed on a LumiImager (Roche Molecular Biochemicals).
Prenylation of Rab Proteins Expressed in Cultured
Cells--
HEK293 cells or NIH3T3 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% (v/v) fetal calf serum in a 5% CO2 atmosphere at 37 °C. Cells were transfected
with pCMV vectors containing the indicated Rab constructs using
LipofectAMINE PLUS (Life Technologies, Inc.) according to the
manufacturer's instructions. One hour after transfection, the cells
were changed to medium containing 10 µM lovastatin and
200 µCi of [3H]mevalonolactone (Mev) (2.95 Ci/mmol)
with or without 10 µM GGTI-298, a specific inhibitor of
GGTase I (provided by S. Sebti, Moffitt Cancer Center, Tampa FL) (29).
After 18 h at 37 °C, the cells were washed three times with
Hanks' balanced salt solution and disrupted in 200 µl of ice-cold
lysis buffer (20 mM HEPES, pH 7.3, 20 mM
MgCl2, 150 mM NaCl, 0.75% Nonidet P-40
supplemented with protease inhibitors). All subsequent steps were
carried out at 4 °C. Particulate material was removed by
centrifugation of the lysates at 10,000 × g for 5 min,
and the epitope-tagged Rab proteins were immunoprecipitated by
incubation with a mouse monoclonal antibody against the Myc epitope for
2 h. Immune complexes were collected by a 1-h incubation with
protein A-Sepharose beads coupled to goat anti-mouse IgG. The beads
were washed 3 times with lysis buffer, then the Myc-tagged Rab proteins
were eluted in immunoprecipitation sample buffer (50 mM
Tris-HCl, pH 6.8, 1.4 mM
To determine the subcellular distribution of the prenylated Rab
proteins, the metabolic labeling, as described above, was started
3 h after transfection, and cells from three 100-mm dishes were
pooled. Cell lysates were centrifuged at 100,000 × g
for 30 min at 4 °C to obtain cytosol and membrane-enriched
fractions. The Myc-tagged Rab proteins were then immunoprecipitated
from each fraction and analyzed as described above.
Carboxymethylation of Rab Proteins Expressed in 293 Cells--
Starting 3 h after transfection, cells were switched
to methionine-free Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 40 µCi/ml
L-[methyl-3H]methionine (PerkinElmer Life
Sciences) and incubated at 37 °C. After 18 h, the cells
were harvested and lysed as described in the preceding section.
One-tenth of the cell lysate was subjected to SDS-PAGE and immunoblot
analysis to confirm MycRab1B expression. The Myc-tagged Rab proteins
were immunoprecipitated from the remaining cell lysate as described
above. After SDS-PAGE and fluorography, the dried gel was cut into
0.5-cm sections, and the amount of [3H]methanol released
by alkaline hydrolysis of protein O-methyl esters in each
gel slice was determined as described by Clarke et al.
(30).
Assay for Rab Interaction with REP in Cultured Cells--
HEK293
cells were co-transfected with a vector encoding T7-tagged REP-1
(pCMVREP1) and different Myc-tagged Rab1B constructs. After
transfection, cells were maintained in medium containing an inhibitor
of isoprenoid synthesis (10 µM lovastatin) to prevent prenylation and promote the accumulation of nascent Rab·REP
complexes. After 48 h, cells from 2 parallel 100-mm cultures were
pooled, and cytosolic fractions were subjected to size-exclusion
chromatography on a Superose-12 column, essentially as described by
Overmeyer et al. (23). The elution positions of the T7-REP
and MycRab1B were determined by immunoblot analysis of the column
fractions using antibodies against the epitope tags.
Interaction of Rab Proteins with FLAG-GDI Immunofluorescent Localization of Expressed Rab Proteins in 293 Cells--
Cells were seeded in 60-mm dishes containing laminin-coated
coverslips and were transfected with the specified vectors.
Approximately 24 h later, the coverslips were processed for
immunofluorescence microscopy. Cells were fixed and permeabilized with
cold methanol for 15 min or fixed with cold 3.0% paraformaldehyde then
permeabilized by incubation for 2 min with 0.1% (v/v) Triton X-100 in
PBS. Mouse or rabbit antibodies against the Myc epitope were used to
detect the Rab1B constructs. Where indicated, the cells were
co-incubated with a monoclonal antibody against GM130 (1:25 dilution)
(Transduction Laboratories, Lexington, KY), a polyclonal antibody
against Rab6 (1:200 dilution) (Santa Cruz Biotechnology, Santa Cruz,
CA), or a polyclonal antibody against calreticulin (1:150 dilution)
(Affinity Bioreagents, Golden, CO). Rhodamine-conjugated goat
anti-mouse IgG (Calbiochem) and fluorescein isothiocyanate-conjugated
goat anti-rabbit IgG (Sigma) were used at a 1:100 dilution as the
secondary antibodies. Photomicrographs were obtained with a Nikon
Eclipse E800 fluorescent microscope equipped with a digital camera.
Pseudo-coloring and merging of images were performed with Image-Pro
Plus software (Media Cybernetics, Silver Spring, MD).
Assay for Post-translational Processing of the LDL
Receptor--
Cultured 293 cells were co-transfected with pLDLR17,
which encodes the human LDL receptor (31), and each of the indicated pCMVRab1B constructs. Twenty-four hours later, cells were
pulse-labeled for 30 min with 100 µCi of [35S]Easy-Tag
ExpressTM protein-labeling mix (PerkinElmer Life Sciences) in 1.0 ml of
methionine-free Dulbecco's modified Eagle's medium (DMEM) followed by
a 2-h chase in DMEM plus 10% fetal bovine serum supplemented with 200 µM methionine and 200 µM cysteine. Cells from parallel cultures were harvested immediately after the pulse or
after the 2-h chase, and the radiolabeled LDL receptor was immunoprecipitated as described by Castellano et al. (32).
A REP Binding-deficient Rab1B Mutant Can Be Prenylated after
Changing the C-terminal Motif--
Mutations in the predicted
To verify that the results of the cell-free assays accurately
predicted the ability of the C-terminal CC The addition of a CLLL Motif to the C terminus of Rab1B Does Not
Affect the Interaction with REP--
The absence of prenylation of
Rab1B(Y78D)CLLL by REP/GGTase II in the cell-free assay (Fig. 1)
suggested that the addition of CLLL motif to the Y78D mutant allowed
prenylation by GGTase I but did not restore REP binding. This was
confirmed in intact cells, where the CLLL versions of Myc-tagged
Rab1B(wt) or Y78D were co-expressed with T7-tagged REP1. As shown in
Fig. 3, size-exclusion chromatography of
cytosol obtained from cells expressing MycRab1B-CLLL revealed a typical
high molecular weight Rab·REP complex, similar to that previously
reported for MycRab1B(wt) (23). In contrast, MycRab1B(Y78D)CLLL was
unable to form a stable complex with T7-REP1, consistent with our
earlier observations with MycRab1B(Y78D) containing the normal
double-cysteine motif (23).
The Y78D Mutation Prevents Interaction of Rab1B with
GDI--
Sequence alignments of REP and GDI reveal four major
conserved regions between these two proteins (36), consistent with some
functional similarities. Both proteins associate with prenylated Rab
proteins in the GDP-bound conformation and can deliver them to
membranes (10, 23, 26, 37, 38). However, GDI cannot replace REP in the
prenylation reaction (38). For the current studies, the possibility
must be considered that GDI could associate with the nascent
Rab1B(Y78D)CLLL after prenylation by GGTase I and serve as an escort
protein in place of REP. To address this issue, we used an established
co-immune precipitation assay (26, 39) to compare the abilities of the
Myc-tagged Rab constructs to interact with FLAG-GDI The Prenylated Form of Rab1B(Y78D)CLLL Can Associate with
Cell Membranes--
In mammalian cells, prenylated Rab proteins are
found in specific membrane compartments and in the cytosol, where they
exist in a complex with GDI (10). Immunoblots from cells expressing MycRab1B and MycRab1B-CLLL showed a typical distribution of the expressed proteins between cytosol and membrane compartments (Fig. 5), consistent with their ability to
undergo prenylation by one or both geranylgeranyltransferases (Figs. 1
and 2). In contrast, MycRab1B(Y78D) was localized entirely in the
cytosolic fraction, as expected in light of its inability to undergo
REP-dependent prenylation. Most notably, the addition of
the CLLL motif to the C terminus of Y78D allowed a significant portion
of the expressed protein to localize to the membrane fraction (Fig.
5).
Because earlier studies have indicated that under conditions of
transient Rab overexpression a significant pool of cytosolic Rab
protein remains unmodified (26), we repeated the subcellular partitioning studies, tracing the fate of [3H]Mev-labeled
MycRab1B instead of total MycRab1B (Fig.
6). As expected, no
[3H]Mev-labeled protein was detected when MycRab1B(Y78D)
was immunoprecipitated from the soluble or membrane fractions of
transfected 293 cells. However, when the same protein was converted to
a substrate for GGTase I by the addition of the CLLL motif,
radiolabeled protein was clearly detected. The partitioning of the
prenylated Rab1B(Y78D)CLLL resembled that of the wild-type Rab1B, with
the majority of the [3H]Mev-labeled protein present in
the membrane fraction. Interestingly, these studies also revealed that
changing the C terminus of wild-type Rab1B from CC to CLLL caused a
significant shift in its subcellular distribution. Specifically, the
percent of total [3H]Mev-labeled protein in the cytosolic
fraction increased from less than 10% in the case of Rab1B to more
than 65% in the case of Rab1B-CLLL (Fig. 6). A possible explanation
for the increased proportion of prenylated Rab1B-CLLL in the cytosol is
provided by the studies of Shen and Seabra (40), who reported that
mono-geranylgeranylated Rab1A forms a more stable complex with REP than
the di-geranylgeranylated protein. For Rabs with a double-cysteine
motif, this presumably allows the mono-prenylated intermediate to
remain tightly associated with REP in the cytosol until
geranylgeranylation of the second cysteine is complete. The lower
affinity of the doubly-prenylated form of the Rab for REP would then
allow the GTPase to dissociate from the escort protein when an acceptor
membrane is encountered. In the context of this model, we would expect
to see an increased cytosolic pool of mono-prenylated Rab1B-CLLL but
not Rab1B(Y78D)CLLL, since the latter cannot interact with REP and is
prenylated exclusively by GGTase I.
Immunofluorescent Localization of Rab1B in Intracellular
Membranes--
From the preceding observations we conclude that
MycRab1B(Y78D)CLLL can associate with membranes after prenylation by
GGTase I in intact cells. This apparently occurs through a mechanism that does not require the formation of a REP or GDI carrier complex. We
next carried out a study using immunofluorescence microscopy to
determine whether or not the CLLL-modified versions of Rab1B were
delivered to the same subcellular compartments as the wild-type Rab1B.
Cells expressing MycRab1B(wt) exhibited a juxta-nuclear staining
pattern that showed significant overlap with proteins known to function
in the Golgi compartment; i.e. GM130 (41) and Rab6 (42)
(Fig. 7). MycRab1B(wt) also showed
partial co-localization with a resident ER protein, calreticulin (43)
(Fig. 7). This is typical of the localization reported for Rab1 in
previous studies (5, 6, 26).
To directly compare the localization of the MycRab1B-CLLL constructs
with the localization of the wild-type protein in the same cells, we
carried out a co-transfection study in which Rab1B(wt) was tagged with
the HA epitope and the CLLL constructs were tagged with the Myc
epitope. Preliminary studies were first performed in which cells were
co-transfected with Rab1B(wt) constructs bearing the different epitope
tags. These studies established that the localization pattern of Rab1B
was not changed by the HA epitope (not shown). The images in Fig.
8 demonstrate that MycRab1B-CLLL (mono-prenylated but still competent to bind REP) had an
immunofluorescent localization pattern nearly identical to that of
HA-Rab1B(wt). The localization pattern of MycRab1B(Y78D)CLLL
(mono-prenylated, but incompetent to bind REP) was similar to that of
HA-Rab1B(wt) but also contained a peripheral component that extended
beyond the perinuclear region in a pattern reminiscent of calreticulin. From these observations we can conclude that MycRab1B(wt),
MycRab1B-CLLL, and MycRab1B(Y78D)CLLL were all targeted to ER/Golgi
membranes, although the latter construct must arrive in this
compartment by a mechanism that does not depend on REP or GDI escort
functions.
MycRab1B-CLLL and MycRab1B(Y78D)CLLL Undergo C-terminal Proteolytic
Processing and Carboxymethylation in HEK293 Cells--
Proteins
modified by GGTase I typically undergo additional modifications
consisting of the removal of the three amino acids distal to the
prenylated cysteine followed by C-terminal carboxymethylation of the
exposed prenyl cysteine residue (44). The results shown in Fig.
9 demonstrate that volatile
[3H]methyl groups were incorporated into the
immunoprecipitated MycRab1B-CLLL and MycRab1B(Y78D)CLLL when these
proteins were expressed in 293 cells incubated with
[methyl-3H]methionine. In contrast, no specific
incorporation was detected in MycRab1B or MycRab1B(Y78D), which
terminate with a typical Rab double-cysteine motif (CC) that does not
undergo carboxymethylation (45, 46). Accumulating evidence indicates
that the prenyl-cysteine carboxymethyltransferase resides in the ER
(47, 48). Therefore, these findings provide additional evidence that
Rab1B(Y78D)CLLL was delivered to membranes of the ER, independent of
interaction with REP or GDI.
The Y78D Mutation Abrogates the Activity of a Dominant-negative
Rab1B Mutant--
To determine whether the mono-prenylated Rab1B
constructs bearing the CLLL motif and/or the Y78D substitution might
interfere with ER In the present study we have examined the requirement for REP in
the membrane delivery of Rab1B in intact cells by using four different
Myc-tagged Rab1B constructs as follows. (i) Rab1B(wt) binds to REP and
is di-geranylgeranylated exclusively by GGTase II. (ii) Rab1B(Y78D)
cannot bind REP and therefore fails to undergo prenylation. (iii)
Rab1B-CLLL binds to REP and is geranylgeranylated on the single
cysteine by GGTase II, but it can also be modified in a REP-independent
manner by GGTase I. (iv) Rab1B(Y78D)CLLL cannot bind REP but can be
geranylgeranylated by GGTase I. Our results show that whereas
Rab1B(Y78D) partitioned exclusively in the cytosol, both Rab1B-CLLL and
Rab1B(Y78D)CLLL accumulated in membrane fractions of transfected cells
(Figs. 5 and 6). The subcellular distribution patterns of these
proteins resembled that of the wild-type Rab1B (Figs. 7 and 8).
Moreover, they underwent additional modifications typical of
CAAX-motif proteins (proteolytic removal of the terminal LLL
tripeptide and carboxymethylation, Fig. 9) that require enzymes
localized in the ER (48, 52). It is well established that under normal
circumstances both REP and GDI function in the delivery of prenylated
Rab proteins to intracellular membranes (38, 40, 53, 54). However,
since the Y78D mutation prevents Rab1B from binding to REP or GDI
(Figs. 3 and 4) (23), we conclude that protein interactions mediated by
these carrier proteins are not absolutely required for targeting of
Rab1B to intracellular membranes, provided that the C terminus can be
geranylgeranylated by an alternative mechanism (GGTase I).
It remains to be determined precisely how the mono-geranylgeranylated
form of Rab1B(Y78D)CLLL is delivered to membranes in the absence of REP
or GDI. However, it seems likely that the initial targeting of this
protein to the ER may follow the same path as newly prenylated Ras and
Rho GTPases. In this regard, the recent studies of Dai and
co-workers (48) suggest a model wherein the prenylated cysteine,
in the context of the CAAX motif, may serve as a signal
structure that targets proteins to a receptor on the ER membrane, so
that AAX-trimming and carboxymethylation of different GTPases can be completed by a common set of enzymes. Proteins destined
for peripheral membranes (e.g. Ras, RhoA, Rac1) are then sorted by mechanisms yet to be defined (55), whereas proteins that
normally function in the endomembrane compartment (in this case, Rab1B)
remain there.
The recent studies of Allan et al. (56) provide insights
into the role of Rab1 in the vesicle budding and fusion machinery of ER
There are two obvious possibilities that could account for the ability
of the Y78D mutation to eliminate the inhibitory activity of
Rab1B(S22N). The first is that REP (or GDI) plays a cooperative role
with the Rab1 GTPase to promote its interaction with the COPII docking
complex or nucleotide exchange factors at the vesicle membrane.
According to this model, the Y78D mutation, by preventing Rab1B from
associating with REP (23) or GDI (Fig. 4), indirectly prevents the
dominant-negative mutant from binding to its protein targets even
though it can reach the ER membrane by virtue of the prenylated
CAAX motif. Although studies of the yeast REP, Mrs6p,
suggest that REP may indeed bind to proteins in ER/Golgi membranes
(59), there is presently no direct evidence that cooperative interactions mediated by REP are absolutely required for functional Rab1B association with the COPII complex. On the contrary, in vitro experiments have shown that recombinant Rab1B can support ER
2 helix of Rab1B (i.e.
Y78D) that abolishes REP and GDI interaction without disrupting
nucleotide binding or hydrolysis. These studies demonstrate that
replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL
motif permits this protein to be prenylated by
geranylgeranyltransferase I but not II both in cell-free enzyme assays
and in transfected cells. Subcellular fractionation and
immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL,
which remains deficient in REP and GDI association is, nonetheless,
delivered to the Golgi and endoplasmic reticulum (ER) membranes. When
the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the
resulting monoprenylated construct suppressed ER
Golgi protein
transport. However, when the Y78D mutation was added to the latter
construct, its inhibitory effect on protein trafficking was lost
despite the fact that it was localized to the ER/Golgi membrane.
Therefore, protein interactions mediated by the
2 helical domain of
Rab1B(S22N) appear to be essential for its functional interaction with
components of the ER
Golgi transport machinery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2-helix (e.g. Tyr-78
Asp) completely prevent association with REP (23). By
changing the two C-terminal cysteine residues of Rab1B(Y78D) to a CLLL
motif, we were able to convert the protein to a substrate for
geranylgeranyltransferase type I (GGTase I), which can modify monomeric GTPases in the absence of REP. Despite its inability to
associate with REP or GDI, the Rab1B(Y78D)CLLL construct was delivered
to ER membranes. However, when a dominant-negative mutation was
introduced into the same protein, it failed to suppress ER
Golgi
transport. Thus, although membrane targeting of Rab1B can occur in the
absence of REP, it appears that protein interactions mediated either
directly by the Rab1B
2 helical domain or indirectly by formation of
the REP·Rab1B complex are essential for functional association
of Rab1B with the vesicular transport machinery.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CC), which lacks the two terminal cysteine residues,
MycRab1B(wt) and MycRab1B(Y78D), were generated as described previously
(23, 24). Two additional constructs involving an amino acid
substitution (S22N) or replacement of the two C-terminal cysteine
residues with CLLL were created by polymerase chain reaction
modification of the rab1B cDNA using Pfu
polymerase (Stratagene, La Jolla, CA) and appropriate oligonucleotide primers. The cDNAs were subcloned into pCMV5 (25) for expression in
mammalian cell lines and into pET17b (Novagen, Madison, WI) for
expression in Escherichia coli. The sequences of all
constructs were confirmed by dye-terminator cycle sequencing using an
ABI 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Several of the constructs were subsequently subcloned into a pCMV5 vector that
had been modified by polymerase chain reaction to encode an in-frame HA
epitope tag (YPYDVPDYA) instead of the Myc tag at the N terminus of the
expressed protein. Vectors encoding T7 epitope-tagged REP (pCMVREP1)
and FLAG-tagged GDI
(pCMVGDI
) were generated as described
previously (23, 26).
-mercaptoethanol, 2.0% SDS,
30.0% glycerol, 0.025% bromphenol blue). One-tenth of each
immunoprecipitate was subjected to immunoblot analysis to compare the
recoveries of different Rab constructs, whereas the remainder of the
sample was analyzed by SDS-PAGE and fluorography to measure
[3H]Mev incorporation.
in Intact
Cells--
HEK293 cells were co-transfected with a pCMV vector
encoding FLAG-tagged GDI
and the specified Myc-tagged Rab1B.
Twenty-four hours after transfection, cells from a 100-mm culture were
harvested, and soluble fractions were prepared as described (26).
One-tenth of this fraction was subjected to SDS-PAGE and immunoblot
analysis to check for expression of FLAG-GDI and MycRab1B. The
remaining sample was subjected to immunoprecipitation with anti-FLAG
affinity beads to detect Rab proteins bound to FLAG-GDI
, as
described in detail by Wilson et al. (26).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2
helix in the Switch-2 domain of Rab1B (e.g. Tyr- 78
Asp)
disrupt the ability of the GTPase to interact with REP, without
impairing GTP binding or hydrolysis (23). Consequently the same
mutations prevent REP-dependent prenylation of Rab proteins
by GGTase II (23, 33). We hypothesized that an
2 helix mutant might
be used to determine whether or not REP is required for the delivery of
Rab1B to intracellular membranes, provided that the GTPase could be
prenylated by an alternate REP-independent mechanism. To achieve this
goal, we took advantage of our previous finding that Rab8, which ends
with a C-terminal
CAAL2 prenylation
motif similar to that in the Rac and Rho GTPases, can be modified
either by the REP-dependent GGTase II or by the REP-independent GGTase I, which does not require the formation of a
REP·Rab complex (33). Hence, the two C-terminal cysteine residues of
Rab1B(wt) and Rab1B(Y78D) were replaced with CLLL to form a GGTase I
recognition motif (34, 35). Cell-free assays were used to compare the
prenylation of the various recombinant Rab1B proteins by GGTase I
versus GGTase II. As seen in Fig.
1, Rab1B(wt), which ends with a CC motif,
is a substrate only for GGTase II. The addition of the CLLL motif to
the C terminus of this protein permits it to be prenylated by either
GGTase I or GGTase II. Separate studies in which REP was omitted from
the reaction mixture showed that prenylation of Rab1B(wt) or Rab1B-CLLL by GGTase II was entirely dependent on the presence of the escort protein (data not shown). On the other hand, the addition of REP to the
GGTase I reaction neither permitted prenylation of Rab1B(wt) nor
enhanced prenylation of Rab1B-CLLL by GGTase I (data not shown). Most
importantly, the studies in Fig. 1 show that by substituting the normal
CC motif with CLLL, the REP binding-deficient Rab1B(Y78D) mutant could
be prenylated in a REP-independent manner by GGTase I.
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Fig. 1.
Prenylation of recombinant Myc-Rab1B proteins
by GGTase I versus GGTase II. Aliquots of
E. coli lysate containing the indicated recombinant
Myc-Rab1B proteins were geranylgeranylated in cell-free reactions. The
top panel depicts an immunoblot showing the amount of
MycRab1B present in 25% of each prenylation reaction. The bottom
panel shows the corresponding fluorograph of the remaining 75% of
the reaction, demonstrating the [3H]geranylgeranyl
pyrophosphate incorporation into the same protein.
CLLL substitution to
permit prenylation of the Rab1B(Y78D) mutant in intact cells, Myc-tagged Rab1B constructs were overexpressed in 3T3 cells, and the
relative incorporation of [3H]mevalonate into
immunoprecipitated proteins was compared (Fig. 2). Mevalonate is an isoprenoid precursor
that is incorporated into the geranylgeranyl pyrophosphate substrate
used by either GGTase I or GGTase II. Incorporation of this
precursor was measured in the presence and absence of GGTI-298, a
specific inhibitor of GGTase I (29). Several general conclusions can be
drawn from the results. First, when both GGTase I and GGTase II were
active (no inhibitor), Rab1B(Y78D) with the normal CC motif was not
labeled by [3H]Mev, whereas the same protein with
the added CLLL motif was clearly prenylated. Second, the prenylation of
Rab1B(Y78D)CLLL was completely prevented by the inhibitor, GGTI-298.
Taken together, these results confirm that the modification of
Rab1B(Y78D)CLLL in vivo is catalyzed by GGTase I, as
indicated by the cell-free assay (Fig. 1). Surprisingly, the wild-type
Rab1B-CLLL, which has only a single cysteine available for
modification, showed a greater incorporation of [3H]Mev
per unit protein than the wild-type Rab1B with the original CC motif.
However, it is important to bear in mind that this type of study does
not provide a stoichiometric analysis of prenylation, because
overexpression results in the accumulation of a large pool of
non-prenylated Rab1B in the transfected cells (26). Thus, the increased
incorporation of [3H]Mev into Rab1B-CLLL, compared with
Rab1B, may reflect the fact that two different GGTase enzyme systems
are able to prenylate the Rab1B-CLLL, whereas only the REP/GGTase II
system can prenylate Rab1B with the CC ending.
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Fig. 2.
Prenylation of Myc-Rab1B proteins expressed
in cells in the presence or absence of a GGTase I inhibitor.
Parallel cultures were transfected with each of the indicated Myc-Rab1B
constructs. The Myc-tagged proteins were immunoprecipitated from cell
lysates after an 18-h incubation with [3H]Mev in the
presence or absence of 10 µM GGTI298, a specific
inhibitor of GGTase I. In the upper panel one-tenth of each
immunoprecipitate was subjected to immunoblot analysis. In the
middle panel the remainder of the immunoprecipitate was
analyzed by fluorography to visualize the prenylated proteins. The
ratios of 3H (relative units) per unit of immunodetectable
protein (ECL signal) are indicated in the bottom panel and
are expressed as percent of the ratio determined for MycRab1B(wt) in
the absence of inhibitor. cys represents a MycRab1B construct that
had both of the terminal cysteine residues removed.
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Fig. 3.
Gel filtration analysis of MycRab1B
constructs coexpressed with T7-REP in 293 cells. 293 cells were
co-transfected with plasmids encoding the indicated Myc-Rab1B construct
and T7-REP. The cells were maintained in medium containing 10 µM lovastatin for 48 h after transfection, then cell
lysates were analyzed by gel filtration fast protein liquid
chromatography, as described under "Materials and Methods." The
individual fractions were subjected to SDS-PAGE and immunoblotted using
monoclonal antibodies against the Myc and T7 epitopes of the expressed
proteins. The graphs represent quantification of the
125I-labeled IgG from PhosphorImager
analysis.
in transfected
293 cells. Fig. 4 shows that MycRab1B and
MycRab1B-CLLL were effectively co-immunoprecipitated with FLAG-GDI,
whereas the Y78D mutants, regardless of the C-terminal cysteine motif,
were not associated with FLAG-GDI. These observations provide direct
evidence that the Switch-2 region of Rab1B is important for interaction
of the GTPase with GDI as well as with REP, implying that conserved
regions in GDI and REP are involved in binding the
2 helix of the
Rab protein. These findings are also important for the localization
studies described in the following sections, because they indicate that
any membrane targeting of Rab1B(Y78D)CLLL must occur by a mechanism
that does not depend on REP or GDI serving as a carrier.
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Fig. 4.
Co-precipitation of Rab1B proteins with
FLAG-GDI. Parallel cultures were co-transfected with plasmids
encoding FLAG-GDI and one of the indicated MycRab1B constructs.
Anti-FLAG affinity resin was mixed with the cell lysates to isolate
proteins associated with the expressed GDI. Proteins in one-tenth of
the cell lysate, before addition of the affinity resin (panel
A) and the all of the eluate from the anti-FLAG resin (panel
B), were subjected to SDS-PAGE and immunoblot analysis using the
antibodies indicated at the left of each panel.
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Fig. 5.
Subcellular fractionation of 293 cells
expressing MycRab1B proteins. Parallel cultures were transfected
with each of the indicated Myc-Rab1B constructs. Cell lysates were
fractionated by centrifugation at 100,000 × g. The
cytosolic (C) and membrane (M) fractions were
immunoblotted for Myc-tagged proteins. Chemiluminescent signals,
quantified by scanning with a LumiImager, are expressed as a percent of
the total MycRab1B detected in both fractions.
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Fig. 6.
Subcellular distribution of prenylated
Myc-Rab1B proteins. Parallel cultures were transfected with each
of the indicated Myc-Rab1B constructs. The cells were incubated with
[3H]Mev for 18 h, then the Myc-tagged proteins were
immunoprecipitated from membrane and cytosolic fractions. The
upper panel shows the fluorograph of the immunoprecipitated
proteins to visualize the prenylation. The bottom panel is a
graph of the data quantified by densitometer analysis. C,
cytosolic fractions; M, membrane fractions.
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Fig. 7.
Immunofluorescent localization of
MycRab1B. 293 cells were transfected with the expression
vector encoding MycRab1B(wt). The next day the cells were fixed and
co-stained with a Myc antibody and the indicated Golgi (Rab6 or GM130)
or ER marker (calreticulin) antibody, followed by fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine-conjugated
goat anti-mouse IgG as described under "Materials and Methods."
Gray scale images were pseudo-colored to correspond to the
red (rhodamine) and green (fluorescein
isothiocyanate) fluorescence, with yellow indicating the
overlapping regions.
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Fig. 8.
Co-localization of CLLL mutants with
HA-Rab1B(wt). 293 cells were co-transfected with expression
vectors encoding HA-Rab1B(wt) and either MycRab1B-CLLL or
MycRab1B(Y78D)CLLL. The next day the cells were fixed and co-stained
with Myc polyclonal and HA monoclonal antibodies followed by
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and
rhodamine-conjugated goat anti-mouse IgG as described under
"Materials and Methods." Gray scale images were
pseudo-colored to correspond to the red
(rhodamine) and green (fluorescein
isothiocyanate) fluorescence, with yellow indicating the
overlapping regions.
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Fig. 9.
Carboxymethylation of MycRab1B proteins
expressed in 293 cells. Parallel cultures were transfected with
each of the indicated MycRab1B constructs. The cells were incubated
with [methyl-3H]methionine for 19 h, then the
Myc-tagged proteins were immunoprecipitated (IP) and
separated by SDS-PAGE. The dried gel lanes were cut into 0.5-cm
sections, and a vapor-phase equilibration assay was used to measure the
tritium incorporation into base-hydrolyzable methyl esters. The
graphs show the amount of volatile tritium released from the
gel slices. The upper left panel is an immunoblot showing
the MycRab1B collected in one-tenth of each immunoprecipitate. The
arrow at the top of each panel marks the gel
slice containing the immunodetectable MycRab1B.
Golgi transport, we co-expressed these proteins
together with the human low density lipoprotein receptor (LDLR) in 293 cells. The LDLR undergoes O-glycosylation in the medial
Golgi compartment, resulting in a shift in its electrophoretic mobility on SDS gels from a sharp band at ~120 kDa (the immature, ER form) to
a poorly resolved band between 160 and 170 kDa (32, 49, 50). As shown
in Fig. 10, when either Rab1B(wt),
Rab1B-CLLL, Rab1B(Y78D), or Rab1B(Y78D)CLLL was co-expressed with the
LDLR, processing of the radiolabeled receptor to the mature
O-glycosylated form was readily detected by pulse-chase
analysis of the immunoprecipitated protein. Thus, the Y78D constructs
do not impair the function of endogenous Rab1-dependent ER
Golgi transport pathways. Previous studies have shown that Rab
proteins bearing amino acid substitutions at the position equivalent to
Ser-17 in Ha-Ras are locked in the inactive GDP state because they have
a greatly reduced affinity for GTP but not GDP (6, 7, 51). Introduction
of such mutations into Rab1A (S25N) or Rab1B(S22N) causes these
proteins to act as dominant suppressors of Rab1 function in cultured
cells, so that ER
Golgi protein trafficking is arrested (6). To
determine whether the mono-prenylated form of Rab1B (S22N) would still
be able to suppress ER
Golgi trafficking, we converted its C
terminus to the CLLL motif. As shown in Fig. 10, the mono-prenylated
Rab1B(S22N)CLLL suppressed LDLR processing just as well as the
Rab1B(S22N) with the normal CC motif (Fig. 10). However, when the Y78D
mutation was inserted into the Rab1B(S22N)CLLL construct, the
dominant-negative effect of the S22N mutation on ER
Golgi
trafficking of the LDLR was lost. Thus, even though MycRab1B-CLLL and
MycRab1B(Y78D)CLLL show similar localization (Fig. 8), only the former
construct suppresses ER
Golgi trafficking when the S22N
substitution is introduced.
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Fig. 10.
Effect of expression of various MycRab1B
constructs on LDL receptor processing. The indicated MycRab1B
constructs were co-expressed with the LDLR in 293 cells. 24 h
after transfection, the cells were pulse-labeled for 30 min with
[35S]Met, then chased with excess cold methionine and
cysteine for 2 h. Immunoprecipitation of the LDLR was analyzed by
SDS-PAGE and fluorography. m indicates the migration
position of the mature O-glycosylated LDLR, whereas
i indicates the position of the immature form of the
receptor found in the ER.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Golgi transport. Specifically, it appears that Rab1 in its active
GTP-bound state may recruit a tethering protein, p115, to the coat
protein complex II (COPII) on budding ER vesicles. The GTPase then
promotes the assembly of a functional SNARE
(N-ethylmaleimide-sensitive factor attachment protein
receptor) complex required for vesicle fusion with the Golgi acceptor
compartment (56). An important question raised by the present studies
is whether the functional interaction of Rab1B with the COPII complex
is affected by the specific nature of the post-translational
modifications (di-geranylgeranylation versus
mono-geranylgeranylation and carboxymethylation) or the mode of
delivery of Rab1B to the ER/Golgi membranes (REP-mediated versus prenyl CAAX-mediated translocation from
GGTase I). We approached this question by examining the effects of the
C-terminal CLLL modification and Y78D substitution on the activity of
the dominant-negative mutant, Rab1B(S22N), in intact cells. The
biological activity of this mutant depends on prenylation (7) and
presumably involves competition with endogenous Rab1 for binding to
nucleotide exchange factors or other docking proteins in the COPII
complex on the budding transport vesicle (7, 57, 58). Our results show that Rab1B(S22N)CLLL is fully capable of suppressing ER
Golgi transport of the LDL receptor (Fig. 10), implying that the relevant Rab1B(S22N) protein interactions can be supported by
mono-geranylgeranylation and carboxymethylation instead of
di-geranylgeranylation. However, when the Y78D mutation was introduced
into Rab1B(S22N)CLLL, its inhibitory effect on LDL receptor trafficking
was lost.
Golgi transport when added to perforated cells in monomeric form
rather than as a REP or GDI complex (4). The alternative explanation
for the loss of inhibitory activity that occurs when the Y78D mutation
is combined with the S22N mutation would postulate that the
2 helix
in the predicted Switch-2 region of Rab1B is not only critical for
interaction with REP and GDI but is also involved in the association of
Rab1B with docking proteins or exchange factors on the budding
transport vesicle. Support for this view comes from earlier studies of
chimeric Rab proteins showing that the
2 helix of Rab5 is one of the
domains that affects its localization and functional specificity (60).
Thus, it will be interesting to determine how amino acid substitutions
in the
2 helix of Rab1B may affects its ability to interact with
components of the COPII complex. The functional evaluation of such Rab
mutants has been hindered by the fact that they cannot be prenylated
and delivered to membranes by the REP-dependent GGTase II
pathway. However, the present study demonstrates that this problem can by circumvented by changing the C-terminal motif to one that can be
recognized by GGTase I.
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ACKNOWLEDGEMENT |
---|
We are grateful to Miguel Seabra for providing purified REP and GGTase II, Patrick Casey for providing purified GGTase I, and Said Sebti for providing GGTI-298. We also thank Deborah Heitzman for technical assistance with preparation of the HA-tagged Rab constructs.
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FOOTNOTES |
---|
¶ To whom correspondence should be addressed: Dept of Biochemistry and Molecular Biology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4100; Fax: 419-383-6228; E-mail: wmaltese@mco.edu
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M101511200
This work was supported by National Institutes of Health Grant CA34569 (to W. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 CAAL represents a cysteine residue followed by two aliphatic amino acids and a terminal leucine residue.
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ABBREVIATIONS |
---|
The abbreviations used are: ER, endoplasmic reticulum; GDI, guanine nucleotide dissociation inhibitor; GGTase, geranylgeranyltransferase; REP, Rab escort protein; HEK, human embryonal kidney; Mev, mevalonate; LDL, low density lipoprotein; LDLR, LDL receptor; COPII, coat protein complex II; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; WT, wild type.
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