Modification of Catalytically Active Phospholipase D1 with Fatty Acid in Vivo*

Maria Manifava, Jane Sugars, and Nicholas T. KtistakisDagger

From the Department of Signaling, Babraham Institute, Cambridge CB2 4AT, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Phospholipase D1 (PLD1) was covalently labeled with 3H when expressed transiently in COS cells and immunoprecipitated following labeling of the cells with [3H]palmitate. Labeling of PLD1 was abolished by treatment with hydroxylamine at neutral pH, indicating that the fatty acid is linked via thioester to the enzyme. In pulse-chase studies the label persisted over a 3-h chase, indicating a slow rate of turnover. A catalytically inactive point mutant of PLD1 that changes serine at position 911 to alanine (S911A) was partially but not entirely redistributed to the cytosol, and it contained no detectable palmitate label. Similarly, N- and C-terminal domain fragments of the protein, encompassing in combination the entire coding region and all expressed to levels comparable with the wild type protein, showed no label with palmitate. Treatment of immunoprecipitated PLD1 with hydroxylamine diminished catalytic activity to background levels in a dose response manner that paralleled the removal of label from [3H]palmitate-labeled protein. We suggest that modification of PLD1 with palmitate is related to its catalytic activity and may be an important requirement for the function of this enzyme.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Phospholipase D (PLD)1 hydrolyzes phosphatidylcholine to generate phosphatidic acid and choline (1). In mammalian cells, two isoforms of PLD have been identified based on sequence homology to plant and yeast enzymes PLD1 and PLD2 (2, 3). In addition to differences in primary sequence, the two PLD isoforms differ also in intracellular localization and with respect to their ability to be regulated by a variety of activating proteins and a lipid. Whereas PLD1 is activated in vitro by two small GTP-binding proteins ADP-ribosylation factor (Arf) and Rho, as well as by protein kinase C and by the lipid phosphatidylinositol 4,5-bisphosphate, PLD2 in vitro is constitutively active and only requires phosphatidylinositol 4,5-bisphosphate. In terms of localization, PLD1 is found mainly in intracellular membranes, whereas PLD2 is on plasma membrane. Based on its localization and activation requirements, PLD1 is a good candidate to mediate those aspects of vesicular traffic in which a "PLD activity" has been implicated from earlier studies (4-6).

Although PLD activity responsive to the known activators is evident in Golgi-enriched membranes from a variety of cell lines, endogenous PLD1 protein is difficult to detect, and it has not been purified to homogeneity from any source. We and others have begun studying the localization, activity, and regulation of PLD1 following overexpression, either transiently or in inducible cell lines. In this work we identify fatty acylation as a post-translational modification of PLD1. This modification is undetectable in a catalytically inactive mutant and in fragments of the protein that together encompass the entire coding sequence. Removal of palmitate by hydroxylamine renders PLD1 catalytically inactive, suggesting an important role for this modification in PLD1 function.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

PLD1 Expression Plasmids, Mutagenesis, and Transfection-- The human PLD1a gene was generously supplied by Dr. Paul Sternweis in expression vector pCMV5. In this vector, PLD1a expression is very poor by several criteria. PLD1a was excised from pCMV5 with HpaI/XbaI and subcloned into pCMV3, which is an expression vector with a longer, more potent cytomegalovirus promoter (7). Point mutations in hPLD1 were constructed in pCMV5 using the Quick-change kit (Stratagene) and subcloned into pCMV3 using HpaI/XbaI. Mutants were constructed in duplicate, and both were analyzed. The N-terminal PLD fragment (SHS) was constructed by digesting PLD1a with HpaI/HindII and subcloning the 1400-base pair fragment into pCMV3. Similarly, the C-terminal 2100-base pair PLD1a fragment (LHS) was excised with HindII/XbaI and subcloned into pCMV3. Two other terminal fragments were produced using the EcoRI sites at positions 936 and 1069 of PLD1a giving rise to a N-terminal 936-base pair fragment (SES) and a C-terminal 2540-base pair fragment (LES). Transfection into COS cells was done using the DEAE-dextran/chloroquine protocol. Transfection efficiencies were in the 25-50% range.

Antibodies-- The peptides MSLKNEPRVNTSALQC (N-terminal) and CLPSVGTKLVPMEVWT (C-terminal) of PLD1 were synthesized on a Biosearch 9500 peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and coupled by the thiol group of cysteine to the amino groups of purified protein derivative of Tuberculin (PPD) using Sulfo-SMCC (Pierce). Conjugates were injected into Bacillus Calmette-Guerin vaccinated rabbits. Two rabbits were used for each peptide, and positive sera were obtained after the first bleed. The antibodies have been used both as crude sera and after affinity purification against the relevant peptides (for immunofluorescence).

Labeling of PLD1 in Vivo-- Labeling experiments were done with transfected COS cells in 6-well plates. For 35S TranSlabel labeling, the cells were washed three times with DMEM without methionine and cysteine and were incubated in this medium for 30 min at 37 °C to deplete the intracellular pool of methionine and cysteine. Medium containing 200 µCi/ml of Tran 35S-label (ICN, Irving, CA) was added to the cells for 1 or 2 h at 37 °C. At the end of labeling the cells were processed for immunoprecipitation as described above. Labeling with [3H]palmitate was done as follows: [3H]palmitate at 1 mCi/ml in ethanol was concentrated under a stream of nitrogen such that the final ethanol concentration in the labeling medium would be less than 0.5%. Cells to be labeled were washed with serum-free DMEM and incubated in DMEM containing 10% dialyzed serum and 200 µCi/ml palmitate for the indicated times. For chasing the incorporated palmitate, cells were washed once and incubated in DMEM with 10% serum and 150 µM nonradioactive palmitate.

Immunoprecipitation of Transiently Expressed PLD1-- Most immunoprecipitation experiments were carried out with transfected COS cells in 6-well plates. The volumes given are per well. Cells were washed twice with ice-cold PBS, and all remaining PBS was removed by aspiration. Lysis was with 1 ml of buffer A (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM EDTA, 1% Nonidet P-40, 0.6 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml trypsin inhibitor) for 15 min at 4 °C. At the end of lysis, cells were scraped with a rubber policeman and transferred to Eppendorf tubes for centrifugation at 17000 × g for 10 min at 4 °C. The supernatant was removed and incubated in a rotator for 30 min at room temperature with 2 µl of pre-immune antibody and 50 µl of 10% protein A-Sepharose that was prepared in buffer B (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Tween-20, 0.02% sodium azide). At the end of this preclearing step, the lysate was centrifuged at 1000 × g for 5 min at 4 °C. The supernatant was incubated with 3 µl of anti-PLD1 serum for 1 h in a rotator, plus an additional 30 min with 50 µl of 10% protein A-Sepharose. The immunoprecipitates were then washed four times with buffer B, and once with PBS, before the beads were collected for analysis. For immunoprecipitation from large scale transfections (for activity assays), cells were plated in 150-mm plates, the initial spin was in a glass Corex tube, and all volumes were adjusted proportionately.

Subcellular Fractionation-- 40 h post-transfection, COS cells were removed from the plates by trypsin and washed once in DMEM containing 10% serum, once in DMEM, and once in PBS containing protease inhibitors. The washed pellet was resuspended on ice in Hepes/KCl buffer (50 mM Hepes, pH 7.2, 90 mM KCl) and homogenized with 25 strokes in a steel homogenizer. The homogenate was spun at 2500 rpm, and the supernatant (post nuclear supernatant) was centrifuged in an SW60 rotor at 100,000 × g for 60 min at 4 °C. The supernatant from this spin is termed the soluble fraction. The pellet (termed membranes) was resuspended in Hepes/KCl. Both were flash frozen and stored at -70 °C.

Treatment of PLD1a with Hydroxylamine from Labeled Samples-- After labeling and immunoprecipitation of PLD1, the labeled protein was resolved by SDS-PAGE. The gels were fixed for 2 h and were then treated overnight with 1 M hydroxylamine, pH 7.0, or with 1 M Tris-HCl, pH 7.0, as a control. After these treatments the gels were washed briefly with water and were incubated with M salicylic acid for 30 min to enhance the radioactive signal before drying and autoradiography. For analysis of the radioactive lipid by TLC, the washed beads at the end of immunoprecipitation were incubated for 1 h with 1 M hydroxylamine or Tris to release the lipid from PLD1. The supernatant from this incubation was extracted and analyzed by TLC as described previously.

Treatment of PLD1a with Hydroxylamine for Activity Assay-- After immunoprecipitation from large scale transfections the beads were incubated for 30 min with the indicated amounts of hydroxylamine or Tris both at pH 7.1. The beads were washed twice with PBS and were then incubated with liposomes for measuring PLD1 activity.

Immunoblotting-- Samples for transfer onto nitrocellulose were resolved on 7.5% SDS-polyacrylamide gels on the day they were obtained. Following overnight transfer at 150 mA, the nitrocellulose was blocked with 5% defatted milk in PBS for 30 min at room temperature. PLD1 antibodies were used at 1:5000 dilution for 2 h at room temperature in milk/PBS. The blots were washed in PBS and incubated with secondary antibodies for 30 min at a dilution of 1:10000 in 1% BSA/PBS. The blots were again washed in PBS and developed using chemiluminescence. No detergents were used during immunoblotting because they interfere with PLD1 detection.

Immunofluorescence-- Cells were plated on glass coverslips 22 h post-transfection. At 40 h post-transfection the cells were washed twice in PBS and fixed for 5 min in methanol kept at -20 °C. At the end of fixing, the cells were incubated in 1% BSA/PBS for 30 min to overnight and were stained for indirect immunofluorescence using PLD antibodies at 1:200 dilution in BSA/PBS for 30 min at room temperature, followed by fluorescent second antibodies for 30 min at room temperature. Between antibody incubations the cells were washed with BSA/PBS. No detergents were used during immunofluorescence because they interfere with PLD1 detection.

Assay for PLD Activity-- We modified the protocol originally developed by Brown and Sternweis (8) as follows. Appropriate amounts of lipid solutions in chloroform were combined and dried under a nitrogen stream. The dried film was resuspended and sonicated in 0.1% defatted BSA in PBS to give liposomes at these final concentrations: dipalmitoyl phosphatidylcholine, 3 µM (Sigma); phosphatidyl-D-myo-inositol-4,5-bisphosphate from bovine brain, 5 µM (Calbiochem); phosphatidylethanolamine from bovine liver, 33 µM (Sigma); and [14C]dipalmitoyl phosphatidylcholine, 500 nCi/ml (Amersham Pharmacia Biotech). The beads from the final immunoprecipitation wash were collected by brief centrifugation and were incubated with the liposomes for 30 min at 37 °C. Where indicated, GTPgamma S at 20 µM and recombinant Arf6 (2 µg/assay sample, a kind gift of Dr. Alex Brown, Cornell University) were added to the reaction immediately before incubation at 37 °C. Throughout the reaction at 37 °C, the samples were vortexed gently every 5 min. At the end of incubation, lipids were extracted and analyzed by TLC as described before. The amount of phosphatidic acid formed was estimated by densitometric scanning of the films.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

To study the function of PLD1 in vivo, and especially those aspects of the enzyme implicated in intracellular transport, we have raised peptide-based antibodies against the two terminal regions of PLD1, and we have made recombinant plasmids encoding full-length PLD1, parts of PLD1, or point mutants. Reagents that will be discussed in this work are shown in Fig. 1.


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Fig. 1.   PLD-related reagents used in this work. Polyclonal antibodies against peptides corresponding to the N- and C-terminal sequence of PLD1 were raised in rabbits. In addition, by taking advantage of EcoRI and HindII sites in the sequence of the PLD1 gene, we have generated N (SES and SHS) and C (LES and LHS) domain fragments. Finally, a point mutant changes serine at position 911 to alanine (S911).

When expressed transiently in COS cells, PLD1 is largely membrane-bound, despite the lack of a transmembrane domain or of recognizable consensus sequences specifying modification by lipid moieties (3). We examined the possibility that PLD1 is nevertheless modified covalently with lipid by labeling COS cells expressing the protein with [3H]palmitate, immunoprecipitating, and analyzing the immunoprecipitates by autoradiography. Following such an experiment, PLD1 is labeled specifically with tritium (Fig. 2A). Labeling for 2 h results in stronger signal than labeling overnight (Fig. 2A, compare lanes 2 and 5), reflecting the fact that the half-life of PLD1 in COS cells is short and incorporation of palmitate into cells is linear for only the first 5-6 h of labeling (results not shown). The nature of the tritium linkage on PLD1 was explored by treating resolved PLD1 with neutral hydroxylamine, a condition known to cleave thioester bonds (9). Labeling of resolved PLD1 was completely sensitive to neutral hydroxylamine (Fig. 2A) and resistant to Tris, suggesting a linkage to a cysteine residue. A portion of the lysates used for immunoprecipitation in Fig. 2A were resolved and immunoblotted (Fig. 2B). It can be seen that both PLD1 and SHS accumulate to the same level in COS cells.


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Fig. 2.   Palmitoylation of PLD1. A, COS cells transfected with a control plasmid (-) or with plasmids encoding full length PLD1 (PLD) or an N-terminal fragment (SHS) were labeled overnight or for 2 h with [3H]palmitate. Following lysis and immunoprecipitation with N-terminal antibodies, the samples were resolved on identical SDS-polyacrylamide gels. After electrophoresis and fixing, one gel was treated with 1 M Tris-HCl, pH 7.0 (- hydroxylamine), and the other with 1 M hydroxylamine, pH 7.0 (+ hydroxylamine), before autoradiography. Shown is a 30-day exposure. B, lysates from the samples shown in A were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with N-terminal antibodies.

We expanded the analysis of labeling with [3H]palmitate to various fragments of PLD1 expressed separately in COS cells and to a catalytically inactive point mutant that was originally constructed by Sung et al. (10) and changes serine at position 911 to alanine. After transfection into COS cells, labeling was done either with [35S]methionine or with [3H]palmitate and immunoprecipitation with both N- and C-terminal-specific antibodies. With methionine labeling, it can be seen that all proteins are synthesized to comparable levels (Fig. 3, top panels), and the ability to be immunoprecipitated is strictly dependent on the type of antibody used. In addition, hydroxylamine treatment does not change the amount of label in any of the relevant bands (Fig. 3, top panels, compare - hydroxylamine with + hydroxylamine). Equally clear is the result with palmitate labeling: the only protein that incorporates label is the wild type PLD1, and again this label is sensitive to hydroxylamine treatment. We point out that, although in general the N-terminal antibodies immunoprecipitate better than the C-terminal antibodies, this alone does not explain why the amount of tritium-labeled PLD1 immunoprecipitated with the C-terminal antibodies is so much less than that immunoprecipitated with the N-terminal antibodies (Fig. 3, bottom right panel). We have not explored this difference further.


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Fig. 3.   Relative palmitoylation of wild type PLD1, fragments, and S911A mutant. COS cells transfected with the indicated PLD-related constructs were labeled either with 35S TranSlabel or with [3H]palmitate as indicated for 2 h, and lysates were prepared. Lysates from each sample were divided in half, and each was immunoprecipitated with N- or C-terminal antibodies as indicated. After immunoprecipitation the samples were resolved on duplicate SDS-polyacrylamide gels, one of which was treated with Tris-HCl, pH 7 (- hydroxylamine), and the other with hydroxylamine, pH 7 (+ hydroxylamine), before autoradiography. Shown is a 60-day exposure. S/A stands for S911A; the rest of the abbreviations are as described in the legend to Fig. 1.

The absence of detectable label in the fragments suggests that palmitoylation of PLD1 may not be dependent simply on the availability of a relevant amino acid sequence but in addition may relate to the catalytic ability of this protein. This is strengthened by our observation that the S911A mutant (which contains all potential relevant cysteine residues) incorporates no palmitate. We examined the accumulation of this mutant after transfection into COS cells and its activity in our hands (Fig. 4). S911A is synthesized and accumulates to very comparable levels with the wild type protein in COS cells (Fig. 4A and results not shown), and it can be immunoprecipitated as efficiently. It is different from the wild type protein in electrophoretic mobility in that it does not reveal a faint higher molecular mass form of the protein as does the wild type protein, an observation originally made by Sung et al. (10) (Fig. 4A, compare lanes 1 and 4 with lanes 2, 3, 5, and 6). This upper band has been proposed to represent a covalent intermediate with phosphatidic acid in the hydrolytic cycle of PLD1 (10), but we have not seen formation of such an intermediate during catalysis in vitro.2 In agreement with Sung et al. (10), the S911A mutant has no hydrolytic activity after immunoprecipitation from COS cells (Fig. 4B) or in vivo (data not shown). We have also examined the subcellular distribution of S911A. By immunofluorescence, the wild type PLD1 when overexpressed to reasonable levels shows a distribution that is perinuclear and is characterized by numerous punctate structures (Fig. 5A). In contrast, the S911A mutant shows no punctate structures, and it has a more diffuse distribution suggestive of relocation to the cytosol (Fig. 5A). In parallel experiments, distribution was assessed biochemically, and it largely confirmed the microscopic data. Whereas PLD1 was primarily found with the membrane fraction (Fig. 5B, lane 3), about half of the S911A mutant was in the cytosolic pool (Fig. 5B, lane 5).


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Fig. 4.   Expression and catalytic activity of S911A. A, COS cells were transfected with wild type PLD1 or with two independent isolates of the S911A point mutant. Following lysis and immunoprecipitation, both lysates and immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with N-terminal antibodies. Note the absence of higher molecular mass eight band in S911A. B, COS cells were transfected with control plasmid (-) or with plasmids encoding wild type PLD1 or S911A. After immunoprecipitation and extensive washing, the beads were incubated with GTPgamma S and 14C-labeled phosphatidylcholine in the context of phosphatidylethanolamine and phosphatidylinositol 4,5-bisphosphate lipids with or without recombinant Arf6 for 30 min at 37 °C. At the end of incubation lipids were extracted and analyzed by TLC. PC, phosphatidylcholine; PA, phosphatidic acid; O, origin.


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Fig. 5.   Immunofluorescence and fractionation of PLD1 and S911A. A, COS cells were transfected with wild type PLD1 (PLDwt) or with S911A (PLDS911A) and plated on coverslips for immunofluorescence. Shown are representative examples of cells expressing the two proteins and stained with N-terminal antibodies. B, COS cells were transfected with wild type PLD1 or with S911A. Post nuclear supernatants (PNS) were produced and further fractionated by sedimentation into membrane (mem) and soluble (cyt) fractions. Membranes were resuspended to the same volume as the soluble fraction, and equal aliquots from each fraction were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with N-terminal antibodies.

The data on the S911A mutant can be interpreted in two ways that are not mutually exclusive: either modification of PLD1 with palmitate is part of the requirement for membrane attachment as is the case for many other proteins (11) or membrane attachment is partially a consequence of catalytic ability (perhaps as simple as binding the substrate lipid), which in turn depends to some extent on palmitoylation. Because membrane attachment seems to depend on additional mechanisms unrelated to palmitoylation and restricted to specific regions of PLD1,3 it was easier to address the second possibility alone. In the cases where palmitoylation can be shown to have a functional role, depalmitoylation with hydroxylamine has been used to probe the function of several proteins such as rhodopsin, the asialoglycoprotein receptor and phospholipid scramblase (12-14). In the same vein, we treated immunoprecipitated PLD1 with hydroxylamine and assayed its catalytic activity (Fig. 6). Hydroxylamine treatment abolished PLD1 activity (over 85% reduction by densitometry), both unstimulated and stimulated with Arf6 (Fig. 6A, compare lanes 5 and 6 with the other lanes). When the concentration of hydroxylamine was varied, we found a linear reduction of PLD1 activity as a function of hydroxylamine concentration (Fig. 6B, bottom panel). (The overall reduction in activity observed with both Tris and hydroxylamine treatments (Fig. 6B, bottom panel, compare lane 1 with the other lanes) is explained by the fact that some PLD is lost from the beads during the additional washes (Fig. 6B, top panel, compare lane 1 with the other lanes).) We chose the highest concentration of Tris or hydroxylamine (1 M) to follow the activity of the treated enzyme as a function of incubation at 37 °C (Fig. 6C). It can be seen that although the Tris-treated enzyme displays linear activity as a function of incubation at 37 °C, the enzyme that was treated with hydroxylamine was essentially inactive, and even at the longer incubation times its activity was reduced compared with the Tris-treated sample by 85% (Fig. 6C, compare lanes 1-4 with lanes 5-8). It is of course possible that in addition to fatty acid removal, hydroxylamine treatment changes the structure of PLD1 sufficiently to account for reduction in catalytic activity. We cannot rule this possibility out based on our data. However, when the ability of hydroxylamine to remove the palmitate label was assayed as a function of concentration (Fig. 6D), we found a very good correspondence between reduction in catalytic activity and palmitate removal (compare lanes 7-10 in Fig. 6B, bottom panel, with lanes 5-8 in Fig. 6D, top panel). We also point out that treatment with hydroxylamine had no effect on electrophoretic mobility of PLD1 or on its immunoreactivity (Fig. 6B, top panel, and Fig. 6D, bottom panel).


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Fig. 6.   Effect of depalmitoylation on PLD activity. COS cells were transfected with PLD1. A, after immunoprecipitation and extensive washing, the beads were divided into four equal aliquots. One aliquot (PBS) was kept on ice. The other three were incubated for 30 min at room temperature with PBS (PBS*), 1 M hydroxylamine, pH 7 (hydroxyl.), or 1 M Tris-HCl, pH 7 (Tris). The beads were then washed twice with PBS, again divided in two, and assayed for PLD activity as described above with or without Arf6. The production of phosphatidic acid was measured by TLC after extraction of the lipids. B, after immunoprecipitation and extensive washing, the beads were divided into 11 equal aliquots and treated as indicated for 30 min at room temperature. The beads were then washed twice with PBS and assayed for PLD activity by TLC shown in the bottom panel. One-tenth of the actual activity reaction was removed and mixed with sample buffer, and its PLD1 content was determined after electrophoresis and immunoblotting (shown in the top panel labeled blot). C, after immunoprecipitation and washing, the beads were divided into two equal aliquots. One aliquot was treated with 1 M Tris-HCl, pH 7.0, and the other was treated with 1 M hydroxylamine, pH 7.0, for 40 min at room temperature. The beads were then washed twice with PBS and divided into four aliquots. PLD substrate with Arf6 and GTPgamma S was added to each aliquot, and the assay for PLD activity was carried out at 37 °C for the indicated times. At the end of incubation, lipids were extracted, and phosphatidic acid formation was measured by TLC. Note that the total amount of PLD1 protein per sample for this experiment was three times that of PLD1 used in other experiments to account for the loss of protein from the beads during the washing steps. D, cells were labeled for 2 h with [3H]palmitate and lysed, and PLD1 was immunoprecipitated. After washing, the beads were divided into eight equal aliquots and treated for 30 min at room temperature as indicated (sample labeled PBS was treated on ice, the one labeled PBS* at room temperature). The beads were then washed twice with PBS and resolved by SDS-PAGE on duplicate gels. One gel, containing one-tenth of the sample, was transferred to nitrocellulose and probed with PLD antibodies (blot). The other gel, containing the rest of the sample, was fixed, enhanced, dried, and exposed for autoradiography (3-H palm). Shown is a 20-day exposure. PC, phosphatidylcholine; PA, phosphatidic acid.

How transient is the modification with palmitate on PLD1? If it represented a covalent lipid intermediate in catalysis, it would be expected to be short-lived because unstimulated PLD1 is catalytically active in COS cells.2 If, on the other hand, it were a permanent modification that facilitates catalytic activity, it would be expected to last for as long as the newly synthesized enzyme. Labeling of PLD1 in parallel with [35S]methionine or with [3H]palmitate and chasing for various times revealed that the half-life of tritium label is comparable with the label with 35S (Fig. 7). This suggests that under our experimental constraints, modification with palmitate is long-lived. It is possible that under conditions of stimulation or inhibition of the enzyme, modification may vary.


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Fig. 7.   Rate of turnover of PLD1 labeled with [35S]methionine or with [3H]palmitate. COS cells transfected with PLD1 were plated on 6-well plates in duplicate. 40 h after transfection half of the samples were labeled for 2 h with 35S TranSlabel and the other half were labeled for 2 h with [3H]palmitate. At the end of 2 h one set of samples was placed on ice (0 min chase). For the rest, the labeling medium was replaced with medium containing methionine, cysteine, and 150 µM palmitate, and the samples were returned to 37 °C for the indicated times. At the end of incubation the cells were lysed, and PLD1 was immunoprecipitated and resolved on duplicate gels and fixed overnight. One gel was treated with 1 M Tris-HCl, pH 7.0 (- hydroxylamine), and the other was treated with 1 M hydroxylamine, pH 7.0 (+ hydroxylamine). Shown are the 18-h exposure for [35S]methionine (35-S met) and the 30-day exposure for [3H]palmitate (3-H palm).

In conclusion, we have identified a modification on PLD1 that is consistent with palmitoylation: it is evident after palmitate labeling, and it is sensitive to neutral hydroxylamine. The low levels of PLD1 expression (even using the strong promoter described here) combined with the weakness of the tritium label have precluded us so far from establishing the identity of the fatty acid unambiguously, and we are currently in the process of exploring other detection methods. Interestingly, this modification is present only on catalytically active PLD1. It is absent from the catalytically inactive mutant S911A that replaces a serine thought to be involved in initiating catalysis with an alanine. Complementary to this, wild type PLD1 is rendered catalytically inactive by pretreatment with neutral hydroxylamine. The simplest interpretation of our data is that modification of PLD1 with fatty acid facilitates catalysis, perhaps by enabling the enzyme to interact better with its phospholipid substrate in situ, and that the region of PLD1 around serine 911 is probably involved in the binding of the fatty acid.

    ACKNOWLEDGEMENTS

We thank Alex Brown for the gift of Arf6 and for advice on the exogenous substrate assay and Maurine Linder for bringing to our attention many apocrypha of the fatty acylation literature. In addition, we thank our colleagues Phil Hawkins and Len Stephens of the Signaling Program for making available their expression vectors.

    FOOTNOTES

* This work was supported by the Biotechnology and Biological Sciences Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-1223-496000, Ext. 323 or 459; Fax: 44-1223-496030; E-mail: nicholas.ktistakis{at}bbsrc.ac.uk.

The abbreviations used are: PLD, phospholipase D; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GTPgamma S, guanosine 5'-O-(thiotriphosphate); SHS, short HindIII sense; LHS, long HindIII sense; SES, short EcoRI sense; LES, long EcoRI sense.

2 M. Manifava, unpublished observations.

3 J. Sugars, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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