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Vol. 14, Issue 5, 1882-1899, May 2003
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Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, London SW7 2AZ, United Kingdom
Submitted October 10, 2002;
Revised December 23, 2002;
Accepted January 23, 2003
Monitoring Editor: Suzanne Pfeffer
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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and propose that the mistargeting of Rabs to the ER results from
loss of targeting information. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Seabra, 1998). Prenylation is
required for proteins to associate with cellular membranes. In the absence of
prenyl modification, the affected proteins remain cytosolic and are unable to
function properly.
Three distinct protein prenyltransferases have been identified in
eukaryotic cells: protein farnesyl transferase (PFT), protein geranylgeranyl
transferase type-I (PGGT), and Rab geranylgeranyl transferase (RGGT)
(Casey and Seabra, 1996). The
first two enzymes are also called CAAX prenyltransferases due to
their ability to recognize proteins containing a CAAX motif on their
carboxyl terminus. In this motif, C is a cysteine, A is an aliphatic
amino acid and X is a methionine, serine, glutamine, or alanine for
PFT substrates (e.g., Ras and nuclear lamins), or a leucine or phenylalanine
for PGGT substrates (e.g., Rho and Rac). Unlike the CAAX
prenyltransferases, RGGT only recognizes Rab substrates in the context of a
1:1 complex with a Rab escort protein (REP)
(Andres et al., 1993;
Anant et al., 1998;
Seabra, 2000). REP recognizes
newly synthesized Rabs and presents them to RGGT, which then catalyzes the
transfer of GG groups to Rabs (Seabra,
2000; Thoma et al.,
2001b,c).
Alternatively, newly synthesized Rabs can associate with a preformed REP:RGGT
complex (Thoma et al.,
2001a)
After prenylation in the cytosol, CAAX-containing GTPases are
first targeted to the endoplasmic reticulum (ER) and only subsequently to
their site of action (Choy et
al., 1999; Apolloni et
al., 2000; Michaelson
et al., 2001). This common ER entry site seems to be a
consequence of the specific localization of the two enzymes that modify the
CAAX-GTPases downstream of the prenyltransferases
(Dai et al., 1998;
Schmidt et al.,
1998). These are a prenyl-CAAX protease (Rce1 in mammals,
RCE1 or AFC1 in Saccharomyces cerevisiae) that
cleaves the last three amino acids (AAX) in the CAAX motif
and an isoprenyl cysteine carboxyl methyltransferase (Icmt in mammals,
STE14 in S. cerevisiae) that methylates the newly exposed
prenylated cysteine. The CAAX motif alone, when fused to EGFP, is
able to target this otherwise soluble protein to the ER and Golgi
(Choy et al., 1999).
Transport of the CAAX proteins from the ER to the plasma membrane
requires a second signal within the hypervariable region of these proteins,
which is palmitoylation for H-Ras and N-Ras, or a polybasic amino acid
sequence for K-Ras (Hancock et
al., 1991). This second signal is responsible for different
trafficking routes. H-Ras and N-Ras are targeted to the plasma membrane via
the classical exocytic pathway, whereas K-Ras travels via an alternative path,
which seems to be microtubule dependent
(Choy et al., 1999;
Apolloni et al.,
2000). These mechanisms seem to apply to Rho proteins except that
some Rho proteins associate with the cytosolic protein RhoGDI, which adds an
additional level of complexity to Rho membrane association
(Michaelson et al.,
2001).
Much less is known about how Rab proteins associate with membranes and the
role of the prenylation motif in the process. There are six different
carboxyl-terminal motifs on the 60 known human Rab proteins, XXXCC,
XCCXX, XX- CXC, CCXXX, XXCCX and
XCXXX (Pereira-Leal and Seabra,
2000,
2001). Most Rab proteins
contain two carboxyl-terminal cysteines, both of which undergo
geranylgeranylation (Farnsworth et
al., 1994). One example is Rab5a (terminating in the sequence
XCCXX), a ubiquitous Rab involved in the regulation of early
endocytic traffic (Zerial and McBride,
2001). Activated Rab5a interacts with >20 proteins, reflecting
the complexity of its mechanism of action
(Christoforidis et al.,
1999). Proposed functions for Rab5 include promoting homotypic
early endosome fusion, regulating clathrin-dependent endocytosis at the plasma
membrane, integrating signal transduction with endocytosis, and regulating the
motility of early endosomes along microtubules
(Tall et al., 2001;
Zerial and McBride, 2001).
Another example of a doubly geranylgeranylated Rab is Rab27a (terminating in
the sequence XXCXC), a cell-type specific Rab that localizes
to melanosomes, melanin-containing granules within melanocytes, and other
lysosome-related organelles (Marks and
Seabra, 2001). Rab27a regulates the transport of melanosomes to
the periphery of melanocytes via interaction with melanophilin and myosin Va
(Matesic et al.,
2001; Fukuda et al.,
2002; Hume et al.,
2002; Provance et
al., 2002; Strom et
al., 2002; Wu et
al., 2002). In cytotoxic T lymphocytes, Rab27a regulates a
late step in the regulated secretion of lytic granules
(Haddad et al., 2001;
Stinchcombe et al.,
2001). Consequently, mutations in Rab27a result in partial
albinism and immunodeficiency with hemophagocytic syndrome, a disease called
Griscelli Syndrome (Seabra et
al., 2002).
Intriguingly, some Rabs possess only one prenylatable carboxyl-terminal
cysteine. Most, but not all, mono-cysteine–containing Rabs contain
sequences that do not conform to the CAAX motif. For example, Rab13
possesses a CSLG motif, which is unlikely to serve as substrate for the
CAAX prenyl transferases. Conversely, Rab8 contains a CVLL motif,
which may be geranylgeranylated by both RGGT and PGGT in vitro. Nevertheless,
the majority of Rab8 seem to be geranylgeranylated in vivo via the REP/RGGT
pathway (Wilson et al.,
1998).
The functional significance for the existence of different prenylation
motifs and their role in Rab membrane targeting remains obscure.
Geranylgeranylation itself is essential for membrane attachment but must serve
as a rather nonspecific signal as all Rabs are geranylgeranylated. Replacement
of the di-cysteine GGCC carboxyl-terminal sequence in Rab1b for a
mono-cysteine motif CLLL did not seem to affect targeting of Rab1b to the
endoplasmic reticulum (ER) and Golgi apparatus, suggesting that the
prenylation motif is not important for Rab targeting
(Overmeyer et al.,
2001). Carboxyl methylation could potentially confer specificity
to the membrane association of Rabs because only those containing the
XXCXC motif are methylated by isoprenylcysteine
methyltransferase (Icmt) (Newman et
al., 1992; Giannakouros
et al., 1993; Li and
Stahl, 1993; Smeland et
al., 1994; Bergo et
al., 2001). However, loss of Icmt activity (and consequently
loss of Rab methylation) does not lead to mistargeting of Rab6 in
Icmt—/— cells
(Bergo et al., 2001).
The present studies were designed to address the importance of the prenylation
motif in Rab membrane targeting.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The myc-epitope EQKLI-SEEDL was introduced into pCMV7
(Andersson et al.,
1989) (Hume and Seabra, unpublished data). Human Rab5a cDNA was
subcloned into pCMV7-myc after PCR amplification to introduce KpnI
and BamHI sites. The myc-Rab5aCVLL construct was obtained by
introducing the CVLL sequence into pCMV7-myc-Rab5a by using the Quickchange
mutagenesis kit. pEGFP-Rab27a was prepared as described previously
(Hume et al., 2001).
This construct served as a template to generate pEGFP-Rab27aCVLL by using the
Quickchange site-directed mutagenesis kit. The myc-epitope fused with
Rab27aCVLL cDNA generated by PCR was subcloned into the XhoI site of
the eukaryotic expression vector pCAGGS, which contains the chicken
-actin promoter with CMV-IE enhancer, and the rabbit -globin
poly(A) signal (Barral et al.,
2002). The pEGFP-CVLL and pEGFP-CVIM were obtained by replacing 12
base pairs within the stop codon region of the multiple cloning site of
pEGFPC3 vector (GAT CTA GAT AAC) for the appropriate base pairs using the
Quickchange site-directed mutagenesis kit (Hume and Seabra, unpublished data).
Similar constructs were also donated by Mark Philips (New York University
School of Medicine, New York, NY). pEGFP-Cdc42 was a gift from Alan Hall
(University College, London, United Kingdom). Bovine GDI cDNA was
amplified using Pfu DNA polymerase from a pCB6 vector (a gift from J.
Gruenberg, University of Geneva, Geneva, Switzerland), digested with
EcoRI and XhoI, and ligated into pGEX-4T1 vector. The
sequence of all inserts was confirmed by sequencing.
Recombinant Proteins
Recombinant His6-Rab5a, His6-Rab27a, and
His6-KRas proteins were produced in Escherichia coli and
purified by nickel Sepharose-affinity chromatography as described previously
(Seabra and James, 1998). The
plasmid pGEX-4T3-Rac1 was a gift from Melanie Cobb (University of Texas
Southwestern Medical Center, Dallas, TX). Recombinant GST-Rac1 and
GST-GDI were expressed in BL21 cells and purified on
glutathione-agarose. The recombinant protein prenyltransferases (RGGT, PGGT,
and PFT) and REP were prepared by infection of Sf9 cells with recombinant
baculoviruses encoding each subunit of the desired enzyme and purified by
nickel Sepharose-affinity chromatography as described previously
(Armstrong et al.,
1995; Seabra and James,
1998). All recombinant proteins were snap frozen in small aliquots
and stored at —80°C until use.
In Vitro Prenylation Assays
In vitro prenylation of His6-Rab proteins in 25-µl reaction
volumes was performed as described previously
(Seabra and James, 1998). For
the RGGT assays, 10 µM Rab was incubated with 25 nM RGGT and 5 µM
[3H]geranylgeranylpyrophosphate (GGPP) in the presence of various
concentrations of REP (0–6 µM) at 37°C for 10 min. For the PGGT
assays, various concentrations of the small GTPases (0–10 µM) were
incubated with 20 nM PGGT and 5 µM [3H]GGPP at 37°C for 10
min. For the PFT assays, various concentrations of the small GTPases
(0–10 µM) were incubated with 40 nM PFT and 1.2 µM
[3H]FPP at 37°C for 15 min. In all of the prenylation
experiments, the [3H]isoprenyl transferred to the small GTPases was
measured by scintillation counting as the precipitable radioactivity after
filtration of the reaction mixtures onto 1.2-µm glass fiber filters.
Antibodies
The monoclonal antibodies were used at the following dilutions:
anti-transferrin receptor (Zymed Laboratories, South San Francisco, CA)
(1:100), anti-human golgin-97 (Molecular Probes, Eugene, OR) (1:100),
anti-EEA1 (Transduction Laboratories, Lexington, KY) (1: 50), anti-TRP-1 (ID
Labs, Glasgow, United Kingdom) (1:200), anti-c-myc clone 9E10 (Oncogene
Research Products, San Diego, CA) (1:50), and anti-Rab5a (Transduction
Laboratories) (1:2000). The polyclonal antibodies used were from StressGen
(Victoria, British Columbia, Canada): anti-calnexin (1:5000 for immunoblotting
and 1:250 for immunofluorescence) and anti-Rab5 (1:2000 for immunoblotting).
The monoclonal anti-ERGIC-53 antibody (1:100 dilution) was a gift from
Hans-Peter Hauri (University of Basel, Basel, Switzerland).
Cell Culture and Transfection
HeLa and human embryonic kidney (HEK)293 cells were cultured in DMEM
supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin
G, and 100 U/ml streptomycin at 37°C with 10% CO2. Melan-c
cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine
serum, 2 mM glutamine, 0.1 mM 2-mercaptoethanol, 200 nM phorbol 12-myristate
13-acetate (Calbiochem, San Diego, CA), 100 U/ml penicillin G, and 100 U/ml
streptomycin at 37°C with 10% CO2. Cells used in
immunofluorescence experiments were grown on 24-well plated coverslips for 24
h, transfected, and fixed after 24 h (HeLa and HEK293 cells) or 48 h (melan-c
cells). Cells for subcellular fractionation were grown in 10-cm dishes,
transfected, and homogenized 24 h after transfection. HeLa cells were
tranfected with the liposomal transfection reagent Effectene (QIAGEN,
Valenica, CA), whereas melan-c and HEK293 cells were transfected with FuGENE6
(Roche Diagnostics, Indianapolis, IN) according to manufacturers'
instructions.
Subcellular Fractionation
After transfection, HeLa cells and HEK293 cells were washed with
phosphate-buffered saline (PBS), scraped, transferred to 15-ml tubes, and
washed once more with PBS. Cells from one dish were resuspended in 450 µl
of either homogenization buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol, and Roche mini complete protease inhibitor cocktail) or RabGDI
extraction buffer (25 mM HEPES-KOH, pH 7.2, 75 mM KOAc, 5 mM EGTA, 5 mM MgOAc,
1.8 mM CaCl2, 1 mM dithiothreitol, 1 mM GDP, and Roche mini
complete protease inhibitor cocktail). Homogenization was achieved by repeated
passage through a 25-gauge needle. Cell debris were removed by centrifugation
at 200 x g for 5 min, and the postnuclear supernatant was
subjected to ultracentrifugation at 100,000 x g by using TLA45
Beckman rotor. The resulting pellet (P100), containing the membrane fraction,
was resuspended in an equivalent volume of supernatant (S100), which contained
the cytosolic fraction. These fractions were either directly subjected to
electrophoresis on 12.5% SDS-PAGE gels followed by immunoblotting as described
previously (Barral et al.,
2002) or used in subsequent RabGDI extraction assays as described
below.
Triton X-114 Partition
Triton X-114 partition was performed as described previously
(Seabra et al.,
1995). Briefly, lysates obtained from cells transfected with the
appropriate vector were adjusted to 1% Triton X-114 and placed on ice for 5
min. Samples were then incubated at 37°C for 5 min, and the phases were
separated by centrifugation at 20,000 x g for 5 min. The
detergent phase (lower) was removed and the aqueous phase was readjusted to 1%
Triton X-114 and the above-mentioned procedure was repeated. The two detergent
phase fractions were combined and the volume adjusted to equal that of the
aqueous phase. SDS-sample buffer was added to both phases and samples were
boiled for 5 min. Equivalent amounts of detergent and aqueous phases were
loaded onto 12.5% SDS-PAGE gels, electrophoresed, blotted onto polyvinylidene
difluoride membranes, and detected with anti-Rab5a monoclonal antibodies.
Immunofluorescence and Confocal Microscopy
After transfection with the various EGFP-Rab constructs, the cells were
washed in PBS and permeabilized for 5 min in K-PIPES buffer containing 0.05%
saponin. Cells were then fixed with 3% paraformaldehyde for 15 min, washed
three times with phosphate-buffered saline, and mounted onto coverslips. When
antibody detection was applied, the cells were further incubated for 15 min in
phosphate-buffered saline containing 0.5% bovine serum albumin and 0.05%
saponin. The subsequent immunodetection steps were performed in this solution.
The cells were incubated with the primary antibody for 30 min and washed three
times, followed by incubation of the secondary antibody conjugated to
tetramethylrhodamine B isothiocyanate and washed as described above. The
coverslips were mounted in ImmunoFluor medium (ICN, Basingstoke, Hants, United
Kingdom) and the fluorescence was visualized using a DM-IRBE confocal
microscope (Leica, Wetzlar, Germany). Images were processed using TCS-NT
software (Leica) associated with the microscope and Adobe Photoshop 4.0
software. All images presented are single sections in the z-plane and
are representative of at least 80% of the transfected cells in the coverslip.
The derivation, culture, and light microscopic analysis of primary melanocytes
was described previously (Barral et
al., 2002).
Drug Treatment and Temperature Shift Experiments
For treatment with brefeldin-A cells were seeded in 24-well plates,
transfected, and incubated 24 h later with 10 µg/ml brefeldin-A for 1 h
before fixation. Cells were then processed for immunofluorescence as described
above. GGTI-298, provided by Said Sebti (University of South Florida)
(McGuire et al.,
1996), is a competitive CAAX peptidomimetic inhibitor
specific for PGGT. For inhibition studies, HeLa or HEK293 cells were seeded in
six- or 24-well plates and transfected 24 h later. The inhibitor was added 4 h
after transfection at a final concentration of 15 µM followed by a 20-h
incubation, permeabilization, and fixation as described above. Temperature
block experiments were performed at 20°C to prevent exit of secretory
proteins from the Golgi apparatus. HeLa cells were seeded in 24-well plates
and grown overnight as described above. The following day, cells were
transfected and 4 h later they were placed at 20°C for 3 h in media
supplemented with 20 mM HEPES buffer (catalog no. 15630-056; Invitrogen,
Carlsbad, CA). Some cells were further incubated at 37°C for 1 h and all
cells permeabilized and fixed as described above.
GDI Extraction Assays
The P100 fraction obtained by subcellular fractionation as described above
from a 10-cm dish was resuspended in 500 µl of RabGDI extraction buffer,
recentrifuged at 100,000 x g, and resuspended in 150 µl of
GDI extraction buffer. GST-RabGDI (0–1.25 µg) was incubated at
37°C for 30 min with P100 (5 µl) in RabGDI extraction buffer in a final
volume of 25 µl and reactions were centrifuged at 100,000 x
g for 30 min. The resulting supernatants were removed and subjected
to electrophoresis on 12.5% SDS-PAGE gels followed by immunoblotting by using
anti-Rab5a monoclonal antibodies.
Generation of Transgenic Mice and Genetic Crosses
All mice described were maintained and propagated under UK Home Office
Project License 70/5071 at the Central Biomedical Services of Imperial
College. Transgenic mouse lines were generated as described previously
(Barral et al., 2002).
Briefly, a 3.4-kb SpeI-BamHI fragment containing
PCAG/mycRab27a/-globin was isolated and microinjected into the pronuclei
of one-cell stage embryos from a C57BL/6JxCBA background collected from
superovulated female mice. Microinjected eggs were transferred at two-cell
stage into the oviducts of pseudopregnant recipient females. Mice born were
identified for incorporation of the transgene by PCR by using genomic DNA
obtained from mouse-tail biopsy and the oligonucleotides JR119
(5'-ATGGAACAAAAACTCATCTCAGAAGAGG) and JR14
(5'-AGTTGAACTTCCCATCAGTGTACTGGTA) corresponding to the myc-tag and the
Rab27a coding sequence, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Wilson et al., 1998).
Additionally, we have generated a Rab5a mutant that contains the K-Ras
farnesylation motif CVIM and a nonprenylatable mutant, Rab5aSSSN, where both
carboxyl-terminal cysteines were replaced by serine residues.
We purified the recombinant Rab5a mutant proteins from E. coli,
which lack the prenylation machinery, and used them as substrates in
established in vitro prenylation reactions
(Figure 1A). Recombinant Rab
proteins except Rab5aSSSN are efficiently prenylated by RGGT in vitro, as
expected (Figure 1B).
Mono-cysteine Rab5a mutants exhibited higher Vmax (our
unpublished data), in agreement with recent data suggesting that transfer of
the first geranylgeranyl group to Rab7 is 4 times faster than the transfer of
the second geranylgeranyl group to Rab7-GG
(Thoma et al.,
2001c). We subjected the same set of proteins to in vitro
prenylation by the CAAX prenyltransferases PFT and PGGT, together
with a known substrate for each enzyme, Rac1 for PGGT and K-Ras for PFT.
Rab5aCVLL and Rab5aCVIM undergo geranylgeranylation by PGGT but are poor
substrates in vitro (Figure
1C). The fact that Rab5a CVLL is a poor substrate for PGGT is in
agreement with data from Wilson and coworkers who have shown that Rab8, a Rab
with an intrinsic CVLL motif, is a poor substrate for PGGT compared with
Cdc42Hs, a natural substrate for this enzyme
(Wilson et al.,
1998). Unlike PFT, PGGT may recognize structural elements in
addition to the carboxyl-terminal CAAX motif
(Yokoyama and Gelb, 2000). As
expected Rab5aCVIM, but not Rab5aCVLL, is modified by PFT
(Figure 1D). Conversely,
neither Rab5aCCSN nor Rab5aCSLG are substrates for PGGT
(Figure 1C) or PFT
(Figure 1D). These results,
summarized in Table 1, suggest
that the mutations resulted in the expected prenylation behavior in vitro.
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Di-cysteine Rab5a Mutants Are Correctly Targeted to Early Endosomes
and Are Functional
We constructed mammalian expression vectors expressing the same Rab5a
mutants fused to the carboxy terminus of EGFP. After transient transfection in
cultured HeLa cells, we performed subcellular fractionation of cellular
lysates. As predicted, all mono- and di-cysteine fusion proteins were
efficiently prenylated as determined by membrane association (Supplemental
Figure 1) and Triton X-114
partition (Supplemental Figure
2, Figure 2).
Nevertheless, all transfections resulted in some degree of soluble proteins,
which probably resulted from the saturation of the cellular prenylation
machinery and of increased association with RabGDI.
|
Next, we asked whether the Rab5 mutants would correctly target to early
endosomes. To address this issue, we performed intracellular localization
studies by immunofluorescence on HeLa cells transiently transfected with the
EGFP-Rab5a constructs. The cells were costained either for transferrin
receptor (TfR), an endosome marker, or with early endosome autoantigen-1
(EEA1), a Rab5 effector protein (Simonsen
et al., 1998). EGFP-Rab5a wild-type (Rab5aCCSN) labels
punctate cytoplasmic structures peripherally distributed throughout the cell
as well as the juxtanuclear region as shown in
Figure 2A, for a cell that is
representative of the population of transfected cells. These structures
colocalize with TfR (Figure 2,
A–C) and EEA1 (our unpublished data), as documented
previously (Bucci et al.,
1992; Mu et al.,
1995; Stenmark et
al., 1996). The EGFP-Rab5aSSSN protein is present in the
nucleus and diffusely throughout the cytoplasm, as observed when EGFP alone is
expressed (our unpublished data). The di-cysteine EGFP-Rab5 constructs exhibit
a pattern very similar to the EGFP-Rab5a wild-type protein, showing extensive
colocalization with TfR (Figure 2,
D–I).
To demonstrate that the EGFP-Rab5a di-cysteine proteins are functional in
addition to being properly localized, we made use of the Q79L mutation in
Rab5a that locks the protein in the active, GTP-bound state. This
constitutively active Rab5a mutant induces the formation of enlarged
endosomes, consistent with its role in homotypic endosome fusion
(Bucci et al., 1992;
Stenmark et al.,
1994). Transient overexpression of EGFP-Rab5aQ79L causes the
redistribution of endosomes from small punctate structures in the cytoplasm to
enlarged vesicles in the juxtanuclear region
(Figure 2, J–L). The
majority of EGFP-Rab5a colocalizes with its effector protein EEA1 in these
enlarged endosomes as expected, although we have also occasionally observed
some vesicular Rab5a staining absent of EEA1
(Figure 2L). When the Q79L
mutation was introduced in two of the di-cysteine constructs, EGFP-Rab5aCGC
and EGFP-Rab5aCCQNI, we could also observe the formation of enlarged early
endosomes, suggesting that these proteins are both correctly targeted and
functional (Figure 2,
M–R).
Mono-cysteine Rab5a Mutants Are Mistargeted and Nonfunctional
Having established that di-cysteine Rab5a constructs are correctly targeted
to endosomes, we then proceeded to analyze the intracellular localization of
the mono-cysteine Rab5a mutants. We transiently expressed the various
EGFP-Rab5a mutants in HeLa cells and costained for TfR. Surprisingly, we
observed that all three mono-cysteine Rab5a mutants displayed a diffuse
pattern with staining of the nuclear envelope and some accumulation in the
perinuclear region (Figure 3, A, D, and
G) rather than the characteristic peripheral punctate appearance
of wild-type Rab5a (Figure 2A).
Labeling of TfR in the same cells revealed a clear separation of signals,
suggesting that the mono-cysteine Rab mutants were mistargeted
(Figure 3, A–I). We then
introduced the Q79L mutation in the context of the prenylation motif mutations
and transiently expressed the mutant proteins as EGFP-fusion proteins in HeLa
cells. We observed that Rab5aQ79L mono-cysteine mutants did not induce the
formation of enlarged endosomes nor colocalize with EEA1
(Figure 3, M–U).
Therefore, the prenylation-induced mistargeting of Rab5a seemed to have
prevented its interaction with EEA1. Furthermore, the similarity of the
staining pattern suggested that all mono-cysteine Rab5a mutants seemed to be
mistargeted to the same intracellular compartment. This suggestion was
confirmed when a myc-tagged version of Rab5aCVLL was coexpressed with
EGFP-Rab5a, EGFP-Rab5aCSLG, or EGFP-Rab5aCVIM. As expected, myc-Rab5aCVLL did
not show colocalization with the small peripheral punctate structures labeled
by EGFP-Rab5a (our unpublished data) but instead showed a nearly complete
overlapping pattern with EGFP-Rab5aCSLG
(Figure 3, J–L) or
EGFP-Rab5aCVIM (our unpublished data). Together, these results suggest that
the mono-cysteine Rab5a mutants are not functional and not correctly targeted
to early endosomes.
|
Rab27aCVLL Does Not Associate with Melanosomes and Is Not
Functional
To rule out the possibility that Rab5a is the only Rab that requires two
cysteines for proper targeting, we performed similar experiments on Rab27a, a
protein that associates specifically with melanosomes
(Marks and Seabra, 2001). We
replaced the ACGC sequence at the carboxyl terminus of Rab27a for CVLL by
site-directed mutagenesis and compared the in vitro prenylation properties and
intracellular targeting of wild-type and mutant proteins. As expected, both
recombinant His-Rab27a wild type and His-Rab27aCVLL are prenylated by REP/RGGT
in vitro, but only His-Rab27aCVLL is geranylgeranylated by PGGT (Supplemental
Figure
3, Figure 3). Next,
EGFP-Rab27a and Rab27aCVLL constructs were expressed in melan-c cells,
melanocytes derived from albino C57BL/6 mice. As expected, EGFP-Rab27a
associates with the limiting membrane of melanosomes as deduced from
comparisons of the staining patterns of Rab27a and TRP1, a melanosome marker
(Figure 4, A–C).
Conversely, Rab27aCVLL does not seem to associate with melanosomes
(Figure 4, G–I), rather
its signal overlaps significantly with that of calnexin, suggesting that the
protein associates with ER (Figure 4,
J–L, compare with D–F). These results support the
Rab5a studies, further suggesting that mono-cysteine prenylation motifs have a
mistargeting effect on Rab proteins.
|
To test the functionality of Rab27aCVLL, we decided to pursue a transgenic
approach taking advantage of a Rab27a null mouse strain, ashen
(Rab27aash). Ashen mice have hypopigmentation as
a result of deficient melanosome transport within skin melanocytes
(Marks and Seabra, 2001). We
have recently shown that the ashen coat color phenotype can be
rescued by crossing with a transgenic mouse line ubiquitously expressing
Rab27a wild type (Figure 5C) (Barral et al., 2002).
When we generated transgenic lines expressing Rab27aCVLL on an ashen
background, we observed little if any coat color correction
(Figure 5B). These results were
confirmed at the cellular level. Primary cultures of melanocytes derived from
ash/ash, —/tgRab27aCVLL mice revealed
clustering of melanosomes in the perinuclear area
(Figure 5D), as observed in
ash/ash melanocytes
(Figure 5E) but not in
ash/ash, —/tgRab27a melanocytes
(Figure 5F)
(Barral et al., 2002).
Furthermore, transfection of an ashen melanocyte cell line with
EGFP-Rab27aCVLL did not result in the redistribution of melanosomes to the
cell periphery (our unpublished data), in contrast with the movement rescue
effect observed upon transfection of EGFP-Rab27a
(Barral et al., 2002).
These results suggest that Rab27aCVLL is not functional in vivo.
|
Mono-cysteine Rab Proteins Are Targeted to Endoplasmic Reticulum
Recent studies on targeting of CAAX-containing proteins revealed
that the initial membrane-insertion event occurs in the ER and that
reporter-CAAX chimeric constructs such as EGFP-CVLL or EGFP-CVIM
associate with and are retained within ER membranes
(Choy et al., 1999).
Indeed, we confirmed that transfected EGFP-CVLL showed extensive
colocalization with calnexin, an ER marker
(Figure 6, A–C). When
myc-tagged Rab5aCVLL was transiently coexpressed with EGFP-CVLL or EGFP-CVIM
in HeLa cells, we observed a good degree of colocalization
(Figure 6, D–F and
G–I). Conversely, transient overexpression of an EGFP-CSLG
construct, which does not conform to the CAAX motif, exhibits a
nuclear and diffuse cytoplasmic staining that is typical of soluble proteins
(Figure 6, J–L). This
distribution contrasts with the pattern exhibited by EGFP-Rab5aCSLG
(Figure
3,Figure 3),
suggesting that the former is unable to undergo prenylation, whereas the
latter undergoes geranylgeranylation via the REP/RGGT pathway. To further
study the subcellular localization of the mono-cysteine Rab mutants, we
stained cells transfected with EGFP-Rab5aCSLG with known organellar markers.
We used the early secretory pathway markers, calnexin (ER), ERGIC-53
(intermediate compartment, IC) and Golgin-97 (Golgi apparatus). We observed a
significant overlap of signals between EGFP-Rab5aCSLG and calnexin
(Figure 6, M–O), and to a
lesser extent ERGIC-53 (Figure 6,
P–R) and Golgin-97
(Figure 6, S–U). Finally,
we treated EGFP-Rab5aCSLG–transfected cells with the fungal metabolite
brefeldin-A, a reagent that causes a dramatic disorganization of the Golgi
apparatus with Golgi markers redistributing to ER membranes
(Lippincott-Schwartz et al.,
1989). In treated cells, the distribution of Golgin-97 became
diffuse, an appearance consistent with its association with the ER
(Figure 6W), whereas the
EGFP-Rab5aCSLG pattern did not change
(Figure 6V). Together, these
studies suggest that mono-cysteine Rab mutants associate with ER
membranes.
|
Rab Proteins Are Recruited to Membranes via a CAAX-independent
Mechanism
The presence of the mono-cysteine Rab mutants in the ER raised the
possibility that they were targeted there via a CAAX-dependent
mechanism. To address this issue, we transfected cells with several
EGFP-fusion constructs in the presence or absence of a highly specific and
effective PGGT inhibitor, GGTI-298
(McGuire et al.,
1996). EGFP-Cdc42 was used as a positive control because it is a
natural substrate for PGGT. The cellular distribution of EGFP-Cdc42 and
EGFP-CVLL changed dramatically upon addition of GGTI-298
(Figure 7, A–D). In the
absence of inhibitor, EGFP-Cdc42 exhibited a Golgi/plasma membrane pattern
(Figure 7A) as expected
(Michaelson et al.,
2001) and EGFP-CVLL exhibited an ER pattern
(Figure 7C; see also
Figure
6). In the
presence of inhibitor, both proteins were observed in the nucleus in
permeabilized cells (Figure 7, B and
D) or in diffuse cytoplasmic and nuclear pattern in
nonpermeabilized cells (our unpublished data). A similar pattern is observed
when EGFP alone and other soluble proteins are expressed, suggesting that the
mentioned EGFP constructs have become soluble. Conversely, the ER-like pattern
of expression of EGFP-Rab5aCVLL, EGFP-Rab5aCSLG, EGFP-Rab5a wild type or
EGFP-HRas did not change in the presence of GGTI-298
(Figure 7). Furthermore,
treatment of GGTI-298 did not affect the proportion of membrane-association or
degree of prenylation as determined by Triton X-114 partition of Rab5aCVLL,
EGFP-Rab5aCSLG, and EGFP-Rab5a (Supplemental
Figure
2, Figure 2). These
results are consistent with the in vitro prenylation experiments (for
EGFP-Rab5aCSLG and wild type) and suggest that these proteins do not depend on
PGGT-mediated geranylgeranylation for membrane association. These results are
especially significant for EGFP-Rab5aCVLL because it is a potential substrate
for PGGT, although we cannot exclude the possibility that the presence of
GGTI-298 shifts prenylation of Rab5aCVLL from PGGT to RGGT. Together, these
results suggest that wild-type and mono-cysteine Rab5a mutants are delivered
to membranes via a REP/RGGT-dependent mechanism.
|
Intracellular Trafficking of Rabs Is Distinct from CAAX-containing
Proteins
We also addressed the possibility that Rab proteins are first delivered to
the ER and then passively follow the secretory pathway until they reach their
target organelle, as demonstrated for H-Ras and N-Ras
(Choy et al., 1999;
Apolloni et al.,
2000). In this model, the presence of the mono-cysteine Rab
mutants in the ER could be due to a failure to progress through the secretory
pathway. To test this hypothesis, we used a temperature shift experiment.
Incubating cultured cells at 20°C blocks protein exit from the Golgi
apparatus, thus resulting in massive accumulation of secretory proteins in
this organelle (Matlin and Simons,
1983; Stinchcombe et
al., 1995). We confirmed this finding with EGFP-HRas, which
accumulated in the Golgi apparatus after 3 h at 20°C
(Figure 8A). Shifting the
cells back to 37°C led to the expression of EGFP-HRas at the plasma
membrane (Figure 8B). The
temperature shift to 20°C did not affect the localization of
EGFP-Rab5aCVLL to the ER (Figure 8,
G–I) or that of EGFP-Rab5a to early endosomes
(Figure 8, D–F). These
results provide for the first time evidence that wild-type Rab proteins do not
follow the same pathway as H-Ras and other CAAX-containing GTPases
and thus argue against the idea that the ER represents the initial point of
membrane insertion of Rabs.
|
Mono-cysteine Rab Proteins Are Able to Interact with GDI
The results mentioned above support the current model that Rabs are
directly targeted to their final destination, but they do not exclude the
possibility that Rabs are initially targeted to the ER by the REP/RGGT pathway
and then retrieved by RabGDI, which mediates specific organelle targeting. In
this model, the presence of the mono-cysteine Rab mutants in the ER could be
due to a failure of RabGDI to retrieve the mono-geranylgeranylated Rabs from
ER membranes. To test this hypothesis, membrane fractions obtained from
transiently transfected cells containing approximately equivalent levels of
Rab5a wild-type and mono-cysteine mutant proteins were incubated with
RabGDIá and then recentrifuged to separate soluble and
membrane-associated proteins. The presence of EGFP-Rab5a in the soluble
fraction suggested membrane extraction by RabGDI. Indeed, increasing
concentrations of RabGDI resulted in increasing amounts of Rab5a in the
soluble fraction (Figure 9, left). Furthermore, Rab5a extraction was promoted by incubation with GDP and
prevented by GTP
S (Figure
9, right). We observed no differences between wild-type and mutant
Rab5aCVLL or Rab5aCSLG in their ability to be extracted from membranes by
RabGDI (Figure 9). Furthermore, cooverexpression of RabGDI with the EGFP-Rab5a mutants did
not lead to endosome targeting of these mutants (our unpublished data). These
results suggest that the ER mistargeting of mono-cysteine Rab5a mutants is not
due to loss of retrieval by RabGDI.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Rab proteins exhibit a variety of mono- and di-cysteine prenylation motifs
(Pereira-Leal and Seabra,
2000,
2001). We decided to use Rab5a
and Rab27a as models to study the influence of those prenylation motifs on the
membrane targeting of Rab GTPases. Replacement of the di-cysteine
carboxyl-terminal prenylation motif in Rab5a by di-cysteine motifs present in
other Rabs, such as CC, CCA, CGC, and CCQNI, had no effect on targeting to
early endosomes. Moreover, these constructs seemed to be functioning properly
because the introduction of the GTPase-deficient Q79L mutation onto the
prenylation motif mutants resulted in the formation of enlarged endosomes as
observed for EGFP-Rab5aQ79L. One of these di-cysteine motifs (CGC) serves also
as a methylation signal. Unfortunately, our results do not shed any new light
into the role of Rab methylation, because Rab5aCGC seemed to be correctly
targeted to early endosomes at steady state. Furthermore, targeting of Rabs
susceptible to be methylated is not affected in cells that lack Icmt activity
(Bergo et al., 2001)
(Gomes and Seabra, unpublished observations).
When the di-cysteine carboxyl-terminal motifs of both EGFP-Rab5a and
EGFP-Rab27a were changed to mono-cysteine motifs, the transiently expressed
proteins were mistargeted and seemed to associate with ER membranes
irrespective of the motif itself (CSLG, CVLL, or CVIM). Furthermore, the
mutants were inactive as determined in cultured cells for Rab5a and in vivo
for Rab27a, presumably as a direct consequence of the mistargeting. Previous
work showed that a Rab1b protein engineered to be prenylated by both RGGT and
PGGT (Rab1bCLLL) is correctly targeted to ER/Golgi membranes and is functional
(Overmeyer et al.,
2001). Although apparently contradictory to the present results,
these observations could be explained by the fact that the target membrane for
the wild-type Rab1b protein and the location for mistargeted
mono-geranylgeranylated mutants is coincidentally the same one, i.e., the ER.
The use of post-Golgi Rabs in this study may have helped in the identification
of mistargeted proteins given the different cellular distributions of the ER
versus endosomes or melanosomes.
Despite a decade of exciting research into the function of Rab proteins,
little is known about the mechanisms regulating their specific targeting to
distinct organelles. One model proposes that newly synthesized and
geranylgeranylated Rabs are delivered by REP to their specific target membrane
(Alexandrov et al.,
1994; Wilson et al.,
1996; Seabra,
1998; Pfeffer,
2001). It is also possible that REP delivers the newly
geranylgeranylated Rab directly to RabGDI in the cytosol, which then mediates
specific targeting of Rabs (Sanford et
al., 1995). Another possibility is that REP delivers Rabs
nonspecifically to ER membranes, which serves as the initial membrane
insertion site. In this scenario, Rabs reach their final destination by
flowing passively via vesicular transport, as proposed for transmembrane
proteins and for the related Ras and Rho CAAX-containing proteins, or
alternatively RabGDI extracts ER-associated Rabs and delivers them
specifically to their target membranes.
The unexpected observation that Rab mono-cysteine mutants were mistargeted
to ER membranes provided us with a new tool to investigate this topic. The
present results further suggest that Rab prenylation and targeting are
distinct from the CAAX-containing proteins. First, we established
that the CSLG motif present in Rab13 is not recognized by the two known
CAAX-prenyl transferases, suggesting that the REP/RGGT pathway is the
only possible route to geranylgeranylation and membrane-association for at
least one mono-prenylated Rab. Second, we demonstrated that a specific PGGT
inhibitor, GGTI-298, has no effect on the membrane association of the
mono-geranylgeranylated Rabs. This result is consistent with a previous study
suggesting that PGGT inhibitors do not affect prenylation and membrane
association of the mono-geranylgeranylated wild-type Rab, Rab8a
(Wilson et al.,
1998). Third, we subjected cells to a 20°C block to prevent
exit of proteins from the Golgi apparatus and showed that the incubation did
not affect the membrane location of either wild-type di-GG-Rab5a to endosomes
or mutant mono-GG-Rab5a to the ER. Our results are most consistent with a
direct pathway for the specific targeting of Rabs, although we cannot
distinguish at present whether targeting is mediated by REP or RabGDI. Future
work will be necessary to clarify these issues.
Why are the mono-GG-Rab mutants unable to stably associate with their
target membranes and instead associate with the ER? One possibility is that
the ER serves as the initial entry site for newly prenylated Rabs (see above)
and that specific targeting is dependent on RabGDI-mediated ER extraction and
correct delivery. Our results are inconsistent with this hypothesis, because
we show that RabGDI is able to extract both mono- and
di-geranylgeranylated Rabs, in agreement with a previous report
(Figure 9)
(Ullrich et al.,
1993). Nevertheless, there remains the possibility that additional
factors required for extraction in vivo, such as the putative Rab recycling
factor, require di-geranylgeranylated Rabs
(Luan et al., 1999).
Another possibility is that membrane targeting of Rabs is partially regulated
by the lipid environment, such that mono-GG-Rabs exhibit lower affinity or are
unstable in the target membranes. A recent report from Gruenberg and coworkers
suggests that changes in the biophysical properties of membranes seem to
influence Rab activity because increases in cholesterol content of late
endosomes results in a stabilization of Rab7 at the membrane
(Lebrand et al.,
2002). The mechanism underlying the cholesterol effect is not
understood, it could be promoting the activation of Rab7 or preventing its
cytosol extraction by RabGDI, or both. Nevertheless, lipid environment is
unlikely to be the sole targeting factor because mono-prenylated Rho proteins,
such as RhoD associate with endosomes
(Murphy et al., 1996;
Michaelson et al.,
2001). A third possibility is that REP forms a tight complex with
the mono-cysteine Rab5a and Rab27a mutants preventing their specific release
into their target organelle (Shen and
Seabra, 1996). However, this does not explain why the mono-GG-Rabs
are able to associate with ER membranes. A further possibility is that
mistargeting to the ER could be a result of methylation after membrane
insertion (Dai et al.,
1998). However, as mentioned above, the lack of Icmt activity does
not affect the targeting of Rabs susceptible to be methylated
(Bergo et al., 2001).
Irrespective of the precise mechanism, our results suggest that the ER is the
default location of prenylated proteins when they do not possess a clear
targeting motif.
The evidence suggesting that the number of geranylgeranyl groups influences the membrane targeting of Rab proteins raises the question why do a few Rabs (such as Rab8a and Rab13) only possess a single prenylatable cysteine. Future studies on the mechanisms underlying REP-mediated targeting of Rab proteins should clarify this and the other issues raised by the present work.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Online version of this article contains supplemental figure materials.
Online version available at
http://www.molbiolcell.org. 
* Present address: Center of Ophthalmology, University of Coimbra, Biomedical
Institute for Research in Light and Image, Azinhaga Sta. Comba, 3000-354
Coimbra, Portugal.
Corresponding author. E-mail address:
m.seabra{at}imperial.ac.uk.
|
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), Rab5aCSLG (
), Rab5aCVIM (
), and the indicated
concentrations of REP. (C) PGGT assay. Each reaction contained 20 nM PGGT, 5
µM [3H]GGPP (3000 dpm/pmol), and the indicated concentrations of
Rac1 (
). (D) PFT assay. Each reaction
contained 40 nM recombinant PFT, 1.2 µM [3H]FPP (11,275
dpm/pmol), and the indicated concentrations of K-Ras (
