(Received for publication, December 7, 1995; and in revised form, January 16, 1996)
From the
Heterotrimeric G proteins are covalently modified by lipids.
Myristoylation of G-protein subunits and prenylation of
subunits are stable modifications. In contrast, palmitoylation of
subunits is dynamic and thus has the potential for regulating protein
function. Indeed, receptor activation of G
increases
palmitate turnover on the
subunit, presumably by stimulating
deacylation. The enzymes that catalyze reversible palmitoylation of
G-protein
subunits have not been characterized. Here we report
the identification of a palmitoyl-CoA:protein S-palmitoyltransferase activity that acylates G-protein
subunits in vitro. Palmitoyltransferase activity is
membrane-associated and requires detergent for solubilization. The
preferred G-protein substrate for the enzyme activity is the
subunit in the context of the heterotrimer. Both myristoylated and
nonmyristoylated G-protein
subunits are recognized as substrates.
The palmitoyltransferase activity demonstrates a modest preference for
palmitoyl-CoA over other fatty acyl-CoA substrates.
Palmitoyltransferase activity is high in plasma membrane and present at
low or undetectable levels in Golgi, endoplasmic reticulum, and
mitochondria of rat liver. The subcellular localization of this enzyme
activity is consistent with a role for regulated cycles of acylation
and deacylation accompanying activation of G-protein signal
transduction pathways.
Signal-transducing G proteins are located on the cytoplasmic
surface of the plasma membrane where they couple receptors to
intracellular effectors. Membrane association of heterotrimeric G
proteins is facilitated by the covalent addition of lipids to G-protein
subunit polypeptides (reviewed in (1) and (2) ). The
carboxyl-terminal cysteine residue is prenylated and methylated on
G-protein subunits. Prenylation is not required for
complex formation, but facilitates subunit and effector interactions.
G-protein
subunits are fatty-acylated. Members of the mammalian
G
family (G
, G
,
G
, and transducin
) contain amide-linked
myristate at the amino terminus. Transducin
(T
) (
)is modified heterogeneously at this site by C14:0, C14:1,
C14:2, or C12:0 fatty acids. Myristoylation also facilitates subunit
and effector interactions. Thioester-linked palmitate is found on most
mammalian G-protein
subunits. G
,
G
, and G
, which are not N-myristoylated, are palmitoylated at one or more cysteine
residues near the amino terminus. G
,
G
, and G
are palmitoylated at a
cysteine residue (Cys-3) adjacent to the amino-terminal myristoylated
glycine.
Reversible post-translational modification is a
well-characterized mechanism for regulating protein activity.
Regulatory cycles of acylation and deacylation of G-protein
subunits may fit this paradigm. Dynamic acylation of G proteins has
been characterized best for G
. Studies of
agonist-induced turnover of palmitate on G
are
consistent with a model where G
is deacylated upon
activation and dissociation from
subunits(3, 4, 5) . Deacylation may be
accompanied by release of G
from membranes, suggesting
a potential role for this process in desensitization of
G
-coupled pathways(5) .
The enzymes responsible
for addition and removal of palmitate from G-protein subunits
have not been identified. G-protein
subunits can be deacylated in vitro by a protein palmitoyl thioesterase that has recently
been purified(6) . However, subsequent cloning of the cDNA
encoding protein palmitoyl thioesterase and characterization of the
gene product revealed that protein palmitoyl thioesterase is a secreted
enzyme(7) , and thus is not likely to be a physiological
regulator of G-protein palmitoylation. Palmitoyltransferase (PAT)
activities have been identified using a number of proteins known to be
palmitoylated as substrates, including viral glycoproteins(8) ,
p21
(9) , and
p59
(10) , but it is not known whether
the substrate specificity of these activities extends to G-protein
subunits. Purification to homogeneity and molecular cloning of
palmitoyltransferase activities have not been achieved to date. Here we
report the initial characterization of a palmitoyl-CoA:protein S-palmitoyltransferase activity highly enriched in plasma
membranes that acylates both myristoylated and nonmyristoylated
G-protein
subunits in vitro.
Purified T and
T
subunits were kindly provided by Dr. Susanne
Mumby (Southwestern Medical Center, Dallas, TX). The preparation of
T
was analyzed by pertussis toxin-catalyzed
ADP-ribosylation supported by T
subunits to ensure
that the preparations were active(14) . Bovine brain
subunits were a gift of Dr. William Singer and Dr. Paul Sternweis
(Southwestern Medical Center). Recombinant
C68S was purified
from insect cells infected with recombinant
1 and
2C68S
viruses(16) . To determine if myristoylated rG
formed a heterotrimer with
C68S, sedimentation analysis
was performed (17) . Myristoylated rG
(0.5
nmol),
C68S (0.5 nmol), or an equimolar mixture was
sedimented through gradients of sucrose (5-20%) in 20 mM NaHepes (pH 8.0), 2 mM EDTA, and 1 mM DTT.
Gradients were subjected to centrifugation in an SW65 rotor (Beckman)
for 16 h at 50,000 rpm and 2 °C. Gradients were separated into 20
fractions and analyzed by SDS-PAGE.
PAT activity was detected in
membranes and detergent extracts. Bovine brain membranes were incubated
with myristoylated rG, bovine brain
subunits (see below), and [
H]palmitoyl-CoA.
Palmitate incorporation into myristoylated rG
was
assessed by SDS-PAGE and fluorography. Palmitate was incorporated into
myristoylated rG
incubated with bovine brain
membranes, suggesting that a palmitoyltransferase activity was present (Fig. 1A, lane 1). (
)Similar
results were obtained when the assay was terminated by precipitation
and protein-bound radioactivity was quantitated by liquid scintillation
counting (data not shown). Far less PAT activity, if any, was detected
in cytosol (Fig. 1A, lane 2), nor was it
released by treatment of membranes with high pH or high concentrations
of salt (data not shown). However, palmitoyltransferase activity was
solubilized by detergent (Fig. 1A, lane 3).
The C3A mutant of myristoylated rG
was not a
substrate in the assay (Fig. 1A, lane 4),
indicating that in vitro acylation is occurring at the
appropriate cysteine residue in the protein.
Figure 1:
Enzymatic acylation of myristoylated
rG. Reactions were carried out as described under
``Experimental Procedures.'' A, aliquots of 20
µg of bovine brain membranes (Mb) (lane 1) or
cytosol (Cyto) (lane 2) protein were assayed for PAT
activity in the presence of myristoylated rG
and
bovine brain
subunits. Bovine brain membranes were extracted
with 2% BigChap, and an aliquot of the extract (XT) (5 µg)
was assayed in the presence (lane 3) or absence (lane
5) of myristoylated rG
plus
or
myristoylated rG
C3A plus
(lane 4).
The samples were subjected to SDS-PAGE and fluorography. Exposure time
was 1 day. B, bovine brain detergent extract (4 µg) was
pretreated at 30 °C for 1 h with 3.2 mg/ml trypsin (column 2) or
3.2 mg/ml trypsin plus 10 mg/ml soybean trypsin inhibitor (column 3).
Trypsin digestion (column 2) was terminated by the addition of 10 mg/ml
soybean trypsin inhibitor prior to the PAT assay. Detergent extract (4
µg) was heat-treated at 100 °C for 1 min (column 4) or
preincubated in 0.05% SDS (column 5) before the PAT reaction. PAT
assays were performed using myristoylated rG
and
bovine brain
subunits as substrate. Samples were subjected
to filter binding and liquid scintillation
counting.
Experiments were
performed to determine if in vitro acylation of G-protein
substrates was indeed enzymatic. Uncatalyzed acylation of substrate
proteins incubated with palmitoyl-CoA in the absence of a source of
enzyme was first reported for rhodopsin (29) and myelin
proteolipid protein(30) . Low levels of palmitate were
incorporated into myristoylated rG in the absence of
enzyme (Fig. 1A, lane 6). Uncatalyzed
acylation was more pronounced if detergent was omitted from the assay
or when the protein was incubated with higher concentrations of
palmitoyl-CoA (data not shown). To confirm that the activity observed
in a detergent extract of membranes required a protein component, we
treated the extract with trypsin. Protease treatment reduced PAT
activity to background levels (Fig. 1B). Soybean
trypsin inhibitor abolished the effect of trypsin. PAT was also
inactivated by boiling or after treatment with SDS, a denaturing
detergent (Fig. 1B). PAT activity is time-dependent; in
the presence of membranes, the assay is linear to 10 min (data not
shown). The activity demonstrated strict concentration dependence on
the source of enzyme, G protein, and palmitoyl-CoA. At high
concentrations of palmitoyl-CoA or G protein, the activity was
saturable (data not shown). Taken together, these data demonstrate that in vitro acylation of myristoylated rG
is an
enzymatic process.
Figure 2:
G-protein substrate specificity of PAT
activity. G-protein subunits (2 µg) were incubated with
bovine brain detergent extract in the presence or absence of bovine
brain
subunits (2 µg) (lanes 2, 4, 6, and 8) or T
subunits purified
from bovine retina (lane 10). G-protein
subunits assayed
were: myristoylated rG
(lanes 3 and 4), nonmyristoylated rG
(lanes 5 and 6), rG
(lanes 7 and 8), and
T
purified from bovine retina (lanes 9 and 10). Assays were analyzed by SDS-PAGE and fluorography as
described under ``Experimental Procedures'' and exposed to
film for 1 day (A), or trichloroacetic acid-SDS precipitates
of the reactions were collected on glass fiber filters and quantitated
by liquid scintillation counting (B).
Myristoylated
rG was a better substrate than nonmyristoylated
rG
(compare lanes 3 and 4 with lanes 5 and 6). The effect of myristoylation on PAT
activity was also observed when rG
and myristoylated
rG
were assayed (data not shown). PAT activity is not
limited to myristoylated substrates. G
is not a
myristoylated protein, but is palmitoylated at Cys-3(4) .
rG
in the presence of
subunits was
palmitoylated in vitro, although not as efficiently as
myristoylated rG
(Fig. 2B, lanes
7 and 8). rG
(C3A) was not a substrate
for PAT activity in vitro (data not shown).
The substrate
specificity of PAT activity in vitro correlates well with our
understanding of G-protein palmitoylation in vivo.
G, G
, and G
are
substrates for PAT; the site of palmitoylation in vitro and in vivo appears to be Cys-3. Myristoylated G
is the optimal substrate for palmitoylation in vivo. In
mammalian cells, expression of a mutant G
lacking the
myristoylation site (G2A) results in a protein that is almost entirely
cytosolic and has undetectable levels of
[
H]palmitate incorporation. If
subunits are co-expressed with G
(G2A), a small
fraction of G
(G2A) is found associated with membranes
and a low level of palmitoylation is observed(27) . In the in vitro assay, myristoylated
subunits were better
substrates than those lacking myristate. However, a moderate level of
acylation of nonmyristoylated G
was observed in the
presence of
subunits. Thus, these characteristics are
similar to what is observed in vivo.
The role of G-protein
subunits in substrate affinity for PAT may be to provide a
mechanism for substrate presentation to PAT.
may facilitate
targeting of the
subunit to the membrane, allowing it to become
acylated by PAT. Although prenylation of G protein
subunits is
required for membrane association of the
complex(1, 2) , it is not required to support in
vitro acylation of myristoylated rG
. Mutation of
the prenylated cysteine residue to serine (C68S) in the
2 subunit
yields a nonprenylated
that heterodimerizes with the
1
subunit(16) . The mutant
(
C68S) binds to
myristoylated rG
, forming a heterotrimer (33) that is acylated in vitro with efficiency similar
to that of the wild type heterotrimer (data not shown). These data
suggest that
provides more than a hydrophobic anchor to bind
to a membrane (or detergent micelle) containing PAT. The palmitoylation
site is contained within the amino-terminal region of the
subunit, which is known to directly interact with
subunits(33, 34, 35) . Indeed,
binding changes G
amino-terminal
structure(33) , perhaps making it a better substrate for PAT.
Myristoylation may also facilitate access to PAT. Studies with
acylated peptides and model membranes have demonstrated that a
myristoyl moiety is not sufficient for high affinity interaction of the
acylated peptide with phospholipid vesicles(36) . However, even
a transient interaction of a myristoylated protein with the membrane
may allow the protein to be recognized by PAT and become palmitoylated.
The dually acylated protein will then have a high affinity for
membranes. Most members of the Src family of protein-tyrosine kinases
have an amino-terminal motif of a myristoylated glycine followed by a
palmitoylated cysteine (37, 38, 39) .
Palmitoylated Src family kinases require prior myristoylation to be
palmitoylated both in vivo(39) and in
vitro(10) . Although we have shown that myristoylation is
not an absolute requirement for recognition of the subunit by the
enzyme, the presence of myristate increases in vitro acylation
of G-protein subunits. Further purification and characterization of PAT
activity is required to resolve whether the same PAT activity acylates
G-protein
subunits and Src family kinases.
Figure 3:
Fatty acyl-CoA chain length specificity of
the in vitro PAT assay. A, PAT activity was partially
purified by gel filtration and ion exchange chromatography as described
under ``Experimental Procedures.'' Acetyl-CoA (C2:0),
lauroyl-CoA (C12:0), myristoyl-CoA (C14:0), palmitoyl-CoA (C16:0),
palmitoleoyl-CoA (C16:1), stearoyl-CoA (C18:0), oleoyl-CoA (C18:1),
linoleoyl-CoA (C18:2), and arachidonoyl-CoA (C20:4) were tested for
their ability to compete with [H]palmitoyl-CoA in
assays of PAT activity. PAT activity was assayed in the presence of 0.4
µM [
H]palmitoyl-CoA with no
additions (first bar) or with a 10-fold molar excess of the
indicated unlabeled acyl-CoA (second through ninth bars) in
the assay. Heterotrimer composed of myristoylated rG
,
and recombinant
C68S was used as the protein substrate. B, concentration dependence of fatty acyl-CoA inhibition of
PAT activity. PAT assays were performed as described in A with
indicated concentrations of nonradioactive acetyl-CoA (
),
arachidonoyl-CoA (
), myristoyl-CoA (
), stearoyl-CoA
(
), and palmitoyl-CoA (
).
Figure 4:
Subcellular localization of PAT in rat
liver membranes. A, rat liver membranes were fractionated as
described under ``Experimental Procedures'' and assayed for
PAT activity. Heterotrimers of myristoylated rGG203A
and
C68S were used as substrates in the assay. The G203A
substitution in E. coli-derived G
increases
its ability to bind to
C68S (44) which has reduced
affinity for G-protein
subunits(16) . (Note, however, at
the concentrations of G-protein subunits in the PAT assay, both wild
type G
and G
G203A form
heterotrimers with
C68S.) The assay was performed as
described except that the reaction was carried out using 15 µg of
membrane protein and the incubation time was 5 min. Specific activities
for PAT activity are expressed as pmol/min/mg. Total PAT activities
(pmol/min) in the subcellular fractions were: plasma membrane, 1.2
pmol/min; Golgi, 0.19 pmol/min; endoplasmic reticulum, none detected;
and mitochondria, 14.8 pmol/min (primarily due to the 45-kDa
mitochondrial protein). Specific activities of the marker enzymes are
expressed as nmol of product/min/mg. B, SDS-PAGE analysis of
PAT activity in subcellular fractions of rat liver. In an independent
fractionation, smooth (SER) and rough (RER)
endoplasmic reticulum were prepared as described (20) , as well
as plasma membrane (PM), cytosol (C), and
mitochondria (M). Note the 45-kDa band migrating above
G
in the assay of mitochondrial PAT
activity.
Studies of receptor-stimulated
turnover of palmitate on G are consistent with the
following model(3, 4, 5) . In the basal
state, the
subunit is palmitoylated and associated with
subunits. Upon ligand binding to the receptor, the
subunit becomes activated and dissociates from
subunits. The
subunit in its GTP-bound form is a substrate for a protein
palmitoyl thioesterase and becomes deacylated. Deactivation of
by
GTP hydrolysis results in its reassociation with
subunits
and coincides with reacylation of the
subunit. These studies do
not discriminate between palmitoylation occurring before or after
inactive
binds to
subunits. Because PAT activity
prefers the
subunit in the context of the heterotrimer as a
substrate, we suggest that palmitoylation occurs after reassociation of
the subunits. Enrichment of PAT activity in plasma membranes suggests
that the subcellular localization of this enzyme allows for rapid
reacylation of the G protein at the plasma membrane and does not
require the G protein to cycle to an intracellular compartment for
reacylation.