1 Departamento de Bioquímica y Biología Molecular, Facultad de
Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
2 Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos
III, Madrid, Spain
* Author for correspondence (e-mail: nacho{at}bbm1.ucm.es )
Accepted 17 May 2002
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Summary |
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Key words: Myristoylation, Caveolin, Palmitoylation, Rafts, Subcellular targeting
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Introduction |
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The partial purification of enzyme activities responsible for
palmitoylation of G subunits
(Dunphy et al., 1996
), N-Ras
(Liu et al., 1996b
) and the
C-terminal regions of Drosophila Ras1 and Ras2
(Ueno and Suzuki, 1997
) has
been reported. However, whereas the former enzymatic activity remains elusive
because of problems with cloning and molecular characterization
(Dunphy et al., 2001
), the
other two palmitoyl transferase activities turned out to be a peroxisomal
3-oxoacyl-CoA thiolase (Liu et al.,
1996a
) and a transferase that did not depend upon the previous
prenylation of the peptide that was used as a substrate
(Ueno and Suzuki, 1997
), a
feature that is required for Ras palmitoylation in vivo
(Hancock et al., 1989
).
Very recently, skinny Hedgehog (ski), a Drosophila
acyltransferase that is able to catalyze the palmitoylation of Hedgehog
(Hh), has been identified (Chamoun
et al., 2001). However, it has been reported that the palmitoyl
group is attached to the N-terminus of Hedhehog on the
amino group of
Cys-24 (Pepinsky et al.,
1998
). This attachment originates through an autoproteolytic
cleavage at an internal site during which the bioactive N-terminal product of
cleavage acquires a C-terminally bound cholesterol molecule. Consequently, it
remains to be seen whether the mammalian orthologs of this acyltransferase are
able to catalyze the incorporation of palmitic acid to other cellular
targets.
Additional acyltransferase activities have been observed and partially
characterized in erythrocyte ghosts and placental membranes
(Das et al., 1997;
Schmidt et al., 1995
;
Veit et al., 1998
) as well as
in brain lysates (Berthiaume and Resh,
1995
), although all of them remain refractory to molecular cloning
and full characterization.
Conversely, Duncan and Gilman showed that palmitoyl-CoA can
stoichiometrically palmitoylate previously myristoylated Gi1 in vitro
in the absence of any cellular acyltransferase with kinetics for the
palmitoylation reaction that resemble those described for palmitoylation in
vivo (Duncan and Gilman, 1996
).
The non-enzymatic palmitoylation of Gs
at Cys3 was also reported
(Mollner et al., 1998
). Magee
and coworkers very elegantly showed that myristoylated N-terminal peptides
from the Yes protein tyrosine kinase, but not their non-myristoylated
counterparts, could be non-enzymatically palmitoylated by palmitoyl-CoA in
vitro (Bañó et al.,
1998
). The palmitoylation reaction was pseudo-enzymatic in the
sense that it depended not only on the previous myristoylation of the
substrate but also on the length of the incubation period, the temperature and
the substrate concentration. Consequently, apparent Km and
Vmax values could be estimated for the peptide substrate.
Other examples of non-enzymatic acylation of proteins reported in the
literature include UDP-glucuronosyl transferase from rat liver
(Yamashita et al., 1995
), the
myelin proteolipid (Bizzozero et al.,
1987
) and rhodopsin (O'Brien
et al., 1987
), as well as cysteine-containing peptides
(Quesnel and Silvius,
1994
).
To investigate whether the cellular transfer of palmitate from
palmitoyl-CoA could occur in a de-novo-designed protein sequence, we
constructed a polypeptide where a consensus N-myristoylation hexapeptide was
followed by the triplet Ala-Gly-Ser repeated nine times and fused to GFP. We
referred to this construct as `linker-GFP'. We avoided any basic residue such
as Lys or Arg within this sequence pattern since that would also promote
membrane targeting and raft localization
(McCabe and Berthiaume, 1999).
Four of the Ser residues in our construct, with a six-residue spacing within
them and located at different distances from the N-terminus, were substituted
by Cys residues (Fig. 1), and
their phenotypes were analyzed. Additionally, 12 residues were introduced
between the last cysteine residue that can be putatively palmitoylated (Cys21)
and the GFP sequence in order to rule out the possibility that the
three-dimensional fold of the GFP might interfere with the palmitoylation
processes. Our data are consistent with the hypothesis that in vivo
palmitoylation of proteins can take place as long as a reactive cysteine
residue is in proximity to the cellular membrane.
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Materials and Methods |
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Construction of the GFP fusion proteins
The general design of the nine mutants described herein consisted of
N-terminal tags fused to the enhanced GFP using a BssHII site in the
pCDNA3 plasmid (Invitrogen, Barcelona, Spain) under the control of the
cytomegalovirus promoter. We performed recursive PCR in order to obtain a
de-novo-designed sequence using six overlapping oligonucleotides and
introducing a NcoI site at the 5' end and a BssHII
site at the 3' end of the desired sequence. This starting sequence,
referred to as linker-GFP, consisted of a consensus myristoylation motif
(MGCTLS) followed by the triplet AGS repeated nine times followed by the GFP
sequence, that is MGCTLS-(AGS)9-GFP
(Fig. 1). Site-directed
mutagenesis was employed in order to introduce the desired mutations. In the
case of the eNOS-GFP construct, we amplified the first 55 residues of
endothelial nitric oxide synthase, introducing novel NcoI and
BssHII sites at the 5' and 3' end, respectively. This
first 55 residues of eNOS include the myristoylation site together with the
two palmitoylated cysteines (Cys15 and Cys26) and is enough to target GFP to
Golgi regions and discrete regions of the plasma membrane
(Liu et al., 1997).
PCR-amplified bands of all the constructs were ligated into the pGEM-T vector
(Invitrogen), and they were subsequently sequenced. Owing to the presence of
NcoI and BssHII sites in pCDNA3, we first double digested
the Gi
1(32 aa)-GFP construct with EcoRI plus XbaI and
ligated the excised fragment into the corresponding sites of a pUC vector
polylinker. All the pGEM-T vectors with linker-GFP, G2A-GFP, C3S-GFP,
G2A/C3S-GFP, C3S/S9C-GFP, C3S/S15C-GFP, C3S/S21C-GFP and eNOS-GFP were then
double digested with NcoI plus BssHII, and the excised band
was subsequently ligated into the corresponding site of pUC-Gi
1(32
aa)-GFP. Finally, all the constructs in pUC were double digested with
EcoRI plus XbaI and the excised bands ligated in the
corresponding sites in pCDNA3. Finally, every plasmid was sequenced to confirm
the desired mutations.
Immunoblot analysis and cellular fractionation
Cellular proteins were resolved by 15% acrylamide SDS-PAGE and transferred
to nitrocellulose membranes. Western blots were incubated for 2 hours in PBS
containing 2% powdered skimmed milk. Subsequently, the nitrocellulose
membranes were incubated overnight with the primary antibodies (typically
1:1000 in PBS), washed and finally incubated for 2 hours with a
horseradish-peroxidase-conjugated goat anti-rabbit antibody (Pierce).
Detection was performed using a ECL detection kit (Amersham Pharmacia
Biotech). Quantification of the intensity of the bands was performed using a
UViband V97 software (UVItec St. John's Innovation Centre, Cambridge, UK).
Fractionation was performed by harvesting the cells and resuspending them in 0.4 ml of PBS supplemented with 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin and 2 mM phenylmethylsulfonyl fluoride (Sigma) as protease inhibitors. The cellular suspensions were then passed several times through a syringe (0.5x16 mm) and were further homogenized with short sonication cycles (3x10 seconds each) on ice. Unbroken cells and cellular debris were eliminated by a 10 minute centrifugation at 3000 g in a table-top microcentrifuge. The samples were then centrifuged for 16 hours at 200,000 g in a SW65 rotor (Beckman) at 4°C. The cellular pellets (particulate fraction) and the supernatants (soluble fraction) were brought up to equal volumes and subsequently analyzed by SDS-PAGE followed by immunodetection with a rabbit anti-GFP antibody.
Metabolic labeling and immunoprecipitation
COS-7 cells transiently transfected with the desired construct were starved
for 1 hour with DMEM in the absence of any FBS 36 hours after transfection.
The monolayers were subsequently labeled for 4 hours with 200 µCi [9,
10-3H]-myristic acid or 200 µCi [9, 10-3H]-palmitic
acid. The radioactive compounds were dried completely under nitrogen and
resuspended initially in 10 µl of DMSO followed by 1 ml of DMEM containing
2% dialyzed FBS plus 0.5% de-fatted BSA (Sigma). The cells were washed with
PBS, scraped and lysed in Ripa buffer (10 mM Tris, 150 mM NaCl, 1% Triton
X-100, 0.1% SDS, 0.1% deoxicholate, pH 7.35) in the presence of protease
inhibitors. After centrifugation of the cell lysate at 6000 g
for 10 minutes at 4°C, the supernatant was incubated with 10 µl of the
rabbit anti-GFP antibody. The mixture was incubated overnight at 4°C, and
then 25 µl of a 1:1 suspension of Sepharose:Protein A-Sepharose beads
(Amersham Pharmacia Biotech) was added to each sample and the incubation was
maintained for 5 hours at 4°C. Immunoprecipitates were centrifugated at
16,000 g in a table top microcentrifuge for 10 minutes, and
the supernatant was removed. Immunoprecipitates were then washed twice in 0.5
ml of ice-cold Ripa buffer. Samples were separated by SDS-PAGE, and the gels
were treated with Enhance solution (Amersham Pharmacia Biotech) in order to
improve the radioactivity signal. The gels were dried in a BioRad vacuum
apparatus and exposed on photography films in a cassette for 4 weeks at
-80°C.
Confocal fluorescence microscopy
Transiently transfected COS-7 cells grown on 0.2% gelatin-coated glass
coverslips were washed two times with PBS and fixed for 15 minutes at room
temperature with freshly prepared 2% paraformaldehyde in PBS. The stock
paraformaldehyde solution was prepared at 4% in PBS and was centrifuged at
16,000 g for 5 minutes at room temperature in a table-top
microcentrifuge in order to remove insoluble material prior to dilution. After
removal of the 2% paraformaldehyde solution, the cells were washed with PBS
and incubated with cold methanol at -20°C for 10 more minutes. The
methanol was removed, and the coverslips were allowed to dry for 5 minutes.
Then, the cells were washed with PBS and the slides were mounted using
Fluoroguard anti-fade reagent (BioRad). The subcellular localization was
observed under a BioRad MRC-1024 confocal microscope equipped with two lasers
using the excitation wavelength of 488 nm for the GFP intrinsic fluorescence
and 543 nm for the Cy3 fluorophore and the BODIPY-TR-ceramide. Double labeling
was performed on transiently transfected COS-7 cells permeabilized with
methanol. Incubation with a rabbit polyclonal anti-caveolin-1 antibody
(Transduction) was done for 1 hour at 37°C in a wet chamber. Next, the
cells were washed with PBS and the anti-rabbit Cy3-labeled secondary antibody
was added. BODIPY-TR-ceramide (1.5 µM in DMEM) staining was done in vivo in
COS-7 transfected cells, and the reagent was added 30-60 minutes before
analysis of the double labeling under the confocal microscope. The live cells
were washed twice with PBS and examined for their subcellular distribution.
Fixing was avoided, since the methanol significantly distorts the
BODIPY-TR-ceramide signal. When the in vivo fluorescence measurements were
done (incubations with ß-methylcyclodextrin), the cells were kept at
37°C using a Peltier system, and Hepes buffer was used all throughout the
experiment. The cycloheximide treatment (100 µg/ml) was performed 24 hours
post-transfection for 2 hours at 37°C.
Preparation of caveolin-enriched low density membrane fractions
Typically, four transiently transfected COS-7 confluent 75 cm2
flasks were scraped and the cells resuspended into 2 ml (final volume) of Mes
buffered saline (MBS) (25 mM MES, pH 6.5, 0.15 M NaCl, 1 mM PMSF plus 1%
Triton X-100) at 4°C. Isolation of the Triton-X-100-insoluble fractions
with a lower number of cells needed the amount of detergent to be scaled down
proportionally. Homogenization of the cells was carried out with a minimum of
10 strokes through a syringe (0.5x16 mm) on ice. The homogenate was then
adjusted to 40% sucrose by the addition of 2 ml of 80% sucrose prepared in MBS
(4 ml in total), and it was placed at the bottom of a Beckman SW40 13 ml
Ultraclear tube. The discontinuous gradient (40-30-5%) was formed by loading 4
ml of 30% sucrose followed by 4 ml of 5% sucrose always in MBS. Separation was
performed at 200,000 g for 18 hours in a SW40 rotor (Beckman)
at 4°C. A light scattering band confined to the interface of the 5-30%
sucrose region was observed that contained the majority of the caveolin-1
protein and excluded most of the cellular proteins. After centrifugation, 1 ml
fractions were collected from the bottom of the tube to yield a total of 12
fractions. After that, 1 ml of cold acetone was added to each tube and the
homogenous mixture was allowed to precipitate overnight at 4°C. The
samples were centrifuged at 16,000 g in a microcentrifuge, and
the protein pellets were allowed to dry for 2 hours to assure the complete
elimination of acetone traces. The protein precipitates were then analyzed by
SDS-PAGE. In addition to caveolin-1, 5'-nucleotidase, a GPI-anchored
protein was also used as a protein marker of caveolin-1-enriched low fluidity
subdomains (Kenworthy and Edidin,
1998). The amount of total protein in each fraction was measured
using a micro-Lowry method. In essence, we first incubated the protein sample
with 2.5 ml of reagent E (25 ml NaOH, 0.2 M plus 25 ml 4% carbonate mixed with
250 µl 1% copper sulfate and 250 µl of 2% tartrate). After 10 minutes of
reaction at room temperature, we added 250 µl of Folin reagent and let the
mixture stand for an additional 30 minutes. Absorbance was determined at 750
and 500 nm, and the values were interpolated in a calibration curve performed
with known quantities of bovine serum albumin.
Co-immunoprecipitation
Transiently transfected COS-7 cells were washed twice with PBS, scraped and
lysed with several strokes of a thin-gauge syringe in Ripa buffer in the
presence of protease inhibitors on ice. Samples were clarified for 1 hour at
4°C using 20 µl of a 1:1 mixture of Sepharose:Protein-A-sepharose.
After clarification, the supernatants were incubated with 10 µl of the
primary antibody (anti-GFP or anti caveolin-1) overnight at 4°C. Then, 20
µl of the Sepharose:Protein-A-sepharose slurry was added and the mixture
was incubated for 2 additional hours at 4°C. The samples were centrifuged
in a table-top microcentrifuge, and the immunoprecipitates were then washed
twice in 0.5 ml of ice-cold Ripa buffer. The resin beads were subsequently
boiled in SDS-PAGE loading buffer and processed in a 15% acrylamide gel. The
SDS-PAGE gel was afterwards transferred to nitrocellulose, and the membrane
was probed with the desired antibodies.
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Results |
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Metabolic radiolabeling of the GFP chimeras was performed using [9,
10-3H]-myristic acid or [9, 10-3H]-plamitic acid
followed by immunoprecipitation of the cell lysates with the anti-GFP antibody
(Fig. 1C). As expected, GFP
alone was neither myristoylated nor palmitoylated, whereas every GFP chimera
that possessed the N-terminal Gly in the context of a consensus myristoylation
sequence (Ser residue at position 6) efficiently incorporated myristic acid
(linker, C3S, C3S/S9C, C3S/S15C, C3S/S21C and eNOS-GFP chimeras). Predictably,
mutation of Gly2 to Ala2 resulted in no myristic acid incorporation (G2A-GFP
and G2A/C3S-GFP chimeras) (Fig.
1C). Efficient incorporation of palmitic acid occurred in the
constructs linker-GFP, C3S/S9C-GFP and C3S/S15C-GFP as well as in the positive
control eNOS-GFP (in addition to the Gi1-GFP construct) (data not
shown). Under identical conditions, a faint band of radioactivity appeared for
the C3S/S21C-GFP chimera, which is indicative of a very low level of
incorporation of palmitate at position 21
(Fig. 1C). Quantification of
this band using the UVI-band software revealed that 18% of the linker-GFP
construct had been incorporated. Non-myristoylated mutant G2A-GFP was unable
to be palmitoylated in spite of the fact that it contains a Cys residue at
position 3. Consequently, efficient S-acylation can occur at positions Cys3
(linker-GFP), Cys9 (C3S/S9C-GFP), Cys15 (C3S/S15C-GFP) and in minimal amounts
at Cys21 (C3S/S21C-GFP) but always in previously myristoylated polypeptides.
Since the four `palmitoylable' Cys residues (Cys3, Cys9, Cys15 and Cys21) were
surrounded by identical amino acids, we must conclude that only the distance
to the N-terminus of the GFP chimera is a determinant for protein
palmitoylation. We could rule out the possibility that the lower incorporation
of palmitic acid at position 21 is caused by its proximity to the GFP
sequence, since 12 amino acids have been introduced between Cys21 and the
first residue of GFP. Previous studies have shown that cysteine residues
located either five (Fyn kinase-GFP construct) or seven amino acids from the
GFP reporter (GAP43-GFP construct) were efficiently palmitoylated
(McCabe and Berthiaume, 1999
).
Note that the myristoylated C3S-GFP construct, which lacks any cysteine
residue in the linker region, is unable to become palmitoylated at any of the
Cys residues present in the GFP sequence itself (Cys48 and Cys70)
(Fig. 1C).
Subcellular localization of the GFP chimeras by confocal
microscopy
To assess whether the N-terminal extensions confer different subcellular
localization information, we transfected COS-7 cells with the various GFP
constructs and inspected their distribution using laser confocal microscopy
(Fig. 2). The linker-GFP mutant
displays a plasma-membrane and focal perinuclear localization with nuclear
exclusion and low cytosolic levels of fluorescence. This phenotype contrasts
with the subcellular distribution of GFP alone, which produces both cytosolic
and a marked nuclear staining; thus linker-GFP distribution strongly suggests
that the acylation of the linker-GFP construct is responsible for the observed
subcellular targeting. Substitution of the Gly residue at position 2 by Ala
(G2A-GFP and G2A/C3S-GFP chimeras), a mutation known to impede myristoylation
(Fig. 1C), restores the
widespread cytosolic and nuclear distribution, with negative staining of
nucleoli observed for GFP alone. Elimination of the putative palmitoylation
site Cys3 and maintaining the myristoylation site (C3S-GFP mutant) results in
a distribution characterized by the nuclear exclusion and the loss of the
plasma membrane staining, with an enrichment in intracellular membranes, in
perinuclear vesicles as well as in the ER
(Fig. 2). The myristoylated and
palmitoylated GFP chimeras C3S/S9C, C3S/S15C as well as the two positive
controls of dual acylation (Gi1 and eNOS) display a similar staining to
the linker-GFP construct; that is, plasma membrane plus distinctive
intracellular vesicular organelles and a complete nuclear exclusion. Finally,
the myristoylated C3S/S21C-GFP chimera, which incorporates palmitic acid very
poorly, exhibits a mixed phenotype reflecting both the linker-GFP and C3S-GFP
construct localizations (Fig.
2).
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Membrane partitioning of the GFP chimeras
To investigate the effect of the various acylation states on the membrane
partitioning of the GFP chimeras, we fractionated the cellular lysates into
Supernatant (S-soluble) and Pellet (P-membrane-associated) fractions after
ultracentrifugation at 200,000 g
(Fig. 3). The presence of the
GFP chimeras was detected using specific antibodies, and the intensity of the
bands was accurately determined using a UViband V97 software. Constructs that
did not incorporate either myristic or palmitic acid, such as GFP, G2A-GFP and
G2A/C3S-GFP, were predominantly soluble. Dually acylated GFP chimeras, such as
linker, C3S/S9C, C3S/S15C, Gi1 and eNOS, partitioned preferentially
into the membrane-associated fractions. (More than 65% of the chimeras were in
membrane-bound fractions; Fig.
3.) The construct C3S-GFP, which is myristoylated but not
palmitoylated, has approximately half of its total immunoreactive protein in
each fraction, and an almost identical distribution is also observed in the
case of the C3S/S21C-GFP chimera. Therefore, as expected, N-terminal
myristoylation increases the overall hydrophobicity of the engineered GFP
chimeras, resulting in an increased association with cellular membranes
(roughly 50% in the P lane), whereas the additive effect of myristoylation
plus palmitoylation results in the majority of the protein associating with
the membrane (Fig. 3).
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Enrichment of the GFP chimeras in Triton-insoluble membrane
rafts
Owing to their elevated cholesterol and sphingolipids content, caveolar
membranes are resistant to extraction at 4°C by nonionic detergents such
as Triton X-100; instead they float on bottom-loaded sucrose density gradients
(Kurzchalia and Parton, 1999;
Simons and Toomre, 2000
). We
inspected the targeting of the various acylated constructs to these
low-fluidity domains preparing 40:30:5% sucrose discontinuous gradients in the
presence of Triton X-100 following a well-defined procedure
(Lisanti et al., 1999
).
Caveolin-1, which is endogenously expressed in COS-7 cells, together with
5'-nucleotidase, a GPI-anchored protein, was used as a marker for these
Triton-X100-insoluble domains (Kenworthy
and Edidin, 1998
; Lisanti et
al., 1999
; Kurzchalia and
Parton, 1999
). Partial (but not complete) localization of the GFP
chimeras to these caveolin-1-enriched domains was only observed for the dually
acylated constructs linker-GFP, C3S/S9C-GFP, C3S/S15C-GFP, Gi
1-GFP and
eNOS-GFP (Fig. 4). The
non-acylated GFP, G2A-GFP and G2A/C3S-GFP proteins were found in fractions 1
through 4 (bottom of the gradient), where most of the cellular proteins were
present (upper panel). Although both the myristoylated C3S-GFP and
C3S/S21C-GFP chimeras had a certain tendency to float towards lower density
fractions (they were present in fractions 1 through 7), they were not
significantly enriched in fractions 8 and 9 accompanying caveolin-1. Thus, the
myristic acid moiety alone confers membrane-interacting properties but not the
capacity to fully interact with the low-fluidity cholesterol-sphingomyelin
enriched domains. Additionally, we used the integrin ß1, a
transmembrane protein known to be excluded from Triton-X100-insoluble rafts
(Nusrat et al., 2000
) as a
specific marker of membrane-associated but caveolae-excluded protein. Integrin
ß1 is concentrated in high-density fractions (40% sucrose,
fractions 1-4) at the bottom of the tube
(Fig. 4).
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Confocal microscopy colocalization studies of the GFP chimeras with
caveolin-1 in transfected COS-7 cells
Next, we investigated whether the subcellular localization of the various
GFP chimeras transfected in COS-7 cells partially overlapped with that of
endogenous caveolin-1 (Fig.
5A). Transiently transfected COS-7 cells were permeabilized and
incubated with anti-caveolin-1 antibodies. The three mutant GFP chimeras shown
on Fig. 5A, linker-GFP, G2A-GFP
and C3S-GFP, are representative of each of the well defined phenotypes. Taking
advantage of the GFP intrinsic fluorescence, together with signal emitted from
the Cy3-labeled secondary antibodies, we inspected the colocalization of the
three chimeras with caveolin-1 that are present endogenously in COS-7 cells.
Caveolin-1 staining was mostly focal and punctate, and it could be observed in
regions close to the plasma membrane and to a lesser degree in intracellular
aggregates; it was always excluded from the cell nucleus
(Fig. 5, middle panels). When
merged with the GFP signal (Fig.
5, right panels; see also supplemental data), there was a
significant overlap (yellow) in distribution with the linker-GFP and G2A-GFP
chimeras at areas close to the plasma membrane (arrows) and no overlap with
the C3S chimera. However, the dually acylated mutant linker-GFP still
exhibited extensive GFP fluorescence in cytosolic regions where the caveolin-1
fluorescence was excluded. A similar codistribution with caveolin-1 in the
plasma membrane and adjacent areas (caveolae) was observed for the other
dually acylated GFP chimeras (data not shown). The coincidence in the
fluorescence signal of the G2A-GFP chimera with caveolin-1 was somehow
expected, since this mutant presents GFP fluorescence in the entire cytoplasm
and nucleus. Interestingly, the merge of the myristoylated, non-palmitoylated
C3S-GFP chimera signal with that of caveolin-1 reveals a crown-like
distribution. Whereas caveolin-1 distribution is centered on plasma membrane
areas, the C3S-GFP fluorescence is selectively excluded from them.
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Intriguingly, dually acylated N-terminal segments of Gi1 could still
associate with caveolin-1 and translocate to the plasma membrane in spite of
the fact that they lack the caveolin-1-interacting motif
(Galbiati et al., 1999
). To
determine whether the targeting to Triton-X100-insoluble domains as well as
the localization of our tagged GFP constructs to caveolae coincided with a
physical interaction between the recombinant proteins and caveolin-1, we
performed coimmunoprecipitation experiments
(Fig. 5B). Immunoprecipitation
of linker-GFP or C3S-GFP transiently transfected COS-7 cells with
anti-caveolin-1 antibodies, and analysis of the immunoprecipitate with
anti-GFP antibodies failed to reveal any positive interaction. Nevertheless,
we were able to detect caveolin-1 as well as a caveolae-associated protein
(the
subunit of Gq) in caveolin-1-immunoprecipitated COS-7 cells
(Fig. 5B, left panel).
Likewise, when we immunoprecipitated linker-GFP or C3S-GFP-transfected COS-7
cells with anti-GFP antibodies, identification of caveolin-1 association in
the immunoprecipitate was unsuccessful
(Fig. 5B, right panel). By
contrast, experiments performed in parallel confirm that the
immunoprecipitation of the GFP constructs was successful. Hence, the most
likely explanation for our results is that dual acylation per se partially
targets the GFP reporter to caveolae owing to the intrinsic low-fluidity
properties of this cholesterol-sphingomyelin-enriched domain, rather than to a
physical interaction with caveolin-1 in COS-7 cells.
Colocalization of chimeric GFPs with the Golgi compartment
To assess whether the various N-terminal fatty acylated sequences contain
different subcellular information that might target the GFP chimeras to the
Golgi apparatus, we used BODIPY-Texas Red-ceramide in vivo as a marker. The
intrinsic fluorescence of the linker-GFP, G2A-GFP and C3S-GFP chimeras in
living cells together with the Golgi staining is shown in
Fig. 6. Internalization of this
Golgi marker occurs via endocytosis, and some of the endosome and areas of the
trans Golgi network can display partial labeling, particularly in our in vivo
staining. The three GFP-tagged constructs colocalize with the Golgi marker in
intracellular membranes adjacent to the nucleus
(Fig. 6A, right panels).
Colocalization of the linker-GFP chimera with BODIPY-TR-ceramide is more
apparent in perinuclear regions, although partial overlap can also be observed
in certain endocytic regions in the proximity of the plasma membrane. On the
other hand, the broad distribution of the G2A-GFP fluorescence in the entire
cytosol and nucleus without any specific staining invalidates any positive
colocalization with the Golgi marker. Mutant C3S-GFP exhibits a clear overlap
with BODIPY-TR-ceramide, which indicates that the myristoylation observed for
this mutant results in Golgi targeting. Additionally, we inspected the
phenotype adopted by the linker-GFP and C3S-GFP chimeras upon incubation with
cycloheximide for 2 hours (Fig.
6B). In both cases, the total fluorescence observed diminishes.
Significantly, in the dually acylated linker-GFP construct, the
plasma-membrane-associated signal is considerably less affected than the Golgi
localization. Hence, this Golgi pool probably reflects a biosynthetic
intermediate of the linker-GFP mutant along the secretory pathway en route to
the caveolae-associated plasma membrane subdomains. In the case of the C3S-GFP
mutant, the Golgi staining becomes significantly reduced, whereas some
ER-associated perinuclear staining is still observed
(Fig. 6B).
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Changes induced in the subcellular localization of the GFP chimeras
induced by cholesterol depletion and by 2-bromopalmitate treatment
We also investigated whether in vivo cholesterol depletion with
ß-methyl cyclodextrin alters the plasma membrane targeting of the dually
acylated chimera linker-GFP (Fig.
7). Inspection of the changes arising from cholesterol removal in
COS-7 cells 36 hours post-transfected with the linker-GFP chimera revealed
that after 10 minutes of incubation with ß-methyl cyclodextrin, the GFP
fluorescence associated with the plasma membrane was altered
(Fig. 7B). Examination of the
live COS-7 cells at 20 and 30 minutes after treatment
(Fig. 7C,D) allows us to
conclude that a redistribution of the caveolar targeting of the mutant is
taking place owing to the sequestration of cholesterol. Whereas the
perinuclear Golgi staining remains basically unaffected by treatment for 30
minutes with the reagent, most of the plasma membrane localization of the
myristoylated and palmitoylated mutant is profoundly modified
(Fig. 7D, arrows). Thus, after
dual acylation and partial translocation to plasma membrane subdomains, the
integrity of the cholesterol-sphingomyelin rafts appears to be requisite for
the correct caveolar association of the dually acylated construct.
|
Recently, 2-bromopalmitate was reported to be an effective inhibitor of
protein palmitoylation in vivo (Webb et
al., 2000). Therefore, we used this reagent to further confirm the
role of palmitoylation in the membrane targeting of the dually acylated GFP
chimeras (Fig. 7E-J). As
expected, the linker-GFP, C3S/S9C-GFP and C3S/S15C-GFP chimeras were
significantly affected by the 2-bromopalmitate treatment, resulting in
phenotypes that strongly resembled that of the C3S-GFP chimera, with most of
the plasma membrane localization being lost. The G2A-GFP and C3S-GFP chimeras
remained unaffected by the treatment (Fig.
7), as did the G2A/C3S-GFP construct (data not shown).
Intriguingly, the reagent also affected the fluorescence associated with
intracellular membranes, resulting in increased vacuolization, which lead to
the appearance of small vesicles that display trapped GFP fluorescence. This
phenotype could be observed both in the singly myristoylated (C3S-GFP,
C3S/S21C-GFP) as well as in the dually acylated chimeras and very probably
reflects some collateral metabolic alteration induced by 2-bromopalmitate
treatment. However, treatment of COS-7 cells with bromopalmitate or
methyl-cyclodextrin under identical conditions did not affect the plasma
membrane staining of integrin
3ß1, a protein
known to be excluded from rafts/caveolae (data not shown).
![]() |
Discussion |
---|
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---|
To date, the mechanisms underlying cellular protein palmitoylation have
remained elusive despite the significant number of identified proteins that
are post-translationally modified through the attachment of a palmitic moiety
to the side chain of a cysteine residue. Unlike the well characterized
myristoylation motif recognized by the enzyme N-myristoyl transferase,
inspection of the N-terminus of dually acylated proteins reveals that no clear
consensus sequence exists for a certain protein for S-acylation with palmitic
acid. Although some palmitoylated cysteine residues are in the proximity of
basic residues (for example, in Hck and Fgr kinases and Gz),
a few others are close to Ser or Thr residues (for example, in Lck kinase,
G
i1, G
0 and Vac8p) or hydrophobic residues
(Fyn, Lyn and Yes kinases, AKAP18, MPSK and eNOS). Remarkably, myristoylation
is frequently a prerequisite for a dually acylated protein to become
palmitoylated, since elimination of the myristic acid aceptor residue, Gly2,
abrogates subsequent palmitoylation (Koegl
et al., 1994
; McCabe and
Berthiaume, 1999
; Galbiati et
al., 1999
). The putative existence of a unique cellular palmitoyl
transferase presupposes that this enzyme must recognize cysteine residues
inserted within different amino acid motifs before catalyzing the addition of
palmitate from palmitoyl-CoA. Moreover, this enzyme should selectively
recognize a very vague sequence motif present only in a myristoylated
substrate and subsequently transfer palmitic acid to the thiol group of a
proximal Cys residue. Conversely, multiple palmitoyl transferases might exist,
each with a different cellular substrate, implicated individually in the tight
regulation of the activity of one dually acylated protein, many of which are
involved in signaling processes (Milligan
et al., 1995
; Dunphy and
Linder, 1998
; Simons and
Toomre, 2000
; Janes et al.,
2000
).
However, since the palmitoylation-depalmitoylation recycling of proteins is
a strictly regulated process, one can envisage that this cycle might be
controlled at the depalmitoylation level
(Dunphy and Linder, 1998;
Resh, 1999
). Interestingly, a
cytoplasmic acyl-protein thioesterase (APT-1) that removes palmitate from G
protein
subunits, p21Ras and eNOS has been recently identified
(Duncan and Gilman, 1998
;
Yeh et al., 1999
), although
the cellular mechanisms of regulation of this esterase remain to be
established. Since APT-1 also hydrolyzes both free palmitoyl-CoA and palmitic
acid covalently bound to proteins, it is conceivable that an increase in
cellular APT-1 activity is accompanied by both diminished protein
palmitoylation and augmented protein depalmitoylation. Nevertheless, further
experiments seem necessary in order to analyze the cellular mechanisms that
regulate protein acylation.
In this study, we have analyzed whether de-novo-designed sequences fused to
the GFP reporter become effectively palmitoylated in vivo. Two other dually
acylated GFP-tagged chimeras - Gi1 and eNOS - were used as positive
controls. In the absence of hydrophobic residues, polybasic signals or
prenylation sites, the palmitoylation of a Cys residue inserted in the
linker-GFP chimera is a direct consequence of its reactivity towards cellular
palmitoyl transferases (enzymatic palmitoylation) or its direct interaction
with Pal-CoA (non-enzymatic palmitoylation). Efficient palmitoylation was
observed at Cys3, Cys9, Cys15 and marginally at position Cys21, which is
indicative that (i) palmitoylation can efficiently occur in a de novo-designed
sequence, (ii) this process is dependent on the proximity of the reactive
thiol to the N-terminus of the protein and (iii) palmitoylation only takes
place on sequences that were previously myristoylated. The degree of
palmitoylation observed was similar to the one displayed by the eNOS-GFP
chimera under identical pulse treatment
(Fig. 1C). It is interesting,
in this context, to note that eNOS becomes N-terminally myristoylated and
subsequently palmitoylated at positions Cys15 and Cys26
(Liu et al., 1995
;
Robinson et al., 1995
).
However, the presence of multiple hydrophobic residues in the proximity of
these palmitoylated cysteine residues are probably responsible for the
attachment of the palmitate in such distant positions, since mutagenesis of
the five Leu residues present within residues 16 and 25 of eNOS abrogate
palmitoylation (Liu et al.,
1997
).
According to our data, myristoylation is a prerequisite for protein
palmitoylation (Fig. 1C),
mimicking the well known dependence of S-acylation on prior membrane
association via myristoyl, prenyl or transmembrane peptide moieties
(Dunphy and Linder, 1998;
Resh, 1999
). Whereas
N-terminal myristoylation is enough to induce the nuclear exclusion and
partial intracellular membrane association of the GFP reporter, dually
acylated proteins are excluded from the cell nucleus and localize to
intracellular Golgi membranes and partially to the plasma membrane
(Fig. 2). Although both
myristoylation and palmitoylation individually increased the partitioning of
the proteins into membranous fractions, the dually acylated proteins were
found almost uniquely in the particulate fractions
(Fig. 3). The increased
hydrophobicity provided by both post-translational acylations resulted in
augmented interaction with cellular membranes, although only palmitoylation
conferred on the GFP chimeras the ability to translocate towards
cholesterol-sphingomyelin-enriched domains, in accordance with the proposed
role of palmitoylation and caveolae localization (Figs
4 and
5). Indeed, plasma membrane
staining of the dually acylated, but not the myristoylated, GFP chimeras
provides support for the unique lipid-interacting properties endowed by the
palmitic moiety (Fig. 5). Since
our constructs lack the consensus caveolin-1-interacting motif
(Couet et al., 1997
), it must
be concluded that palmitate per se conveys on the N-terminal tag the ability
to interact with Triton-insoluble, low-fluidity domains. Significantly, in
spite of the colocalization with caveolin-1 in plasma membrane locations
(Fig. 5), dually acylated
chimeras were unable to physically interact with caveolin-1. This lack of
physical interaction with caveolin-1 has also been observed in short stretches
of dually acylated kinases fused to the GFP reporter as well
(McCabe and Berthiaume,
2001
).
N-terminal myristoylation was sufficient to colocalize the GFP chimeras
with the Golgi marker BODIPY-TR-ceramide, whereas dual acylation lead to both
Golgi targeting and partial colocalization with caveolin-1 (Figs
5 and
6). The coincidence with the
caveolin-1 staining for our myristoylated plus palmitoylated chimeras occurs
mostly in the proximity of the plasma membrane, although, as expected
(Luetterforst et al., 1999),
some Golgi staining could also be observed. Further demonstration of the
localization of the dually acylated constructs to Triton-insoluble membrane
subdomains was achieved using the cholesterol-sequestering agent
ß-methyl-cyclodextrin and 2-bromopalmitate, a known inhibitor of cellular
palmitoylation (Fig. 7).
Interestingly, recent studies carried out with surfactant protein C, a
small protein that contains a polar palmitoylated segment followed by a long
transmembrane stretch, have demonstrated that S-acylation is strongly
dependent on the distance of the palmitoylable cysteine residue from the
hydrophobic transmembrane -helix
(ten Brinke at al., 2002
). In
this context, cysteine residues that are positioned in proximity to the
transmembrane domain are more efficiently palmitoylated than those separated
by a greater distance.
Finally, although our data cannot rule out the existence of cellular
palmitoyl transferase activities, they are consistent with the hypothesis that
certain cellular proteins are non-enzymatically S-acylated, especially if the
reactive cysteine thiol is in the proximity of the membrane environment owing
to a previous lipidic modification (myristoylation or prenylation). In our
case, if palmitoylation was the result of an enzymatic activity, we must then
assume that a cellular palmitoyl-transferase is able to recognize a
non-cellular N-terminal sequence (designed de novo). Alternatively, since
approximately 10% of the cellular palmitoyl-CoA is bound to membranes in COS-7
cells (Bañó et al.,
1998), it is then conceivable that the direct transfer of
palmitate to a reactive cysteine residue might take place. The recent
discovery that Src family tyrosine kinases, G
subunits, GAP43 and Ras
can become heterogeneously acylated on cysteine residues with fatty acids
other than palmitate (Liang et al.,
2001
) also supports the suggestion that non-enzymatic
palmitoylation of proteins might be occurring within cells. Since the thiol of
a cysteine is a good nucleophile, the selective incorporation of one fatty
acid over the other might depend on the availability of the specific
membrane-bound acyl-CoAs in the proximity of the reactive cysteine.
![]() |
Acknowledgments |
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