©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Depalmitoylation of CAAX Motif Proteins
PROTEIN STRUCTURAL DETERMINANTS OF PALMITATE TURNOVER RATE (*)

(Received for publication, December 8, 1994; and in revised form, January 25, 1995)

Jui-Yun Lu Sandra L. Hofmann (§)

From the Department of Internal Medicine and the Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 alpha subunit of the heterotrimeric Gs protein, receptor-linked agonist stimulation causes a change in the half-life of [^3H]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 beta(2)-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.


EXPERIMENTAL PROCEDURES

Materials

[^3H]Palmitic acid (60 Ci/mmol) was from DuPont NEN. TranS-label (>1000 Ci/mmol) was from ICN. Plasmid pAT-rasH was a gift of Dr. Channing Der (University of North Carolina). NIH 3T3 cells were obtained from American Type Culture Collection (ATCC CRL 1658). COS-1 cells were a gift of Dr. Jane Zara (University of Texas Southwestern Medical Center). Antibodies c-H-ras and Y13-259 and Protein G-PLUS agarose were from Oncogene Science. Affinity-purified rabbit IgG was a gift of Dr. Susanne M. Mumby (University of Texas Southwestern Medical Center). Secondary antibodies (horseradish peroxidase-linked anti-rat and anti-rabbit IgGs) were from Amersham Corp. Other reagents were purchased from Sigma.

Plasmids and Mutagenesis

Plasmids were constructed by standard molecular biological techniques(17) . The plasmid Ras(WT) was derived from pAT-rasH (18) by polymerase chain reaction (PCR) (^1)amplification of the entire H-Ras coding region using oligonucleotides (5`-CATGCGGCCGCACGATCATGACAGAATACACAG-3` and 5`-CATTCTAGAATGTTCAAGACAGTCTGTGCA-3`) corresponding to nucleotides 1-16 and 736-759 of the c-H-ras-1 cDNA(19) , followed by subcloning into NotI/XbaI-digested RcCMV (Invitrogen). Mutations were introduced into Ras(WT) by PCR using 3` oligonucleotides with desired point mutations and Ras(WT) as a template to form the plasmids listed in Table 1. PCR inserts were verified by dideoxynucleotide sequencing.



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.

Cell Culture and Transfections

Simian COS-1 cells were maintained in monolayer culture in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B and transfected by electroporation (200 volts/960 µF) at a concentration of 40 times 10^6 cells/ml in maintenance medium containing 40 µg/ml of plasmid DNA and 200 µg/ml of sonicated salmon sperm DNA as described previously(21) . Sodium butyrate (6 mM) was present in the culture medium for 24 h after electroporation and plating.

Metabolic Labeling and Pulse-Chase Experiments

For metabolic labeling with [^3H]palmitate, transfected COS-1 cells (65-72 h post-electroporation) were preincubated for 30 min in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5 mM sodium pyruvate, and 4 times non-essential amino acids (FA medium), then labeled with 200 µCi/ml [^3H]palmitate in fresh FA medium. The labeling medium was prepared as described(22) . To demonstrate turnover of protein-bound palmitate, the cells were pulse-labeled for 15 min with [^3H]palmitate in FA medium, the medium was removed, and the dishes were rinsed four times with chase medium (FA medium containing 100 µM palmitic acid and 1 mg/ml bovine serum albumin (fraction V, Sigma)). Chase medium containing 50 µg/ml cycloheximide was added and the incubations were continued for varying periods of time as indicated. For metabolic labeling experiments involving [S]methionine, COS-1 cells were preincubated in methionine-free medium for 30 min, pulsed with 100 µCi/ml [S]methionine for 15 min, washed four times, and chased with unlabeled medium containing 10 times the normal methionine content.

Immunoprecipitation and SDS-Polyacrylamide Gel Electrophoresis

Cells were washed twice in ice-cold phosphate-buffered saline (PBS), pelleted by centrifugation, and lysed in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM phenylmethylsulfonyl fluoride, pH 7.4). After 10 min on ice, the lysates were centrifuged at 12,000 times g for 10 min, and the supernatants were collected. Aliquots containing 50 µg of total protein were either analyzed directly by SDS-polyacrylamide gel electrophoresis and fluorography or adjusted to a total volume of 0.5 ml in RIPA buffer and incubated with anti-Ras antibody (Y13-259, Oncogene Science, 1 µg) for 16 h at 4 °C, and the immune complexes were collected using 15 µl of protein G-PLUS agarose beads (Oncogene Science). To immunoprecipitate protein A-H-Ras fusion proteins, supernatants were incubated with rabbit IgG-agarose (15 µl of a 50% suspension in RIPA buffer). The resulting immunoprecipitates were washed three times with RIPA buffer and once with 100 mM Tris-HCl, pH 6.8, dissolved in Laemmli (23) loading buffer containing 50 mM dithiothreitol, heated to 75 °C for 3 min, and electrophoresed in 12% SDS-polyacrylamide slab gels(23) . [^3H]Palmitate-labeled proteins were detected by fluorography, which was performed by incubating Coomassie Brilliant Blue-stained gels with ENTENSIFY (DuPont) according to the directions supplied by the manufacturer. Treated gels were dried and exposed directly to Kodak XAR-5 film at -70 °C.

Subcellular Fractionation

Transfected COS-1 cells (two 100-mm dishes/data point) were harvested three days post-electroporation into ice-cold PBS and pelleted at low speed. Cell pellets were suspended in 0.5 ml of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and the cells were lysed by 15 passages through a 25-gauge needle. Nuclei and intact cells were removed by microcentrifugation at 3000 rpm for 5 min. The supernatant was centrifuged for 30 min at 120,000 times g in a T-1270 ultracentrifuge (Sorvall Instruments). The supernatant from this high speed spin was designated the soluble (cytosol) fraction. The resulting membrane pellet was washed, resuspended in 0.25 ml of membrane buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl(2), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and designated the particulate (membrane) fraction. Samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis followed by immunoblotting.

Immunoblotting

Cell lysates containing 1-5 µg of total protein were electrophoresed on 12% SDS-polyacrylamide gel electrophoresis slab gels and the proteins were transferred to nitrocellulose as described previously (24) in a buffer containing 10 mM NaHCO(3) and 3 mM Na(2)CO(3) at pH 9.9 in 20% (v/v) methanol. Filters were blocked for 1 h in PBS-T (0.25% (v/v) Tween 20 in PBS) containing 5% (w/v) dried milk, washed in PBS-T, and incubated for 2 h with the appropriate antibody (either mouse anti-H-Ras, Oncogene Science, catalog number OP23, 0.5 µg/ml or affinity-purified rabbit IgG, 0.1 µg/ml) in PBS-T. The filters were washed and incubated for 30 min with a horseradish peroxidase-conjugated secondary antibody (sheep anti-mouse or donkey anti-rabbit IgG, Amersham) diluted 1:2000 in PBS-T and developed using an enhanced chemiluminescence detection kit (Amersham). Exposure times on Kodak XAR-5 film varied from 3 to 20 s.

Other Methods

Protein concentrations were determined by Bio-Rad D(C) Protein Assay.


RESULTS

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 [^3H]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 [^3H]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 [^3H]palmitate bound to H-Ras in transfected COS-1 cells. The proportion of [^3H]label bound to H-Ras with time (relative to time 0) in H-Ras-transfected cells was determined by densitometric scanning of films from [^3H]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 [^3H]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.




DISCUSSION

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 Gsalpha, the addition of beta blocks AlF(4)-dependent depalmitoylation of Gsalpha(7) ; furthermore, a mutant form of Gsalpha that cannot dissociate from beta does not undergo agonist-stimulated palmitate turnover(29) . These observations are consistent with a model in which the dissociation of beta subunits from the alpha 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.


FOOTNOTES

*
This work was supported by Public Health Service Grant CA-61823 from the National Cancer Institute, the Robert A. Welch Foundation, the Charles E. Culpeper Foundation (of which S. L. H. is a Medical Scholar), and the Texas Higher Education Coordinating Board (Advanced Research Program). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Simmons Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8593. Tel.: 214-648-4911; Fax: 214-648-4940; Hofmann{at}simmons.swmed.edu.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation.


ACKNOWLEDGEMENTS

We thank David Carnahan for technical assistance, Christine Kim Garcia for advice in the preparation of the protein A fusion constructs, and Shari Cundiff and the Simmons Cancer Center Nucleic Acid Core Facility for oligonucleotides.


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