(Received for publication, December 8, 1994; and in revised form, January 25, 1995)
From the
In the present study, we examined the effect of amino acid substitutions on the rate of turnover of palmitate bound to a model ``CAAX'' motif protein H-Ras. These experiments were designed to shed light on the specificity of the process that removes palmitate from prenylated proteins. H-Ras, protein A-Ras fusion constructs, and constructs with amino acid substitutions in the H-Ras hypervariable region were transfected into COS cells, and the turnover rate of palmitate bound to each expressed protein was measured. We found no evidence for strict sequence specificity for palmitate removal, but found a strong inverse correlation between palmitate turnover rate and the degree of membrane association for any given construct, with slower turnover rates associated with stronger membrane binding. These data support a model in which the palmitate turnover rate is determined by access to a depalmitoylating enzyme and argue against a more complex model in which specific recognition of palmitoylated proteins is required.
Many intracellular proteins are modified by the 16-carbon fatty acid palmitate; in most cases, the fatty acid is found on cysteine residues located in close proximity to the inner leaflet of the plasma membrane(1) . Palmitoylated proteins are invariably membrane-bound, and it has been suggested that the palmitate group serves as a membrane anchor or that it may play a role in protein-protein interactions or vesicle fusion. It is also possible that it plays a structural role that is unique to each modified protein.
Palmitate is bound in a high-energy thioester linkage to
the sulfhydryl group of cysteine in proteins, and the reversible nature
of this bond allows for a potentially regulated, dynamic cycle of
palmitate addition and removal. In several instances, the palmitate
group has been shown to be metabolically active, with a t much shorter than that of the polypeptide
chain. Some examples, in which the t
was
measured in pulse-chase experiments, include: the transferrin receptor,
12 h(2) ; ankyrin, 50 min(3) ;
p56
, less than 1 h(4) ; and N-Ras, 20
min (5) . A careful study of palmitoylated proteins in a single
cell type (the erythrocyte) showed a wide variation in palmitate
turnover rates, from less that 30 min to greater than 3 h(6) .
The basis for this variation in palmitate turnover rates among
different proteins is unknown, but in a few instances, palmitate
turnover has been shown to be stimulated by addition of a hormone or
agonist. In the case of the
subunit of the heterotrimeric Gs
protein, receptor-linked agonist stimulation causes a change in the
half-life of [
H]palmitate from 20 min to less
than 2 min, with a concomitant shift of the protein from membrane to
cytosol(7) . In this case, depalmitoylation was suggested as a
mechanism for receptor-linked down-modulation of G protein activation.
The
-adrenergic receptor may also undergo an
agonist-stimulated palmitoylation cycle(8) .
Little is known about the enzymes responsible for palmitoylation and depalmitoylation of proteins. No clear consensus sequence for protein palmitoylation has emerged from the study of amino acid sequences surrounding palmitoylation sites or from limited mutagenesis studies(9) . Palmitate groups are usually found either at the junction of the transmembrane domain and the cytoplasmic portion of intrinsic membrane proteins or in close proximity to plasma membrane lipid targeting signals, such as myristate or prenyl groups(10) . Ras and Ras-related small GTP-binding proteins associate with intracellular membranes by virtue of COOH-terminal lipid modifications(11, 12) , and these modifications are determined by sequences at the COOH terminus (the ``CAAX'' motif, where C is cysteine, A is any aliphatic amino acid, and X is any amino acid) that directs COOH-terminal protein prenylation (farnesylation or geranylgeranylation), removal of the COOH-terminal three amino acids, and carboxymethylation. In addition to the farnesyl modification, Ras proteins require either a palmitate group or a polybasic region for localization to the plasma membrane(13, 14) . These ``second signals'' (a single palmitate group in the case of N-Ras, two palmitate groups in the case of H-Ras, and a polybasic region in the case of K-Ras) greatly enhance the membrane association and transforming ability of these oncogenic proteins(13, 14) . Palmitoylation of Ras requires prior farnesylation(15) , and palmitoylated Ras is found entirely in association with cell membranes (16) . Magee et al. (5) have shown that the palmitoylation of N-Ras is a dynamic event, with a half-life in pulse-chase experiments of about 20 min. Whether the palmitoylation state of Ras is regulated is currently unknown.
In order to learn about the specificity of the process that removes palmitate from prenylated proteins, we systematically examined the effect of amino acid substitutions on the rate of turnover of palmitate bound to a model CAAX motif protein (H-Ras). We found no evidence for strict sequence specificity for palmitate removal, but we found a good inverse correlation between palmitate turnover rate and the degree of membrane association for any given palmitoylated protein construct.
Protein A-Ras fusion constructs were made as follows. Complementary oligonucleotides encoding the final 12 amino acid residues of c-H-Ras (nucleotides 537-575 in (1) )) were annealed and ligated into EcoRI/BamHI-cut pRIT2T (Pharmacia Biotech Inc.) to generate the intermediate plasmid pRIT2T-Ras. (pRIT2T contains protein A IgG-binding domain sequences followed by a polylinker for subcloning of inserts). pRIT2T-Ras was used as a template in a PCR reaction to produce an insert containing Protein A and Ras sequences on a KpnI-BamHI fragment that was subcloned into the expression vector pCMV5(20) . Inserted Ras sequences were verified by dideoxynucleotide sequencing.
Table 1is a list of the expression constructs used in
this study. Fig. 1shows the results of a representative
experiment in which COS cells were transfected with plasmid DNA by
electroporation, labeled with [H]palmitate for 15
min, and harvested varying times after the addition of unlabeled
palmitate. Cell lysates were prepared and subjected to electrophoresis
on SDS-polyacrylamide slab gels and processed for fluorography. Under
these conditions, the expressed H-Ras protein is the major labeled
protein in the cells; no labeled band is seen after transfection with
vector DNA alone or with Ras(C186S) (farnesylation site deleted) or
Ras(C181, 184C) (both palmitoylation sites deleted) (Table 1,
plasmids N and O, data not shown). When wild-type H-Ras is
overexpressed (Fig. 1, upper panel), the half-life of
palmitate bound to the protein is relatively long (approximately 90
min). However, substitution of serine for cysteine at either of the two
palmitoylation sites (C181S, middle panel; or C184S, lower
panel) considerably shortened the half-life observed. The
intensity of the labeling after a 15-min pulse was 2-3-fold
higher as compared with the wild-type, a result consistent with faster
turnover. The experiments presented in Fig. 1were repeated four
to seven times, and the gels were quantitated by densitometry; this
data are shown in Fig. 2. The half-life of palmitate bound to
wild-type H-Ras was 90 min, as compared with 15 min for Ras(C181S) and
20 min for Ras(C184S).
Figure 1:
Turnover of palmitate
bound to H-Ras proteins in COS-1 cells. COS-1 cells were transfected
with Ras(WT) (top panel), Ras(C181S) (middle panel),
or Ras(C184S) (bottom panel), pulse-labeled with
[H]palmitate for 15 min, and chased with an
excess of unlabeled palmitate for varying periods of time as indicated.
Cell lysates (50 µg of post-nuclear supernatant protein) were
subjected to electrophoresis in 12% SDS-polyacrylamide slab gels and
processed for fluorography. Fluorography was carried out at -85
°C for 16 days. Untransfected COS-1 cells yielded no detectable
signals under these conditions (data not
shown).
Figure 2:
Turnover of
[H]palmitate bound to H-Ras in transfected COS-1
cells. The proportion of [
H]label bound to H-Ras
with time (relative to time 0) in H-Ras-transfected cells was
determined by densitometric scanning of films from
[
H]palmitate pulse-chase experiments performed as
described in the legend to Fig. 1. Exposures were used for which
the intensity of the bands was linearly dependent on the radioactivity.
Results are expressed as the mean ± S.E. for four to seven
separate experiments. Closed circles, wild-type H-Ras; closed triangles, H-Ras (C181S); and closed squares,
H-Ras (C184S).
The COOH-terminal 10 amino acids of H-Ras are sufficient to direct palmitoylation and plasma membrane localization when attached to a heterologous protein, protein A(25) . Fig. 3shows the results of pulse-chase experiments in which plasmids A through J in Table 1were transfected into COS cells and the half-life of palmitate bound to each expressed protein was determined from plots similar to that shown in Fig. 2. Each bar represents the half-life (in minutes) of palmitate bound to a Ras protein (open bars) or protein A-Ras fusion protein (closed bars). The first important finding is that replacing all but the final 12 amino acids of H-Ras with protein A had no effect on the rate of palmitate turnover (Fig. 3, plasmid A versus plasmid B, 90 versus 85 min). However, changes in the final 12 amino acids had dramatic effects on palmitate turnover rate, with slightly more dramatic effects seen in the protein A-Ras fusion proteins (plasmids C through J). For example, the substitution of a serine for the palmitoylation site cysteine at positions 181 (plasmids C and D) or position 184 (plasmids E and F) caused a shortening of the palmitate half-life to 20 or 30 min in the case of H-Ras (plasmids C and E) and 15 min in the case of the protein A-Ras fusions (plasmids D and F).
Figure 3:
Effect of COOH-terminal mutations on
palmitate turnover rate of H-Ras (open bars) and protein A-Ras
fusion proteins (closed bars). Plasmids A through J (Table 1) were transfected into COS-1 cells, and the turnover
rate of palmitate bound to the expressed protein was measured as
described under ``Experimental Procedures.'' A P symbol at the indicated amino acid position indicates a palmitate
group present at that site, a dash indicates the absence of a
palmitate group at the site, and an F or G indicates
farnesylation or geranylgeranylation at position Cys,
respectively. Each value represents the mean ± S.E. for three to
seven separate experiments.
We next examined the effect of substituting leucine for serine at the COOH terminus of the H-Ras or protein A-Ras fusion proteins. This substitution results in the recognition of the expressed protein by geranylgeranyltransferase I, resulting in a geranylgeranyl (20 carbons) rather than a farnesyl (15 carbons) group as the COOH-terminal prenyl modification(26, 27) . There was no effect of this substitution on the half-life of palmitate bound to H-Ras and a modest increase in the half-life of palmitate bound to the protein A-Ras fusion protein (compare plasmids A versus G and plasmids B versus H). Similarly, there was a modest increase in the half-life of the monopalmitoylated proteins when geranylgeranyl was substituted for the farnesyl moiety (plasmids C versus I and plasmids D versus J).
Fig. 4tests the effect of further substitutions in the COOH-terminal amino acids of protein A-Ras fusion proteins. Plasmid D is the monopalmitoylated form of H-Ras(C181S), plasmid K contains sequences corresponding to the final 12 amino acids of N-Ras, and plasmids L and M substitute glutamines (plasmid L) or lysines (plasmid M) for amino acid residues 178 through 183 of H-Ras. The most striking finding is that the rapid turnover rate seen for the monopalmitoylated protein A-Ras fusion is virtually unaffected by substitution of a neutral amino acid at any of the remaining H-Ras-specific amino acids upstream of the CAAX motif. (Note that we did not replace the lysine at position 185 with glutamine; however, a proline replaces the lysine at this position in plasmid K without effect.) Substitution of amino acids at positions 178-183 with lysine, a modification designed to mimic the polybasic domain found in K-RasB and other small GTP-binding proteins(28) , caused a marked inhibition of palmitate turnover, as reflected in a long half-life of bound palmitate.
Figure 4: Effect of COOH-terminal mutations on half-life of palmitate bound to protein A-H-Ras fusion proteins. Plasmids D, K, L, and M (Table 1) were transfected into COS-1 cells, and the turnover rate of palmitate bound to the expressed protein A-Ras fusion protein was measured as described under ``Experimental Procedures.'' Each value represents the mean ± S.E. for three to seven separate experiments. Plasmid D contains H-Ras(C181S) sequences, and plasmid K contains wild-type N-Ras sequences fused to protein A as shown in Table 1.
The
increased rate of turnover of palmitate observed for the
monopalmitoylated forms of H-Ras and for the protein A-Ras fusion
proteins could have arisen as an artifact of increased turnover of the
polypeptide. This explanation was ruled out in an
[S]methionine pulse-chase experiment. COS-1
cells expressing plasmids A, C, I, or J (Table 1) were
pulse-labeled for 15 min with [
S]methionine and
incubated for varying periods of time up to 4 h in the presence of
unlabeled methionine and cycloheximide, and the expressed H-Ras or
protein A fusion proteins were collected by immunoprecipitation and
analyzed by SDS-polyacrylamide gel electrophoresis and fluorography.
These experiments showed that the expressed proteins were stable
throughout the period of observation (data not shown); therefore, the
palmitate turnover measured is not a reflection of increased protein
turnover. Immunoblotting of parallel samples used to generate all of
the data in Fig. 3and Fig. 4showed no change in
immunoreactive protein over the time period of the experiments (data
not shown).
For each of the plasmids in Table 1, the relative distribution of the expressed protein between membrane and cytosol was determined. For wild-type H-Ras, about 60% of the protein was membrane associated and 40% was cytosolic (Fig. 5, plasmid A). Previous studies have shown a higher percentage of H-Ras in the membrane (80-90%) (13) ; the lower percentage in our experiments may reflect some saturation of processing due to overexpression in our system (see below). When either palmitoylated cysteine is replaced by serine, there is a dramatic shift of protein from membrane to cytosol (compare plasmid A with plasmids C and D and compare plasmid B with plasmids E and F in Fig. 5). Substitution of a geranylgeranyl group for farnesyl at the COOH terminus did not change the membrane distribution of wild-type H-Ras (plasmid A versus plasmid G), but increased the membrane association of the protein A-H-Ras fusion proteins and the monopalmitoylated H-Ras and protein A-Ras fusion proteins (compare plasmid B versus H, plasmid C versus I, and plasmid D versus J). Fig. 6shows the membrane-cytosol distribution of the same proteins examined in Fig. 4. Protein A-Ras fusion proteins with COOH-terminal amino acids corresponding to H-Ras (C181S) (plasmid D), N-Ras (plasmid K), and polyglutamine (plasmid L), which all showed rapid palmitate turnover, had weak membrane association (about 20% in the membrane and 80% in the cytosol), whereas the protein with the polylysine substitution (plasmid M), which had the slowest palmitate half-life (110 min), was highly membrane associated (about 85%).
Figure 5:
Effect of COOH-terminal mutations on
membrane distribution of H-Ras (open bars) and protein A-Ras
fusion proteins (closed bars). Plasmids A through J (Table 1) were transfected into COS-1 cells, and the distribution
of the expressed protein between membranes and cytosol was measured as
described under ``Experimental Procedures.'' A P symbol at the indicated amino acid position indicates a palmitate
group present at that site, a dash indicates the absence of a
palmitate group at the site, and an F or G indicates
farnesylation or geranylgeranylation at position Cys,
respectively. Each value represents the mean ± S.E. for two to
three separate experiments.
Figure 6: Effect of COOH-terminal mutations on the membrane distribution of protein A-H-Ras fusion proteins. Plasmids D, K, L, and M (Table 1) were transfected into COS-1 cells, and the distribution of the expressed protein between membranes and cytosol was measured as described under ``Experimental Procedures.'' Each value represents the mean ± S.E. for two to three separate experiments. Plasmid D contains H-Ras(C181S) sequences, and plasmid K contains wild-type N-Ras sequences fused to protein A as shown in Table 1.
It is conceivable that if one of the steps involved in Ras processing were rate-limiting, then the membrane/cytosol distribution of a given H-Ras mutant might be influenced by its level of expression, producing misleading results. To address this possibility, we analyzed the relative expression levels of the constructs shown in Fig. 3and Fig. 4by immunoblotting and densitometry. We found no more than a 3-fold variation in expression among the various H-Ras constructs and a 2-fold variation among the protein A-H-Ras fusion proteins. There was no correlation between the relative expression level and either membrane distribution or the palmitate turnover rate presented in Fig. 3and Fig. 4(data not shown).
The palmitate
turnover rates we observed were not a consequence of events occurring
during the preparation of the material for analysis, as the palmitate
bound to Ha-Ras was stable in broken cell preparations. COS-1 cells
transiently expressing the mutant protein were labeled with
[H]palmitate for 15 min, lysed by sonication, and
incubated at 37 °C in PBS or hypotonic buffer. Samples were removed
after different periods of time and analyzed by SDS-polyacrylamide gel
electrophoresis and fluorography. Over a period of 2 h, no significant
loss of labeled palmitate from the protein was detected (data not
shown).
Comparison of the palmitate turnover rates for all of the constructs shown in Fig. 3and Fig. 4with the data for membrane association in Fig. 5and Fig. 6shows a striking correlation. The constructs showing the longest half-lives for bound palmitate are found to a much greater degree in the membrane and the constructs with the shortest palmitate turnover rates show a low degree of membrane association. This correlation is presented graphically in Fig. 7, which is a plot of the percentage of protein that is membrane-bound versus the half-life (in minutes) of palmitate bound to the protein.
Figure 7: Correlation of the half-life of palmitate bound to expressed proteins and the proportion of expressed protein that is membrane bound. Data extracted from Fig. 3Fig. 4Fig. 5Fig. 6for each expressed H-Ras protein (open circles) or protein A-Ras fusion protein (closed circles) is presented.
We examined the effect of amino acid substitutions on the turnover of palmitate bound to a model CAAX motif protein H-Ras. Substitution of all amino acids upstream of residue 177 with protein A sequences had virtually no effect on the palmitate turnover rate. Similarly, replacing all of the remaining COOH-terminal amino acids (with the exception of the prenylated cysteine) with neutral residues had no effect. Therefore, we found no evidence for a protein recognition signal that mediates rapid palmitate turnover. However, three different modifications at the COOH terminus prolonged palmitate turnover: dipalmitoylation versus monopalmitoylation, geranylgeranylation versus farnesylation, and the presence of a polybasic domain. These modifications have all been shown (by ourselves or others) to increase membrane association. A strong correlation between more avid membrane association and slower palmitate turnover was evident in the current study.
Our experiments revealed
a strong correlation between membrane binding and prolonged palmitate
turnover, but they cannot distinguish as to whether membrane
association dictates slower palmitate turnover or whether slower
palmitate turnover dictates membrane association. However, the absence
of a specific recognition signal for depalmitoylation argues against a
model in which depalmitoylation causes release from the membrane as a
primary event. In the case of agonist-stimulated depalmitoylation of
Gs, the addition of
blocks
AlF
-dependent depalmitoylation of
Gs
(7) ; furthermore, a mutant form of Gs
that cannot
dissociate from
does not undergo agonist-stimulated
palmitate turnover(29) . These observations are consistent with
a model in which the dissociation of
subunits from the
subunit increases the access of the palmitate to the deacylating
enzyme, rather than depalmitoylation causing release from the membrane
as a primary event. Our results, showing parallel changes in membrane
association and palmitate turnover rate without strict recognition of
the modified protein, are consistent with this model. We propose that
palmitate turnover is mediated by a nonspecific depalmitoylating enzyme
that is cytosolic or has an active site that is positioned at the
cytosol-membrane interface and that depalmitoylation occurs rapidly
upon access of the palmitoyl-cysteine thioester bond to the cytosolic
compartment. Regulatory proteins would have the potential to deny
access to the depalmitoylating enzyme, thereby stabilizing the
membrane-protein interaction, but specificity would not occur at the
level of the depalmitoylating enzyme. Alternatively, non-enzymatic
depalmitoylation by a cytosolic nucleophile is a formal possibility,
although unlikely given the stability of the thioester bond in broken
cell preparations, as described above.
Whether H-Ras undergoes receptor-coupled palmitate turnover, by analogy to the heterotrimeric G proteins, remains an open question. We did not detect a change in palmitate turnover rate for mutationally activated H-Ras (data not shown). This may have been a consequence of Ras overexpression and loss of normal effector coupling. (The study of the turnover of palmitate bound to endogenous levels of Ras are not currently feasible, due to low levels of expression). Future work will address this question. Furthermore, a detailed knowledge of the biochemical mechanism of protein palmitoylation and depalmitoylation will ultimately depend on the isolation and characterization of enzymes that catalyze these reactions.