(Received for publication, April 22, 1996, and in revised form, February 11, 1997)
From the Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700 009, India
Protein palmitoylation involves the
post-translational attachment of palmitate in thioester linkage to
cysteine residues of proteins. The labile nature of the thioester
linkage makes possible the palmitoylation-depalmitoylation cycles that
have emerged in recent times as additions to the repertoire of cellular
control mechanisms. However, detailed understanding of these cycles has been limited by the lack of knowledge of the transferases and thioesterases likely to be involved. Here, we describe the purification of a protein-palmitoyl acyltransferase (PAT) from human erythrocytes. PAT behaved as a peripheral membrane protein and catalyzed the attachment of palmitate in thioester linkage to the -subunit of
spectrin. On SDS-polyacrylamide gel electrophoresis, PAT appeared as a
70-kDa polypeptide. Antibody against this polypeptide could immunodeplete PAT activity from the crude extract, confirming the
assignment of the 70-kDa polypeptide as PAT. PAT-mediated spectrin
palmitoylation could be inhibited by nonradioactive palmitoyl-, myristoyl-, or stearoyl-CoA. The apparent Km for
palmitoyl-CoA was 16 µM.
A large number of proteins in cells are modified by the covalent
attachment of long chain saturated fatty acid residues (1-3). The two
most common modifications involve acylation with myristate and
palmitate. Myristate is usually attached to an N-terminal glycine via
an amide bond in a relatively stable linkage. Palmitate is usually
attached via a thioester bond to cysteine residues of proteins.
Palmitoylation occurs in membrane proteins with hydrophobic transmembrane segments as well as in hydrophilic proteins such as Ras
(1-4). These lipid modifications have often been implicated in
membrane association of the modified proteins (5). Palmitoylation occurs post-translationally. Moreover, rapid turnover of the
protein-bound fatty acid has been shown for several proteins (6-8).
The recent surge of interest in studying protein palmitoylation is
largely due to the fact that a number of proteins involved in
intracellular signaling are palmitoylated, and this appears to be
regulated by the activation status of these proteins (9-12).
Palmitoylation occurs in, among others, the families of (i) G
protein-linked receptors, (ii) the subunits of heterotrimeric G
proteins, and (iii) the non-receptor tyrosine kinases. Palmitate plays
a role in the membrane association of these polypeptides. Moreover,
recent evidence suggests that this may regulate protein-protein
interactions such as those between growth cone-associated protein 43 and Go in neurons (13). Although evidence that the
interactions of G proteins with effectors are regulated by
palmitoylation is becoming compelling, very little is known about the
enzymes involved in the catalysis of the palmitoylation and
depalmitoylation of these or other proteins.
A clear understanding of the palmitoylation-depalmitoylation cycle as a
cellular control mechanism obviously entails characterization of the
protein palmitoyltransferase(s) and thioesterase(s). A thioesterase has
recently been purified (14), and a corresponding cDNA has been
cloned (15). However, since this thioesterase is a secreted protein,
whereas Ras and G subunits are located on the
cytoplasmic layer of the plasma membrane, these proteins may not be its
physiologic substrates. The protein-palmitoyl acyltransferase (PAT)1 has remained, until lately,
refractory to purification. PAT activity has been demonstrated in
several eukaryotic membranes (16-20) and partially purified from
bovine brain (21) and from rat liver (22).
In our laboratory and that of others, palmitate associated with erythrocyte membrane cytoskeletal proteins has been demonstrated to undergo turnover (23), and palmitoylation has been found to modulate association of erythrocyte protein 4.2 with the membrane (24). These findings necessitated understanding the role of palmitoylation-depalmitoylation cycles in modulating interactions among erythrocyte cytoskeletal proteins, and efforts were made to characterize the PAT from human erythrocytes. This study reports the purification and biochemical characterization of a protein-palmitoyl acyltransferase from human erythrocytes.
[14C]Palmitoyl-CoA and [9,10-3H]palmitic acid were from Amersham International (Buckinghamshire, United Kingdom). Aprotinin, leupeptin, pepstatin, benzamidine, phenylmethylsulfonyl fluoride, dithiothreitol, nonradioactive fatty acyl-CoAs, and Formalin-fixed Staphylococcus aureus bearing protein A were from Sigma. Staphylococcal V8 protease was from Pierce. All other reagents were of analytical grade.
Purification of PATFresh blood was collected from normal healthy volunteers and washed in phosphate-buffered saline, and erythrocytes were packed and lysed in lysis buffer (7.5 mM sodium phosphate, 1 mM Na2EDTA, pH 7.5, containing 30 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml benzamidine, and 20 µg/ml phenylmethylsulfonyl fluoride). Ghosts were pelleted, washed, and extracted with 10 volumes of 0.2 mM Na2EDTA, pH 7.5 (i.e. low ionic strength extraction buffer), containing 20 µg/ml phenylmethylsulfonyl fluoride at 37 °C for 30 min. After centrifugation at 19,000 rpm (SS-34) for 30 min, the pellet was further extracted with an equal volume of KCl extraction buffer (7.5 mM sodium phosphate, 1 mM Na2EDTA, 1 mM dithiothreitol, 1 M KCl, pH 7.5) on ice for 30 min. After centrifugation at 20,000 rpm (SS-34) for 30 min, the supernatant (KCl extract) was dialyzed extensively against 20 mM piperazine buffer, pH 9.5. The dialysate was loaded on a Mono Q HR5/5 column (Pharmacia Biotech Inc.) equilibrated against the same buffer and fitted to a Pharmacia FPLC system. After washing the column, proteins were eluted with an increasing salt gradient (0-1 M NaCl). Fractions were assayed for PAT activity by the immunoprecipitation technique and analyzed by SDS-PAGE. Fractions that appeared homogeneous on SDS-PAGE and showed PAT activity were pooled, neutralized, and processed further.
Fast Desalting ChromatographyMono Q fractions containing
PAT activity were chromatographed on a Pharmacia Fast desalting column
(HR10/10) equilibrated in 100 mM imidazole buffer, pH 7.5, to bring the purified PAT in this buffer. Fractions were finally stored
at 70 °C. Under these conditions, the enzyme gradually lost
activity over a period of 2 weeks.
Spectrin was purified as described
by Bennett (25), stored at 70 °C, and used within 2 weeks.
Purified PAT or spectrin (75 µg) was injected subcutaneously with complete Freund's adjuvant into one male rabbit each. Successive injections of 40 µg each with incomplete Freund's adjuvant were given at the end of the 2nd, 3rd, and 4th weeks, followed by bleeding at the end of the 5th week.
SDS-PAGESDS-PAGE was performed using the discontinuous buffer system of Laemmli (26). Samples were dissolved in 62.5 mM Tris-HCl containing 2% (w/v) SDS, 10% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol, and 0.01% bromphenol blue. Gels were impregnated with 2,5-diphenyloxazole for autoradiography as described by Laskey and Mills (27).
PAT Assay and Analysis by FluorographyRoutinely, spectrin was incubated with PAT and [14C]palmitoyl-CoA in 100 mM imidazole buffer, pH 7.5, at 37 °C as described in the figure legends. Following reaction, samples were boiled in denaturing SDS gel buffer and separated on 7.5% SDS gels, followed by fluorography. To study the nature of PAT-mediated palmitoylation of spectrin, gels containing 14C-palmitoylated spectrin were treated (a) with CHCl3/CH3OH or (b) with 1 M neutral hydroxylamine prior to fluorography. Competition with nonradioactive octanoyl-, decanoyl-, lauroyl-, myristoyl-, palmitoyl-, and stearoyl-CoAs was performed by assaying PAT activity using 40 µM [14C]palmitoyl-CoA and a 2-fold excess of nonradioactive acyl-CoA as described above.
PAT Assay by Immunoprecipitation of SpectrinPAT assay was carried out using 8 µg of spectrin and 40 µM [14C]palmitoyl-CoA (50 mCi/mmol) in 100 mM imidazole buffer, pH 7.5, in a final volume of 30 µl. Incubations were usually carried out for 40 min at 37 °C unless otherwise stated. Following reaction, 20 µl of anti-spectrin antibody was added in a final volume of 100 µl containing 120 mM KCl, 5 mM sodium phosphate, pH 8, 0.5 mM EGTA, 0.5 mM dithiothreitol, 0.1% Triton X-100, and 1 mg/ml gelatin and incubated for 30 min at 25 °C. The amount of antibody and the time of incubation were found to be adequate for immunoprecipitating spectrin in all the assays reported here. This was followed by the addition of 100 µl of 10% Formalin-fixed S. aureus bearing protein A (washed with Buffer A (120 mM KCl, 5 mM sodium phosphate, pH 8, 0.5 mM EGTA, 0.5 mM dithiothreitol, 1 mM EDTA, 0.1% Triton X-100, 1 mg/ml gelatin)) and a further incubation for 1 h at 25 °C.
The S. aureus protein A with the bound antigen-antibody complex was sedimented at 10,000 × g for 3 min in a microcentrifuge and washed three times with 1-ml aliquots of Buffer A. The pellet with bound spectrin was counted in a liquid scintillation counter. For each experiment, a control sample lacking PAT was prepared and treated exactly as described above to correct for any nonspecific acylation of spectrin and any adherence of [14C]palmitoyl-CoA to the S. aureus protein A beads. The counts associated with the beads in these control tubes were subtracted from the counts of the corresponding PAT-containing tubes. The control values did not exceed 10-15% of those obtained in the presence of PAT. The stoichiometry of spectrin palmitoylation was determined using varying amounts of spectrin (2-6 µg), 40 µM [14C]palmitoyl-CoA, and 10 µg/ml PAT. The reaction was allowed to proceed for 60 min, followed by immunoprecipitation of spectrin as described above.
Immunoprecipitation of PATThis was performed essentially as described by Firestone and Winguth (28). Fresh KCl extract (25 µg) was incubated with various amounts of rabbit anti-PAT IgG or preimmune IgG in 30 µl for 1 h on ice in plastic tubes. Then 10 µl of a 10% suspension of prewashed S. aureus protein A was added and incubated for 1 h on ice with occasional shaking. The tubes were centrifuged, and the supernatants (15 µl) were assayed for PAT activity.
Analysis of the Spectrin-bound Labeled Fatty Acid14C-Palmitoylated spectrin was excised from SDS gels. The gel slices were incubated with 1 ml of 1.5 N NaOH at 30 °C for 3 h. The pH was then adjusted to 1-2 with 6 M HCl. The hydrolysate was extracted with CHCl3/CH3OH, and the organic phase was dried under nitrogen. The extracted lipid was analyzed by ascending chromatography on a reverse-phase C18 TLC plate using acetonitrile/acetic acid (1:1, v/v) as the mobile phase. [3H]Palmitic acid and [3H]myristic acid were used as standards. The plate was scraped, and the radioactivity was determined in parts to determine the relative migration of radiolabeled species in the samples.
Labeling of Erythrocytes with [3H]Palmitic Acid and Extraction of Labeled SpectrinHuman erythrocytes were labeled with [3H]palmitic acid, and tightly membrane-associated spectrin was extracted as described by Mariani et al. (4) with slight modifications. After washing the cells three times with phosphate-buffered saline/glucose (10 mM phosphate, 140 mM NaCl, 5 mM KCl, 0.5 mM EDTA, 5 mM glucose, pH 7.4), the cells were incubated twice for 15 min at room temperature with 10 volumes of Buffer B (40 mM imidazole, 90 mM NaCl, 5 mM KCl, 5 mM MgCl2, 15 mM glucose, 0.5 mM EGTA, 30 mM sucrose, 0.3 mM phenylmethylsulfonyl fluoride, 200 units of penicillin G, pH 7.6) containing 0.2% fatty acid-free bovine serum albumin to lower the endogenous pool of fatty acids. The bovine serum albumin-treated cells were washed once with Buffer B containing 5 µM coenzyme A and 5 mM pyruvate (Buffer C). Packed erythrocytes (2 ml) were labeled with 0.5 mCi of [9,10-3H]palmitic acid (54 Ci/mmol) at a hematocrit of 0.3 in Buffer C for 12 h at 37 °C. The labeling was stopped by adding cold phosphate-buffered saline/glucose and washing twice with the same buffer containing 0.2% fatty acid-free bovine serum albumin. Membranes were prepared by hypotonic lysis as described above. KCl-stripped inside-out vesicles were prepared as described by Mariani et al. (4), and tightly membrane-associated spectrin was extracted in the presence of 5 M urea (4) and concentrated with a Millipore Ultrafree microcentrifuge filter (Mr 100,000 cutoff) to 1-2 mg/ml.
Peptide Analyses of SpectrinThe method of Cleveland
et al. (29) was followed. Tightly membrane-associated
spectrin (representing the in vivo palmitoylated form of
spectrin) as well as spectrin palmitoylated in vitro using PAT were electrophoretically separated on a 5% SDS gel. Gel pieces containing the -subunit of spectrin were excised, rehydrated in
Buffer D (0.125 M Tris-HCl, pH 6.8, 0.1% SDS, 1 mM EDTA), and placed in the sample well of a second SDS gel
(15% acrylamide). The spaces of the wells were filled with Buffer D
containing 20% glycerol and S. aureus V8 protease (at an
enzyme/substrate ratio of 1:2). Digestion was allowed to proceed in the
stacking gel for 30 min. Electrophoresis was then continued, and the
gel was treated for fluorography.
Human erythrocyte ghosts have previously been
shown to have PAT activity (20). In this study, ghosts were subjected
to sequential extraction with buffers of increasing ionic strength. PAT
activity was assessed by studying the palmitoylation of spectrin. After extraction with low ionic strength extraction buffer, PAT activity remained associated with the membrane vesicles, and no activity was
detectable in the supernatant. PAT activity could be extracted in
buffer containing 1 M KCl. The 1 M KCl extract
catalyzed the palmitoylation of predominantly the -subunit of
spectrin (Fig. 1). This extract was dialyzed against 20 mM piperazine buffer, pH 9.5, and fractionated on a Mono Q
column as described under "Materials and Methods." PAT activity was
associated with a 70-kDa polypeptide that eluted between 0.7 and 0.9 M NaCl (Fig. 2). Active fractions that
appeared homogeneous on SDS-PAGE and showed an activity
80
pmol/min/ml were neutralized, chromatographed on a Pharmacia Fast
desalting column, and brought to 100 mM imidazole buffer,
pH 7.5. The purification is summarized in Table I. Both erythrocyte ghosts and vesicles remaining after low ionic strength extraction were sources of PAT as assessed by fluorography using spectrin as substrate. However, these could not be included in the
purification table. Quantitative measurements of PAT activity could not
be made with ghosts or vesicles remaining after low ionic strength
extraction since this would necessitate solubilization of the PAT from
the membranes. However, we and others (20) have observed that
detergents such as Triton X-100 used at concentrations usually
necessary for solubilization of membrane proteins (
1%) were
inhibitory for PAT activity. Besides this, ghosts contained substantial
amounts of spectrin, the substrate used in these studies to assess PAT
activity. Therefore, this was an additional impediment in the
quantitation of PAT activity. PAT obtained after Fast desalting chromatography was purified 86-fold over the KCl extract. However, an
assessment of the -fold purification over the ghosts could not be made
for the reasons stated above.
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The assignment of the 70-kDa protein as PAT was confirmed by
immunoprecipitation using antibody raised against this protein. This
antibody precipitated PAT activity from the KCl extract in a
dose-dependent manner (Fig. 3), while
preimmune IgG showed no such effect.
The PAT-catalyzed incorporation of radioactivity into spectrin from
[14C]palmitoyl-CoA was studied as a function of time. PAT
palmitoylated spectrin in a time-dependent manner (Fig.
4A), reaching a plateau after 40 min of
incubation. Palmitoylation of spectrin increased with increasing
amounts of the enzyme (Fig. 5), confirming that the
transfer of the palmitoyl moiety to spectrin is an enzymatic process.
PAT activity in the KCl extract was abolished on heating the enzyme for
5 min at 100 °C (Fig. 1, lane 3). The apparent Km for palmitoyl-CoA was found to be 16 µM (Fig. 4B). It was observed that the
inhibition of PAT activity by nonradioactive fatty acyl-CoAs was
dependent on the chain length of the fatty acyl-CoAs. Octanoyl-,
decanoyl-, and lauroyl-CoAs were poor inhibitors of PAT activity
(<10% inhibition). Among the longer chain fatty acyl-CoAs, when used
in 2-fold excess over radioactive palmitoyl-CoA, the ability to compete
with [14C]palmitoyl-CoA was as follows:
palmitoyl-CoA > myristoyl-CoA > stearoyl-CoA (Fig.
6A). In this connection, it may be mentioned that myristate has been demonstrated to be present in thioester linkage
in platelet proteins (30).
Palmitoylation of Spectrin Occurs via a Thioester Linkage
The
chemical nature of PAT-mediated spectrin palmitoylation was
investigated by soaking the polyacrylamide gel containing palmitoylated
spectrin in CHCl3/CH3OH (Fig. 6B) or
in neutral hydroxylamine. While soaking the gel in
CHCl3/CH3OH had no effect, the bound
radioactive palmitate could be removed by treatment with neutral
hydroxylamine (Fig. 6B), confirming that palmitoylation of
spectrin occurs via a thioester linkage. In a parallel experiment, palmitoylated spectrin was excised from the gel and hydrolyzed. The
hydrolysate was extracted with organic solvents and analyzed by TLC.
The RF value of the sample was identical to that of
standard radioactive palmitic acid, identifying the fatty acid incorporated into spectrin as palmitic acid (Fig. 6C). The
stoichiometry of palmitoylation was determined by immunoprecipitation
of spectrin after labeling with [14C]palmitoyl-CoA.
Incorporation of the palmitoyl moiety into spectrin occurred in a 1:1
molar ratio of fatty acid to spectrin ( +
) (data not shown).
To investigate whether the fatty acid bound to
spectrin palmitoylated in vitro using PAT is incorporated in
the same site(s) as in vivo, peptide maps of in
vivo and in vitro labeled spectrin (-subunit) were
compared using the Cleveland V8 protease digestion technique of gel
slices, followed by fluorography (as described under "Materials and
Methods"). The V8 protease digestion gave one major palmitoylated
peptide in the second dimension SDS-PAGE after fluorography. This was
present in the case of both the in vivo and in
vitro labeled spectrin (migrating with an apparent molecular mass
of 45 kDa) (Fig. 7), suggesting that the same site is
labeled on the protein in both cases.
Our understanding of dynamic protein palmitoylation as a cellular control mechanism has been limited by the lack of detailed knowledge about the enzymology of palmitoylation and the fact that no PAT has been isolated to date. Progress toward the purification of PAT has been hampered by the lack of a rapid and sensitive method of assay, by the absence of an apparent consensus sequence at sites of palmitoylation, and by the relative instability of the enzymes identified so far. Our search for the PAT from human erythrocytes was based on our own observations and that of other laboratories that protein-bound palmitate associated with erythrocyte membrane proteins turns over (8, 23). Moreover, palmitoylating activity has recently been demonstrated in human erythrocyte ghosts (20). Our choice of spectrin as substrate was based on the observations of Mariani et al. (4) that a tightly membrane-associated fraction of spectrin is palmitoylated when human erythrocytes are metabolically labeled with [3H]palmitic acid. Spectrin is an abundant cytoskeletal protein of the erythrocyte that can be easily purified in substantial quantities. In the absence of knowledge on the defined substrate requirement of erythrocyte PAT, spectrin appeared to be a feasible alternative to a peptide substrate that could be used for monitoring the purification of PAT. PAT activity could be extracted from erythrocyte ghosts in buffer containing 1 M KCl. Erythrocyte PAT therefore appeared to behave like a peripheral membrane protein, unlike the PAT identified in bovine brain (21). On the other hand, the rat liver palmitoyltransferase is solubilized from membranes using 150 mM KCl, suggesting that this enzyme, like erythrocyte PAT, may not be tightly bound to membranes. The increase in specific activity of purified PAT in comparison with the KCl extract was 86-fold. PAT was extremely labile and susceptible to proteolysis, as reported earlier by others (21). A 67-kDa band reactive with anti-PAT antibody appeared below the 70-kDa polypeptide in the KCl extract on storage (data not shown). To avoid proteolysis, the purification steps were performed as quickly as possible.
Purified PAT appeared as a 70-kDa polypeptide on SDS-PAGE. The identity of this polypeptide as PAT was confirmed by immunodepletion of PAT activity using antibody against the 70-kDa polypeptide. Nonradioactive palmitoyl-, myristoyl-, and stearoyl-CoAs were competitors of [14C]palmitoyl-CoA, a feature also observed in the case of bovine brain PAT (21).
Dynamic protein palmitoylation represents a recent addition to the repertoire of cellular control mechanisms. Understanding the control of palmitoylation-depalmitoylation cycles necessitates characterization of the enzymes involved in these processes. Purification of the PAT from human erythrocyte membranes has opened up exciting avenues. Whether the plasma membrane-associated PAT from erythrocytes is identical to or different from PAT activities associated with other membranes (21, 22) needs to be evaluated. Dissection of the palmitoylation site of spectrin will provide necessary knowledge of the likely substrate requirements of the PAT purified here. Partially purified bovine brain PAT recognizes the N-terminal "myristoyl-Gly-Cys" sequence of the Src family protein-tyrosine kinases like Fyn (21), while rat liver PAT recognizes a farnesylated and methylated Ha-Ras peptide. Since spectrin lacks these motifs, erythrocyte PAT is likely to represent one of a family of palmitoyltransferases of different substrate specificity and probably of different subcellular localization.