Low molecular mass GTP-binding proteins encoded by the rab gene family play important roles in protein trafficking in
mammalian cells. More than 30 Rab proteins have been identified and
many of these have been localized to discrete vesicle populations or
organelles(1, 2) . Individual Rab proteins have been
implicated as essential mediators of vesicle targeting and/or fusion
events in specific segments of the exocytic and endocytic pathways. For
example, studies employing dominant-negative Rab mutants have
demonstrated that Rab1A and Rab1B function in the transport of
glycoproteins between the endoplasmic reticulum (ER) (
)and cis-Golgi(3, 4, 5) , whereas Rab5 is
required for the fusion of clathrin-coated endocytic vesicles with
early endosomes(6, 7) . It is presently unclear how
Rab proteins interact with the components of the vesicular transport
machinery. However, the most widely held view is that
geranylgeranylated Rab proteins initially associate with membranes
and/or membrane-protein complexes of the budding donor vesicle where
they are activated by exchange of GDP for GTP, facilitated by
proteins(s) that stimulate guanine nucleotide
dissociation(8, 9, 10) . In a manner yet to
be defined, the activated Rab protein helps to ensure correct targeting
of the transport vesicle. As the vesicle fuses with the appropriate
acceptor compartment, GTP hydrolysis is presumed to occur, and the
inactive Rab-GDP cycles off the membrane into a cytosolic pool from
which it can be recruited into additional rounds of transport (reviewed
in (11, 12, 13) ). Mounting evidence suggests
that the recycling of Rab-GDP involves a family of proteins termed
GDP-dissociation inhibitors (GDI's), which form 1:1 complexes
with geranylgeranylated GDP-bound Rab proteins, solubilize them from
membranes, and slow the rate of nucleotide exchange (reviewed in (14) ).
The first Rab-GDI was isolated from bovine brain and
was shown to associate with the GDP form of Rab3A and inhibit
dissociation of bound nucleotide(15, 16) . Subsequent
studies established that post-translational geranylgeranylation of the
Rab protein was required for this interaction (17) and that
bovine Rab3A-GDI can solubilize a variety of other Rab proteins from
cellular membranes(18) . The bovine Rab-GDI is now recognized
as the prototype for a family of mammalian proteins encoded by at least
three distinct genes. The
GDIs include the original bovine
Rab3A-GDI and the closely related rat GDI-
(19) , human
GDI-
(20) , and mouse GDI-1(21) . The
GDIs
are comprised of the rat GDI-
(19) , human
GDI-
(19) , and the recently identified mouse
GDI-
(22) . A third mouse GDI, designated GDI-2, has been
identified(21) . It shares 86 and 95% amino acid identity with
mouse GDI-1 (21) and GDI-
(22) , respectively, and
thus appears to be a distinct gene product.
Geranylgeranylation of
nascent Rab proteins is clearly essential for their entry into the
complex cycle of functional associations with membranes and regulatory
proteins. The modification of Rab proteins is catalyzed by
Rab:geranylgeranyltransferase (also termed GGTase II) (23, 24, 25) . In order for Rab proteins to
serve as substrates for the catalytic 
subunits of GGTase II,
they must first associate with Rab Escort Proteins (REP-1 or
REP-2)(26, 27) . In the absence of detergents,
REP's remain associated with geranylgeranylated Rab proteins
(RabGG) in vitro, suggesting that in addition to
their role in the prenylation reaction, REP's may also serve to
escort newly modified Rab proteins to downstream components of the
vesicular transport machinery(26) . Cytosolic GDIs have been
proposed as potential intermediates that can accept RabGGs
from REPs and deliver them to specific intracellular
membranes(13, 14, 26) . This view is
supported by studies showing that endogenous GDI in reticulocyte
lysates can serve as an acceptor for geranylgeranylated
Rab5(28) , and that pre-formed GDI-RabGG complexes can
deliver functional geranylgeranylated Rab proteins to transport
vesicles in cell-free systems and permeabilized
cells(29, 30, 31) . However, the recent
finding that REP itself can deliver prenylated Rabs directly to
membranes in perforated cells (32) suggests an alternative
possibility that GDI may not be required for the initial delivery of
nascent RabGG to membranes in vivo.
In an effort
to clarify the role of GDI in the initial membrane targeting of
geranylgeranylated Rab proteins in intact cells, we have identified a
Rab1B effector-domain mutant (Rab1B
) that, when
geranylgeranylated, fails to form detectable complexes with GDI in
vitro and in transfected HEK 293 cells. Studies comparing the
subcellular distribution of Rab1B
with that of
Rab1B
indicate that the initial association of the mutant
with intracellular membranes is not significantly impaired. However,
over time, the prenylated Rab1B
fails to accumulate in
the cytosolic compartment. These findings suggest that stable
association of nascent prenylated Rab1B with GDI is not required for
its initial delivery to intracellular membranes, but that GDI is
essential for returning the prenylated Rab protein to the cytosolic
compartment after it enters the vesicular transport cycle.
EXPERIMENTAL PROCEDURES
Mutagenesis of Rab1B
Mutations were introduced
into cDNA encoding Rab1B by means of overlap-extension
PCR(33) , using Taq DNA polymerase (Perkin Elmer) and
pGEM3Zrab1B(34) as the template. The cDNA encoding
Rab1B
was generated as described(35) . The cDNAs
encoding Rab1B
and Rab1B
were modified by
PCR so that the expressed proteins contained an amino-terminal
Myc-epitope (EQKLISEEDL) and were subcloned into pCMV5neo (36, 37) for expression in intact cells. Recombinant
Rab1B proteins that could be purified by Ni
chelation
chromatography were obtained by modifying the rab1B cDNAs by
PCR so that the expressed proteins contained a hexahistidine tag at
their amino termini. The sequences of all rab1B constructs
were verified by the dideoxy chain termination technique, using
Sequenase 2.0 (U. S. Biochemical Corp.).
Epitope-tagging of GDIs
The cDNA encoding bovine
GDI-
(16) was obtained by PCR from a cDNA template,
reverse transcribed from bovine brain mRNA (Clonetech). The PCR product
was cloned into pCRII (Invitrogen) and modified by addition of a 5`
sequence encoding the FLAG epitope (DYKDDDDK). The FLAG-GDI-
construct was then subcloned into
pCMV5neo to generate pCMVGDI
. The cDNA encoding mouse
GDI-2 (21) was obtained from Assia Shisheva (University of
Massachusetts) and subcloned into pTrcHisB (Invitrogen), which had been
modified to include a FLAG sequence immediately upstream of
the EcoRI site. The FLAG-GDI-2 construct was then
cloned into pCMV5.
Expression of Recombinant Rab1B and GDI in E.
coli
DNA sequences encoding Myc-Rab1B, (His)
-Rab1B,
FLAG-GDI-
, and FLAG-GDI-2 were subcloned into pET11a (GDI-
)
or pET17b (Rab1B) (Novagen, Inc.). Expression of Rab1B and
FLAG-GDI-
was induced in Escherichia coli BL21(DE3)pLysS
by incubation of bacterial cultures with 1 mM isopropyl-
-D-thiogalactopyranoside for 1 h. Cell
pellets, obtained by centrifuging 50-ml cultures for 10 min at 4,400
g, were suspended in 0.5 ml of Buffer A (50 mM HEPES, 5 mM MgCl
, 1 mM dithiothreitol, 10 µM GDP) supplemented with 1 mM phenylmethylsulfonyl fluoride, 25 µM leupeptin, 2.8
units of aprotinin. Cell lysates were prepared by freeze-thaw
(-80 °C) and cleared by centrifugation at 15,000
g for 15 min at 4 °C. The supernatant solution was stored
at -80 °C after addition of glycerol (40% v/v). To quantitate
Rab1B in different bacterial lysates, samples were subjected to
SDS-PAGE and immunoblot analysis as described
previously(38, 39) , using an affinity-purified rabbit
polyclonal IgG directed against residues 181-194 of Rab1B (Zymed
Laboratories, South San Francisco, CA) followed by
I-labeled goat anti-mouse IgG (0.45 µCi/ml). Bound
I-IgG was visualized by exposing the blots to Kodak
X-Omat AR film, and radioactivity was quantitated by scanning with a
Molecular Dynamics PhosphorImager.
Affinity Purification of FLAG-GDI
Recombinant
FLAG-GDI-
was purified from bacterial lysate (1 ml) using an
anti-FLAG M2 affinity gel column as described by the manufacturer
(Eastman Kodak). The fraction containing the 55-kDa FLAG-GDI-
was
dialyzed against Buffer A and stored at -80 °C.
Cell-free Assay for the Formation of GDI-Rab
Complexes
Aliquots of E. coli lysate containing equal
amounts of Myc-tagged or His
-tagged Rab1B
or
Rab1B
(determined by immunoblot analysis) were incubated
in Buffer A supplemented with 100 µM GDP, 0.5 mM Nonidet P-40, 80 µg/ml bovine serum albumin, 400 ng of
affinity-purified FLAG-GDI, 200 ng of REP-1, 200 ng of GGTase II

, and 2 µCi of [
H]GGPP (15 Ci/mmol,
American Radiochemical Corp.). Recombinant REP and GGTase II 
were provided by Miguel Seabra (University of Texas Southwestern
Medical Center). After a 1-h incubation at 37 °C, SDS sample buffer
was added to half of the reaction mixture and the extent of Rab1B
prenylation was assessed by SDS-PAGE and fluorography. The other half
of the reaction was added to 50 µl of a 1:1 (v/v) slurry of
anti-FLAG M2 affinity gel (Eastman Kodak Co.) which had been pre-washed
twice with 0.1 M glycine, pH 3, and 4 times with 1 ml of 10
mM NaPO
, 1 mM MgCl
, 200
µM GDP, 0.15 M NaCl, pH 7.4 (phosphate wash
buffer). Samples were mixed with the anti-FLAG resin on an end-over-end
rotator for 30 min at 4 °C and the resin was collected by
centrifugation at 12,000
g for 1 min. The supernatant
solution was removed and the resin was washed 3 times with 1 ml of the
phosphate wash buffer. FLAG-GDI complexes were then eluted with 100
µl of 0.1 M glycine, pH 3, and the eluates were
neutralized by addition of 10 µl of 1 M Tris-HCl, pH 8.
The eluates were mixed with SDS sample buffer and subjected to SDS-PAGE
on 12.5% polyacrylamide gels. Rab protein co-eluting with the FLAG-GDI
was visualized as [
H]GG-labeled protein on the
fluorogram of the dried gel. To check for the presence of
non-prenylated Rab1B in the FLAG-GDI complex, aliquots of eluates from
the anti-FLAG resin were subjected to SDS-PAGE and a
[
-
P]GTP blot overlay assay(40) .
Metabolic Labeling Assay for Geranylgeranylation of Rab1B
Expressed in Mammalian Cells
Transformed human embryonic kidney
(HEK) 293 cells were grown in Dulbecco's modified Eagle's
medium with 10% (v/v) fetal calf serum and plated at a density of 1.8
10
cells/cm
on the day before
transfection. All transfections were carried out using the calcium
phosphate precipitation technique(41) . Three hours after
addition of the DNA, cultures were shocked by exposure to 15% (v/v)
glycerol in phosphate-buffered saline for 30 s and were allowed to
recover for 1 h in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. Cultures (6 cm) were transfected with
15 µg of pCMVrab1B
,
pCMVrab1B
, or
pCMVrab1B
. Parallel cultures transfected
with each Rab1B construct were incubated for either 12 or 24 h in 3 ml
of medium containing 200 µCi/ml
[
H]mevalonolactone (3.4 Ci/mmol), beginning
immediately after recovery from the glycerol shock. During the labeling
period, 10 µM lovastatin was added to the culture medium
to block endogenous isoprenoid synthesis. Cells did not exhibit any
change in morphology or viability during the incubation period,
indicating that the radiolabeled mevalonolactone added to the medium
(approximately 60 µM) was sufficient to satisfy the
cellular requirement for mevalonate (MVA). Labeled cells were harvested
and washed three times with Hanks' balanced salt solution. Cells
were lysed in 100 µl of RIPA (100 mM Tris-HCl, pH 7.4, 2
mM EDTA, 0.1% (w/v) SDS, 0.5% (w/v) deoxycholate, 0.5% (v/v)
Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin, 1 µM pepstatin, 0.3 µM aprotinin) and each lysate was diluted 1:5 by addition of 100
mM Tris-HCl, pH 7.4, 2 mM EDTA, with protease
inhibitors. Insoluble material was removed by centrifugation at 12,000
g for 30 min. Cleared cell lysates were incubated for
2 h at 4 °C with the 9E10 anti-Myc monoclonal antibody (Oncogene
Sciences) at a concentration of 5 µg/ml lysate, and the immune
complexes were collected by addition of protein A-Sepharose coated with
goat anti-mouse IgG (Cappel). The Sepharose beads were pelleted by
centrifugation and washed once with RIPA, twice with 5
diluted
RIPA, and once with 100 mM Tris-HCl, pH 7.4, 2 mM EDTA, 100 mM NaCl. Myc-tagged Rab1B proteins were eluted
from the beads in SDS sample buffer and resolved by SDS-PAGE. Proteins
were transferred to PVDF membrane and tritium incorporation into the
Rab proteins was quantitated with a Molecular Dynamics phosphorimaging
system and ImageQuant software. Each
H value (peak volume)
was then normalized to the amount of immunoprecipitated Myc-Rab1B on
the same blot. This was accomplished by incubating the membrane with
antibody to Rab1B, followed by
I-labeled goat anti-rabbit
IgG. Bound
I-IgG was then quantitated by PhosphorImager
analysis. Before forming a ratio between the tritium and bound
I-IgG values, tritium values were corrected for any
nonspecific radioactivity in the immunoprecipitates by subtracting
corresponding values obtained for Myc-Rab1B
in
parallel [
H]MVA-labeled cultures.
Coexpression Assay for Interaction of Rab1B with GDI in
Intact Cells
HEK 293 cells growing in 10-cm dishes were
transfected with DNA mixtures containing 15 µg of pCMVFLAG-GDI (GDI
or GDI-2) combined with pCMVrab1B
(15 µg), pCMVrab1B
(40 µg),
or pCMVrab1B
(15 µg). At 48 h after
transfection, cultures were harvested and homogenized in 0.5 ml of
lysis buffer (100 mM Tris-HCl, pH 7.4, 0.1 mM GDP, 1
mM MgCl
, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride). Cytosol was obtained by
centrifuging the lysates at 100,000
g for 30 min at 4
°C. A 50-µl aliquot of cytosol was reserved for quantitation of
expressed GDI and Rab1B proteins by immunoblot analysis. The remaining
cytosol was mixed with 100 µl of a 50% suspension of anti-FLAG M2
affinity gel in phosphate wash buffer. Samples were incubated on a
rotating rack for 30 min at 4 °C and the affinity gel was collected
by centrifugation and washed twice with phosphate wash buffer. Bound
FLAG-GDI protein complexes were then eluted by suspending the gel in
100 µl of 0.1 M glycine, pH 3. After removal of the gel by
centrifugation, 10 µl of 1 M Tris-HCl, pH 8.0, was added
to the glycine supernatant solution and the samples were mixed with 25
µl of 5
Laemmli sample buffer. Proteins were resolved by
SDS-PAGE and transferred to PVDF membrane. The upper half of the
membrane was immunoblotted with anti-FLAG antibody (Eastman Kodak) and
I-labeled goat anti-mouse IgG. The lower half of the
membrane was immunoblotted with the antibody to Rab1B and
I-labeled goat anti-rabbit IgG.
Subcellular Distribution of Immunodetectable Myc-Rab1B
Expressed in 293 Cells
Myc-Rab1B proteins were coexpressed with
FLAG-GDI-
as described above. Cells were harvested 48 h after
transfection and homogenized in 0.5 ml of lysis buffer supplemented
with 0.05% Nonidet P-40. Nuclei and unbroken cells were removed by
centrifugation at 500
g for 5 min at 4 °C. The
low-speed supernatant fraction was removed and centrifuged at 100,000
g for 30 min at 4 °C to obtain a crude high-speed
membrane fraction. The latter was solubilized in 100 µl of Laemmli
SDS sample buffer(42) . The high-speed supernatant was
designated the cytosol and was mixed with 100 µl of 5
SDS
sample buffer. 50% of the membrane protein and 25% of the cytosolic
protein were subjected to SDS-PAGE and transferred to PVDF membrane.
Immunoblot analysis was performed with the Rab1B antibody and
horseradish peroxidase-goat anti-rabbit IgG (Bio-Rad).
Chemiluminescence detection was performed using the Amersham ECL kit. To visualize Myc-Rab1B by immunofluorescence, 293 cells were plated
in two-well chamber slides (Lab-Tek) coated with laminin and
transfected with pCMV constructs encoding Myc-Rab1B
,
Myc-Rab1B
, or Myc-Rab1B
. After 18 h,
cells were fixed in 4% (w/v) paraformaldehyde in phosphate-buffered
saline and permeabilized with 0.05% (v/v) Triton X-100 in
phosphate-buffered saline. Immunofluorescence staining of Myc-Rab1B was
performed as described previously(43) .
Subcellular Distribution of Geranylgeranylated Myc-Rab1B
in 293 Cells
Myc-tagged Rab1B proteins (WT, D44N, or
CC)
were transiently coexpressed with FLAG-GDI-
in 293 cells as
described above. Each culture was incubated for 16 h in 3 ml of medium
containing 200 µCi/ml [
H]MVA and 10
µM lovastatin to label the geranylgeranyl groups attached
to newly synthesized proteins. Cells were then harvested, washed three
times with Hank's balanced salt solution, and homogenized in 0.5
ml of lysis buffer without Nonidet P-40. Cells from three identically
transfected 10-cm cultures were pooled for each determination.
Subcellular fractionation was carried out as described above and
cytosol was combined with 300 µl of lysis buffer and 200 µl of
RIPA. The membranes were suspended in 200 µl of RIPA and mixed with
800 µl of lysis buffer. Myc-Rab1B proteins were immunoprecipitated
from the cytosol and membrane fractions, subjected to SDS-PAGE, and
transferred to PVDF. Tritium-labeled Myc-Rab1B proteins were
quantitated by PhosphorImager analysis. To correct for any variations
in Myc-Rab1B recovery in the immunoprecipitates, the same blot was
incubated with antibody to Rab1B and each tritium value was normalized
to the amount of immunodetectable Rab1B (bound
I-IgG). In
a separate study, [
H]MVA-labeled Myc-Rab1B
proteins immunoprecipitated from the membrane and cytosol fractions
were resolved by SDS-PAGE and visualized by fluorography.
Post-translational Processing of
-Amyloid Precursor
Protein (
APP) in 293 Cells
Cells were cotransfected with 2
µg of phCK751 (which encodes
APP
) and
pCMVrab1B
(10 µg),
pCMVrab1B
(10 µg), or
pCMVrab1B
(30 µg) as described previously (43) . Sixteen hours after transfection, the cultures were
pulse-labeled for 10 min at 37 °C with 1 ml of methionine-free
Dulbecco's modified Eagle's medium containing 100 µCi
of [
S]methionine/cysteine
(Tran
S-label, 1100-1200 Ci/mmol, ICN Inc.). Cells
were washed twice with phosphate-buffered saline and subjected to a 1-h
chase in medium containing 2 mM methionine, and 2 mM cysteine. Immunoprecipitation of
APP was then carried out as
described by Dugan et al.(43) . Immunoprecipitates
were analyzed by SDS-PAGE and fluorography. Prior to
immunoprecipitation of
APP, one-tenth of each lysate was retained
for verification of Myc-Rab1B expression by immunoblot analysis.
RESULTS
Identification of a Rab1B Mutant That Fails to
Associate with GDI in Vitro
To explore the potential roles of
GDI versus REP in the delivery of nascent prenylated Rab
proteins to intracellular membranes, we wished to identify a Rab1B
mutant that was capable of undergoing REP-dependent prenylation by
GGTase II (and thus by inference was competent to associate with REP),
but was incapable of forming a stable complex with GDI. To screen for
such mutants, we developed a cell-free immunoprecipitation assay that
measures the association of geranylgeranylated Rab proteins with FLAG
epitope-tagged bovine GDI
(Fig. 1). Recombinant Rab1B was
geranylgeranylated in a reaction mixture containing GGTase II, REP, and
[
H]GGPP. Recombinant FLAG-GDI
was added as
an acceptor for
H-labeled Rab1BGG generated during
the reaction. Upon completion of the incubation, the FLAG-GDI was
collected on anti-FLAG-agarose affinity beads and any associated
H-labeled Rab1BGG was detected by SDS-PAGE and
fluorography. As shown in Fig. 1, when this assay was applied to
Myc-Rab1B
, a substantial portion of the geranylgeranylated
Rab1B was collected with FLAG-GDI
on the affinity beads.
Association of Rab1BGG with the affinity beads was
specifically dependent on the presence of FLAG-GDI. When FLAG-GDI was
omitted from the reaction mixture, neither prenylated Rab1B (detected
by
H fluorography) nor non-prenylated Rab1B (detected by
P-GTP blot overlay) was collected on the anti-FLAG beads.
Consistent with previously published observations, the association of
Rab1B with FLAG-GDI
was entirely dependent on prenylation of the
Rab protein. Thus, when the wrong prenyltransferase was added to the
reaction (e.g. GGTase I instead of GGTase II), there was no
incorporation of [
H]GGPP into the Rab protein,
and no Rab protein was detected in eluates of the anti-FLAG beads by
[
P]GTP overlay (Fig. 1) or immunoblot
assay (not shown).
Figure 1:
Cell-free assay for GDI-Rab complexes.
Recombinant Myc-Rab1B
was geranylgeranylated in reaction
mixtures with or without recombinant FLAG-GDI, as indicated at the top. The FLAG-GDI and any associated
[
H]GG-labeled Rab1B was then collected on
anti-FLAG M2 affinity gel, eluted, and analyzed by SDS-PAGE (see
``Experimental Procedures''). All panels depict regions of
gels or blots between the 21.5- and 30-kDa markers. The fluorogram in
the top row shows the [
H]GG-labeled
Myc-Rab1B present in 25% of the total prenylation reaction mixture. The middle row shows a tritium fluorogram of protein in 50% of the
glycine eluate from the anti-FLAG beads. The bottom panel shows a [
-
P]GTP blot overlay performed
on protein in the remaining 50% of the glycine
eluate.
Using this assay, we screened a number of
Myc-Rab1B point mutants and found that one particular mutant with an
amino acid substitution in the effector domain (D44N) met the criteria
of being competent to undergo prenylation by GGTase II but incompetent
to associate with GDI. Similar results were obtained when the His
tag was substituted for the Myc tag on the Rab substrates. Fig. 2illustrates the results typically obtained when
(His)
Rab1B
and (His)
Rab1B
were compared with respect to their ability to undergo
geranylgeranylation and associate with FLAG-GDI-
in the
coprecipitation assay. As expected on the basis of our previous
attempts to prenylate Rab1B
translation products in
reticulocyte lysates(35) , recombinant Rab1B
was
prenylated less efficiently than Rab1B
. Nevertheless, by
incubating the protein for 1 h with purified REP and GGTase II

, sufficient amounts of
H-labeled
geranylgeranylated Rab1B
were generated so that
association of the prenylated protein with FLAG-GDI on the anti-FLAG
beads should have been readily detected.
Figure 2:
Rab1B
does not
coprecipitate with FLAG-GDI in vitro. Equal amounts of
recombinant (His)
-Rab1B
or
(His)
-Rab1B
were geranylgeranylated and
coprecipitated with recombinant FLAG-GDI
(see ``Experimental
Procedures''). The [
H]GG-labeled Rab1B in
the prenylation reaction and in the FLAG-GDI complex eluted from the
anti-FLAG beads were subjected to SDS-PAGE and fluorography (2-day
exposure).
Rab1B
Is Geranylgeranylated in Intact
Cells
In light of the foregoing results, we decided to extend
our studies of Rab1B
to intact cells. Since
geranylgeranylation of Rab proteins is required for their interaction
with GDI, we first conducted metabolic labeling studies to determine if
Rab1B
could be post-translationally modified in
vivo. Cultured 293 cells were transfected with plasmids encoding
Myc-tagged Rab1B
, Rab1B
, or
Rab1B
and the medium was supplemented with lovastatin
and [
H]MVA. Lovastatin inhibits endogenous MVA
synthesis, so that the [
H]MVA taken up from the
medium constitutes the principal carbon source for synthesis of GGPP
during the period of transient Rab1B expression. To avoid potential
variations in rates of accumulation of the different Rab1B proteins,
[
H]MVA was added immediately after transfection
and cells were collected after 12 or 24 h, with the
[
H]MVA being maintained in the medium throughout
the entire post-transfection incubation period. Upon harvesting the
cells, the expressed Myc-tagged Rab1B proteins were isolated from other
endogenous prenylated proteins by immunoprecipitation with an antibody
directed against the Myc epitope. To control for variations in recovery
of the wild-type and mutant Rab1B proteins in separate cultures, the
amount of [
H]MVA incorporated into the
immunoprecipitated protein was normalized to the total Myc-Rab1B
protein recovered in each sample. This was done by first subjecting the
immunoprecipitated Myc-tagged Rab1B to SDS-PAGE and electrophoretic
transfer to PVDF membrane. The incorporation of
[
H]MVA into the Rab1B protein was then
quantitated by radiometric scanning of the blot. Finally, the same blot
was probed with an affinity purified antibody to Rab1B. By using an
I-labeled secondary antibody, we were able to quantitate
the total immunodetectable Rab1B in each
H-labeled band. As shown in Fig. 3A, immunoprecipitation of
Myc-Rab1B
clearly revealed that the protein was
prenylated in 293 cells. Parallel studies with cells expressing
Myc-Rab1B
, which cannot undergo geranylgeranylation,
showed virtually no
H in the region of the blot containing
Rab1B, confirming that the precipitation with anti-Myc monoclonal
antibody effectively isolated the expressed proteins from endogenous
Rab1B and the host of other small GTPases which are heavily labeled
with [
H]MVA in 293 cells. Determination of the
H/
I ratio in the immunoprecipitated Rab1B in
two separate transfection/metabolic labeling experiments allowed a more
quantitative comparison of the geranylgeranylation of Myc-Rab1B
versus Myc-Rab1B
(Fig. 3B). After 12 or 24 h of continuous
labeling, the amount of [
H]MVA incorporated per
unit of immunodetectable Rab1B was approximately 35-50% lower in
the case of Rab1B
compared to Rab1B
. Thus,
as predicted by the studies carried out in vitro, the D44N
mutant is not prenylated as efficiently as Rab1B
when
overexpressed in intact cells. However, we were encouraged by these
findings, since they demonstrated that (a) Rab1B
is clearly capable of undergoing a significant degree of
REP-dependent prenylation by GGTase II in vivo, and (b) sufficient [
H]geranylgeranylated
Rab1B
accumulates in transfected 293 cells to permit an
analysis of the fate of the prenylated mutant with respect to its
associations with GDI and cellular membranes.
Figure 3:
Prenylation of Myc-Rab1B proteins
expressed in mammalian cells. Parallel cultures of HEK 293 cells were
transfected with pCMVrab1B
,
pCMVrab1B
, or pCMVrab1B
and the cells were incubated with [
H]MVA
for 12 or 24 h. Myc-tagged Rab proteins were then immunoprecipitated
from the cell lysates, subjected to SDS-PAGE, and transferred to PVDF
membrane. Panel A shows the results of PhosphorImager analysis
for one set of blots (24 h samples). The lower panel shows the
[
H]MVA-labeled Myc-Rab1B proteins and the upper panel depicts the results after probing the same blot
with antibody to Rab1B, followed by
I-labeled goat
anti-rabbit IgG. Panel B shows the quantitative results from
two separate experiments in which the [
H]MVA
incorporated into the immunoprecipitated Myc-Rab1B
or
Myc-Rab1B
was expressed as a ratio to the
immunodetectable Rab1B (i.e. bound
I-IgG) in
each sample.
Rab1B
Fails to Form Detectable Complexes
with GDIs in Transfected Cells
Based on the results of cell-free
assays (Fig. 2) we predicted that prenylated Rab1B
would be unable to associate with GDI in the cytosolic fraction
of intact cells. To test this prediction, Myc-tagged versions of
Rab1B
, Rab1B
, or Rab1B
were transiently coexpressed with FLAG-tagged GDI-
in 293
cells. The FLAG-GDI-
was subsequently collected from the cytosol
on anti-FLAG affinity beads and the eluates were subjected to
immunoblot analysis for the presence of Rab1B. As shown in Fig. 4, a prominent band corresponding to Myc-Rab1B (28 kDa) was
readily detected in eluates from the anti-FLAG affinity beads collected
from samples where Myc-Rab1B
was coexpressed with
FLAG-GDI-
. No Rab1B signal was observed in eluates from samples
where Myc-Rab1B
was expressed without FLAG-GDI-
,
ruling out nonspecific association of the overexpressed Myc-Rab1B with
the anti-FLAG beads. When FLAG-GDI-
was coexpressed with
Myc-Rab1B
, which lacks the carboxyl-terminal cysteine
prenylation sites, no detectable Myc-Rab1B signal was observed at 28
kDa, confirming that the interaction of Rab1B with FLAG-GDI-
was
dependent on geranylgeranylation of the Rab protein. Finally, in
agreement with the cell-free studies described earlier, we were unable
to detect any Myc-Rab1B signal associated with the FLAG-GDI-
complex collected from cells where Myc-Rab1B
was
coexpressed with FLAG-GDI-
(Fig. 4). It should be noted
that longer exposures of the blots revealed a minor 26-kDa band
corresponding to endogenous (i.e. untagged) Rab1B in these
samples. However, even at these long exposures, we did not see a band
at 28 kDa corresponding to Myc-Rab1B
.
Figure 4:
Rab1B
does not form a
detectable complex with FLAG-GDI-
in intact 293 cells.
Myc-Rab1B
, Myc-Rab1B
, or
Myc-Rab1B
were transiently coexpressed with or
without FLAG-GDI-
in HEK 293 cells as indicated below each panel.
The FLAG-GDI complexes were collected from cell lysates using anti-FLAG
affinity (see ``Experimental Procedures''). Panel A shows the results of immunoblot analysis performed on aliquots of
the cytosol before addition of the anti-FLAG resin. The primary
antibody applied to the upper segment of the blot (45-66 kDa) was
the anti-FLAG monoclonal, while the antibody applied to the lower
segment (21.5-30 kDa) was the affinity purified antibody to
Rab1B. Panel B shows the results when the same analysis was
performed on aliquots of the glycine eluates from the anti-FLAG
affinity resin. For all of the blots shown, the detection of bound
primary IgGs was accomplished with secondary
I-labeled
IgG (18 h exposure).
To determine
whether the forgoing observations might be specific for the interaction
between Rab1B
and the
-form of GDI, we performed a
similar study wherein Myc-Rab1B
was coexpressed with
FLAG-GDI-2. As shown in Fig. 5, the D44N mutant also failed to
form a detectable complex with FLAG-GDI-2, suggesting that the change
induced by the D44N substitution impairs the ability of Rab1B to
associate with all forms of GDI.
Figure 5:
Rab1B
does not form a
detectable complex with FLAG-GDI-2 in intact 293 cells.
Myc-Rab1B
, Myc-Rab1B
, or
Myc-Rab1B
were transiently coexpressed with or
without FLAG-GDI-2 in HEK 293 cells as indicated below each
panel. The FLAG-GDI complexes were collected from cell lysates using
anti-FLAG affinity gel. Immunoblot analyses for FLAG-GDI and Rab1B were
performed on aliquots of cytosol (Panel A) or the eluates from
the anti-FLAG affinity resin (Panel B) as described in the
legend to Fig. 4. It should be noted that the FLAG-GDI-2
exhibits an electrophoretic mobility similar to that observed for
FLAG-GDI-
(approximately 55 kDa), whereas previous reports have
observed untagged GDI-2 or GDI-
migrating at 45-46
kDa(57, 58) . The basis for this discrepancy is
presently unknown.
Rab1B
Is Delivered to Intracellular
Membranes
The preceding results suggest that if GDI plays an
essential role in the delivery of nascent prenylated Rab proteins to
intracellular membranes, there should be a depletion of Rab1B
in the membranes of cells expressing this mutant. To test this
hypothesis, we performed immunofluorescence localization of the
Myc-tagged Rab1B proteins expressed in 293 cells (Fig. 6). When
cells were stained with the antibody to the Myc epitope, most of the
Myc-Rab1B
appeared to be concentrated in punctate
perinuclear structures, consistent with the expected localization of
Rab1B in ER and Golgi membranes. In contrast, cells expressing
Myc-Rab1B
exhibited a diffuse cytoplasmic staining
pattern, reflecting the inability of this mutant to associate with
membranes. The staining pattern observed for Myc-Rab1B
was not discernably different from that observed for
Myc-Rab1B
.
Figure 6:
Immunofluorescence localization of
Myc-Rab1B in HEK 293 cells. The indicated Myc-tagged Rab1B proteins
were transiently coexpressed with FLAG-GDI-
in parallel cultures
of HEK 293 cells. One day after transfection the cells were fixed and
stained with anti-Myc monoclonal antibody and fluorescein
isothiocyanate-goat anti-mouse IgG (see ``Experimental
Procedures'').
To obtain additional information about the
localization of Rab1B
, we carried out immunoblot
analyses of cytosol and membrane fractions obtained from 293 cells that
were transiently expressing Myc-Rab1B
,
Myc-Rab1B
, or Myc-Rab1B
(Fig. 7). The results confirmed that Myc-Rab1B
was localized predominantly in the cytosol, whereas a significant
portion of the Myc-Rab1B
was localized in the membrane
fraction. The absence of Myc-Rab1B
in the high-speed
membrane fraction is noteworthy because it suggests that potential
aggregates of overexpressed non-prenylated Myc-Rab1B do not partition
in this fraction. In agreement with results of the immunofluorescence
analysis, the proportion of Myc-Rab1B
localized in the
membrane fraction was not noticeably different from that observed in
cells expressing the wild-type protein. Taken together, these
observations suggest that the apparent inability of Rab1B
to form stable complexes with GDI does not prevent delivery of
the mutant protein to intracellular membranes.
Figure 7:
Subcellular distribution of Myc-Rab1B in
293 cells. Membrane and cytosolic fractions were prepared from separate
cultures of 293 cells that were coexpressing the indicated Myc-Rab1B
proteins with FLAG-GDI-
. Aliquots of each fraction were subjected
to SDS-PAGE and were immunoblotted with antibody against
Rab1B.
Geranylgeranylated Rab1B
Does Not
Accumulate in the Cytosol
The conclusion that newly synthesized
Rab1B
can be targeted to membranes without forming a GDI
complex assumes that the behavior of this mutant with respect to the
coexpressed FLAG-GDIs reflects a similar inability to form stable
complexes with any endogenous GDIs that may be present in the 293 cell
line. As discussed previously, GDI is thought to play a key role in
returning geranylgeranylated Rab proteins to the cytosol after they
have hydrolzyed GTP in conjunction with vesicle fusion with the
appropriate acceptor compartment. Hence if Rab1B
is
incapable of associating with all GDIs (both epitope-tagged and
endogenous), one would predict that any geranylgeranylated
Rab1B
that is initially delivered to intracellular
membranes would be unable to recycle. Consequently, at steady state,
the cytosol should be markedly depleted of prenylated
Rab1B
. The immunoblot of total expressed Rab1B depicted
in Fig. 7shows that transfected 293 cells harbor a substantial
cytosolic pool of Myc-Rab1B
. However, this type of
analysis does not discriminate between geranylgeranylated Rab1B and
non-prenylated (i.e. non-functional) protein that may
accumulate in the cytosol if overexpression of Rab1B overloads the
REP/GGTase II machinery. To obtain a more precise indication of the
membrane partitioning and recycling of the geranylgeranylated pool of Rab1B, we labeled the prenyl groups attached to nascent
proteins by incubating 293 cells in medium containing
[
H]MVA for the first 16 h after transfection. The
radiolabeled Myc-tagged Rab1B was then immunoprecipitated from the
cytosol and membrane fractions and analyzed by SDS-PAGE and
fluorography.As shown in Fig. 8A, prenylated
Myc-Rab1B
and Myc-Rab1B
were both readily
detected in the membrane fraction. However, in contrast to the
wild-type protein, there was no [
H]MVA-labeled
Myc-Rab1B
in the cytosolic pool. In a separate
experiment, the immunoprecipitated Rab proteins were transferred to
PVDF membranes and tritium was quantitated by PhosphorImager analysis.
The same blots were then probed with an antibody to Rab1B to estimate
the relative amount of Rab1B collected in each of the
immunoprecipitates. The results, which are depicted in Fig. 8B, confirmed that the lack of a tritium signal in
the cytosolic D44N sample was not due to an absence of Myc-Rab1B
protein in the immunoprecipitate. The results also indicated that the
specific radioactivity of the membrane associated pools of Rab1B
and Rab1B
(i.e. the ratio of
H to bound
I-IgG) were comparable (Fig. 8B), suggesting that if aggregated non-prenylated
protein contributes to the total Rab1B detected in the membrane
fraction, the proportion of such protein is not substantially greater
in cells expressing the D44N mutant than it is in cells expressing
wild-type Rab1B. Parenthetically, the results of this experiment also
show that the relative amount of [
H]MVA
incorporated per unit of immunodetectable Myc-Rab1B
was
substantially higher in the membrane fraction than in cytosol. Thus, it
is reasonable to conclude that much of the immunodetectable Myc-Rab1B
in the cytosol from transfected 293 cells is not post-translationally
modified. This reinforces the importance of the
[
H]MVA metabolic labeling studies, which can
specifically track the fate of the geranylgeranylated pool of Rab1B.
Figure 8:
Geranylgeranylated Rab1B
accumulates in the membrane fraction but is absent from the cytosol.
The indicated Myc-Rab1B proteins were coexpressed with FLAG-GDI-
in 293 cells that were labeled with [
H]MVA for 16
h. Myc-tagged Rab1B proteins were immunoprecipitated from the membrane
and cytosol fractions and subjected to SDS-PAGE. In the experiment
depicted in Panel A, the immunoprecipitated proteins were
subjected to fluorography. In a separate experiment depicted in Panel B, the amount of [
H]MVA
incorporated into each of the immunoprecipitated Myc-Rab1B proteins was
quantitated by PhosphorImager analysis. The amount of immunodetectable
Myc-Rab1B present in each sample was then determined by incubating the
same blot with antibody to Rab1B and
I-labeled goat
anti-rabbit IgG. The graphs at the bottom of the figure show
the results obtained when the
H values were normalized to
the amount of bound
I-IgG.
Overexpression of Rab1B
Does Not Impair ER
Golgi Trafficking in 293 Cells
We used the
post-translational maturation of the Alzheimer's
-amyloid
precursor protein (
APP
) to assess the consequences
of overexpression of Myc-Rab1B
on ER
Golgi
trafficking of glycoproteins in 293 cells.
APP undergoes an
increase in its apparent molecular mass on SDS gels after it is
translocated from the ER to the Golgi apparatus and undergoes O-glycosylation(44, 45) . We have previously
shown that when
APP
is transiently coexpressed with
a Rab1B mutant with reduced affinity for guanine nucleotides
(Myc-Rab1B
), the Golgi-dependent post-translational
maturation of
APP is blocked(43) , consistent with the
established ability of such Rab mutants to suppress the function of
their endogenous counterparts(3, 5) . The pulse-chase
studies depicted in Fig. 9show that when Myc-Rab1B
was coexpressed with
APP
at a level comparable
to that observed in cells expressing Myc-Rab1B
, there was
no detectable inhibition of
APP
maturation. In
contrast, Myc-Rab1B
was a potent inhibitor of
APP
maturation, even when expressed at a much lower
level than the WT and D44N proteins.
Figure 9:
Overexpression of Rab1B
does not impair Golgi-dependent post-translational processing of
APP. Cells that were transiently coexpressing
APP
with the indicated Myc-Rab1B proteins were pulse-labeled with
[
S]methionine and harvested immediately (0 h) or
after a 1-h chase. In Panel A the radiolabeled
APP was
immunoprecipitated and subjected to SDS-PAGE and fluorography.
Positions of the mature (fully processed) and immature (incompletely
processed) forms of
APP
are indicated by the symbols m and i, respectively. To check the expression of
each Myc-Rab1B protein, equal aliquots from each of the
``chase'' lysates used for immunoprecipitation of
APP
were immunoblotted with antibody against Rab1B (Panel B). Both
the expressed Myc-tagged Rab1B (upper band) and the endogenous
Rab1B (lower band) were detected in these
blots.
DISCUSSION
Recent studies have suggested two alternative models to
describe how REP and GDI may function in the delivery of nascent
geranylgeranylated Rab proteins to intracellular membranes and in the
recycling of Rab proteins once they are engaged in the vesicular
transport machinery. One model predicts that REP remains tightly
associated with RabGG upon completion of the prenylation
reaction and then escorts the modified Rab protein directly to the
appropriate intracellular membrane compartment(32) . According
to this model, the primary function of GDI would be to solubilize and
recycle GDP-bound prenylated Rab proteins after they have begun to
function in vesicular transport. An alternative view originates from
the finding that GDI can serve as an acceptor of prenylated Rab
proteins from the REP-GGTase II enzyme complex in cell-free systems (28) (also see Fig. 1and Fig. 2). It envisions
that GDI not only functions in Rab recycling, but also mediates initial
membrane targeting of nascent Rab proteins by serving as an obligatory
intermediate between the translation/prenylation machinery and the
acceptor membrane. In the present study we have attempted to
discriminate between these possibilities by first identifying a Rab1B
mutant (Rab1B
) that undergoes REP-dependent
geranylgeranylation but does not form a complex with GDI, and then
examining the fate of this protein in transfected 293 cells. Our
results support a model in which delivery of newly synthesized and
geranylgeranylated Rab1B
to intracellular membranes can
be accomplished without the formation of a stable Rab-GDI complex,
implying that this function is performed by REP or other unidentified
accessory proteins (Fig. 10). However, our metabolic labeling
studies also show that without the capability to associate with GDI,
prenylated Rab1B
fails to accumulate in the cytosolic
compartment. This provides a direct in vivo confirmation of
the proposed role of GDI in returning prenylated Rab proteins to the
cytosol during the vesicular transport cycle (Fig. 10).
Figure 10:
Hypothetical roles of REP and GDI in the
initial membrane targeting and recycling of Rab proteins. Our results
with Rab1B
indicate that: 1) nascent Rab proteins are
geranylgeranylated and then delivered directly to intracellular
membranes without the obligatory formation of a Rab-GDI complex. The
properties of REP suggest that it may perform this function. The
prenylated Rab protein then participates in a round of vesicular
transport, with GTP hydrolysis presumably occurring at the end point of
the cycle (vesicle fusion with the acceptor membrane). Our results
indicate, that 2) the eventual return of the geranylgeranylated
GDP-bound Rab protein to the cytosol is absolutely dependent on its
ability to associate with GDI.
Geranylgeranylation of Rab1B
We
previously reported that Rab1B proteins bearing mutations in the
effector domain are not efficiently geranylgeranylated when translated
in rabbit reticulocyte lysates(35, 46) . However, in
the present study we found that recombinant
(His)
Rab1B
incubated with REP and GGTase II

in vitro could be geranylgeranylated to levels
approximating 40-50% of those attained in similar reactions with
Rab1B
(see Fig. 2). Similarly, when
Myc-Rab1B
was transiently overexpressed in 293 cells,
the incorporation of [
H]MVA per unit of
immunodetectable protein was 60-70% of that observed for the
wild-type protein (Fig. 3). One possible explanation for these
different findings is that Rab1B
has an altered affinity
for either REP or the GGTase II catalytic components. Thus, when
relatively high concentrations of substrate protein or enzyme
components are combined in vitro, or when Rab1B
is overexpressed for 12-24 h in transfected cells, it is
possible to obtain significant accumulation of geranylgeranylated
protein. While a detailed examination of this issue is beyond the scope
of the present study, two important inferences can be drawn: first,
since prenylation of Rab proteins by GGTase II is completely dependent
on the formation of a Rab-REP
complex(24, 26, 27) , the reasonably
efficient prenylation of Rab1B
compared to Rab1B
in transfected 293 cells implies that the effector-domain mutant
is capable of productive interaction with REP in vivo. Second,
since prenylation of Rab proteins is sensitive to conformational
perturbations (35, 47, 48, 49) and
requires occupancy of the guanine nucleotide binding site(50) ,
it is highly probable that the pool of Myc-Rab1B
that
has undergone geranylgeranylation is not grossly misfolded or
aggregated.
Inability of Rab1B
to Associate with
GDI
Although Myc-Rab1B
can undergo
geranylgeranylation, thereby meeting one of the major criteria for
interaction of Rab proteins with
GDI(17, 51, 52) , we were unable to
demonstrate the formation of stable complexes between this mutant and
epitope-tagged GDI-
or GDI-2 in cell-free systems and intact
cells. It is important to emphasize that the coprecipitation assay used
in these studies readily detects complexes formed between both forms of
GDI and Myc-Rab1B
(Fig. 1, Fig. 4, and Fig. 5), as well as a variety of other epitope-tagged Rab1B
point mutants. (
)Thus, the disruption of Rab1B interaction
with GDI appears to be very specifically related to the introduction of
the D44N substitution in the predicted
2/loop-2 junction of the
effector domain. This finding agrees with a previous study of Rab6, in
which the exchange of an 11-amino acid segment of the effector domain
of Rab6 with the corresponding region from H-Ras rendered Rab6
incapable of being solubilized from membranes by recombinant
GDI(48) .The molecular basis for the disruption of GDI
interaction with geranylgeranylated Rab1B
is presently
unknown. Although incomplete prenylation of the mutant (i.e.,
incorporation of only one geranylgeranyl group) is possible, this
probably would not explain the absence of GDI-Rab1B
complexes, since previous studies have shown that
mono-geranylgeranylated Rabs can be incorporated into GDI
complexes(18, 52) . Another possibility is that the
D44N alteration prevents nucleotide binding. However, several lines of
evidence suggest that this is unlikely: first, studies of Rab3A (53) and Ypt1 (the yeast homolog of Rab1) (54) have
shown that substitutions at the position equivalent to Asp
do not significantly affect nucleotide binding. Second, in blot
overlay assays we have found that recombinant Myc-Rab1B
binds [
-
P]GTP to the same extent as
Myc-Rab1B
.
Third, as mentioned above,
prenylation of Rab proteins by REP/GGTase II requires that the Rab
substrate contain bound nucleotide. Thus, the effective prenylation of
Myc-Rab
in intact 293 cells (Fig. 3) implies that
guanine nucleotide binding is not significantly compromised. It remains
possible that Rab1B
is able to bind GTP but, because of
altered GTPase activity, is unable to assume the GDP state that
interacts preferentially with GDI. Indeed, Becker et al.(54) have shown that Ypt1
is not stimulated
by GAP activity to the same extent as the wild-type protein. Although
we cannot completely discount this possibility, it would be difficult
to reconcile this notion with the fact that recombinant
Myc-Rab1B
fails to associate with GDI even when prepared
and prenylated in buffers containing exclusively GDP (Fig. 2).
Moreover, we have found that when [
-
P]GTP
is bound to Myc-Rab1B
on nitrocellulose membranes, the
rate at which the mutant protein converts the bound GTP to GDP is
essentially the same as for Myc-Rab1B
.
We
favor the hypothesis that the
2/loop-2 domain represents a site of
direct interaction between Rab1B and GDI, and that conformational
alterations conferred by the D44N substitution directly interfere with
association of the two proteins.
Delivery of Newly Prenylated Rab Proteins to
Intracellular Membranes
The present studies show that when
recombinant GDI is added to a cell-free prenylation reaction containing
REP and GGTase II, the GDI is capable of serving as a soluble acceptor
for nascent Rab1BGG ( Fig. 1and Fig. 2). This
observation is consistent with a recent report describing the transfer
of newly prenylated Rab5 to GDI in reticulocyte lysate(28) .
However, our studies with the D44N mutant strongly suggest that
transfer of nascent Rab1BGG to GDI is not an obligatory
intermediate step for delivery of the newly prenylated protein to
intracellular membranes in intact cells. Three separate experimental
approaches provided evidence that the delivery of Myc-Rab1B
to membranes was not impaired by the inability of this mutant to
form stable complexes with GDI in 293 cells. First, the pattern of
immunofluorescence staining of Myc-Rab1B
was not
substantially different from that of Myc-Rab1B
, whereas
the same technique easily revealed the failure of Myc-Rab1B
to associate with intracellular membranes (Fig. 6).
Second, comparable amounts of Myc-Rab1B
and
Myc-Rab1B
were detected in membrane fractions by
immunoblot analysis, whereas the same fractions were devoid of
overexpressed Myc-Rab1B
(Fig. 7). Third, using
a metabolic labeling approach that specifically traces the fate of the
geranylgeranylated pool of Rab1B, we found that comparable amounts of
prenylated (i.e. [
H]MVA-labeled)
Myc-Rab1B
and Myc-Rab1B
could be
immunoprecipitated from membranes obtained from cells expressing these
proteins (Fig. 8). The general immunofluorescence staining
pattern for Myc-Rab1B
was consistent with ER/Golgi
localization and gave no indication that the mutant was grossly
mistargeted (e.g. to peripheral vesicles or plasma membrane).
However, the inability of immunofluorescence light microscopy and
simple centrifugation steps to resolve ER membranes, transitional
vesicles, and Golgi subcompartments leaves open the possibility that
the precise membrane localization of Myc-Rab1B
was
different from that of Myc-Rab1B
. Indeed, as discussed
below, one might predict that the failure of Rab1B
to
undergo GDI-mediated recycling at the end of each round of ER
Golgi transport might result in its disproportionate accumulation in
the acceptor compartment (i.e. the cis-Golgi).
Role of GDI in Recycling of Prenylated Rab
Proteins
Our studies of the subcellular distribution of
geranylgeranylated Rab1B
in 293 cells (Fig. 8)
highlight the key role of GDI in maintaining a steady-state cytosolic
pool of prenylated Rab proteins. However, insofar as the same studies
also suggest that GDI may not function as an obligatory soluble
intermediate in the delivery of newly prenylated Rab proteins to
intracellular membranes, they raise a perplexing question for future
investigation. That is, if the GDIs are able to accept prenylated Rab
proteins from REP in cell-free systems, how might REP be able to
deliver newly prenylated Rab proteins to membrane acceptors in vivo without competitive interference from the relatively abundant
cytosolic pool of GDI? One possibility is that nascent Rab proteins
associated with REP in intact cells are in the GTP-bound state and thus
are not in the correct conformation for recognition by GDI. This seems
unlikely in light of studies suggesting that GGTase II preferentially
modifies Rab proteins that are in the GDP-bound state(50) .
Another possibility is that upon completion of the prenylation reaction
the REP-RabGG complex associates with as yet unidentified
accessory proteins that participate in site-specific membrane targeting
of individual Rab proteins, while simultaneously masking RabGG from GDI. Finally, it is conceivable that not all of the GDI in
intact cells is in an active state that is competent to bind prenylated
Rab proteins, so that the potential for GDI to compete with REP for
binding of nascent RabGG is limited. In this regard, reports
of phosphorylation (55) and isoelectric shifts of GDIs (56) are intriguing, since they suggest that post-translational
modifications may be involved in regulating the activity of the GDI
pool in vivo.
ER
Golgi Transport in Cells Overexpressing
Myc-Rab1B
We have been unable to detect any
perturbation of glycoprotein trafficking in 293 cells overexpressing
Myc-Rab1B
(Fig. 9), using an established
pulse-chase assay that measures the Golgi-dependent processing of
coexpressed
APP
in this cell line(43) .
These findings agree with those of Tisdale et al.(3) ,
who observed that Rab1B
had no effect on vesicular
stomatits virus-G protein processing in HeLa cells. Since these assays
readily reveal trans-dominant suppression of ER
Golgi transport
by Rab1B mutants that have reduced affinity for guanine nucleotides (e.g. N121I) or are predominantly in the GDP state (e.g. S22N) (3, 43) (see Fig. 9), the
APP
processing studies conducted with Rab1B
further support
the assumption that the D44N mutation does not markedly impair the
ability of Rab1B to bind GTP. In light of these findings, we speculate
that geranylgeranylated Myc-Rab1B
delivered to
intracellular membranes is competent to support a single round of
vesicular transport between the ER and Golgi complex. The inability of
the D44N mutant to undergo GDI-mediated recycling might therefore have
little overall impact on ER
Golgi transport in transfected cells
that are continuously delivering newly prenylated Rab1B
to the donor compartment (i.e. the ER). If
Rab1B
does in fact accumulate in the acceptor
compartment (i.e. the Golgi complex) as a result of its
inability to associate with GDI, this apparently does not interfere
with subsequent rounds of vesicle fusion as might be expected if the
mutant were to irreversibly occupy key components of the docking/fusion
machinery. Further insight into this issue should be forthcoming as we
learn more about the precise subcellular localization and properties of
Rab1B proteins with alterations in the
2/loop-2 region.