The First 5 Amino Acids of the Carboxyl Terminus of Phosphatidylinositol Transfer Protein (PITP) alpha  Play a Critical Role in Inositol Lipid Signaling
TRANSFER ACTIVITY OF PITP IS ESSENTIAL BUT NOT SUFFICIENT FOR RESTORATION OF PHOSPHOLIPASE C SIGNALING*

(Received for publication, November 1, 1996, and in revised form, February 26, 1997)

Shuntaro Hara Dagger , Phil Swigart , David Jones and Shamshad Cockcroft §

From the Department of Physiology, University College London, London WC1E 6JJ, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Phosphatidylinositol transfer protein (PITP) is essential for phospholipase C signaling and for constitutive and regulated vesicular traffic. PITP has a single lipid-binding site that can reversibly bind phosphatidylinositol (PI) and phosphatidylcholine (PC) and transfer these lipids between membrane compartments in vitro. The role of the carboxyl terminus was examined by comparing wild-type PITPalpha with PITPalpha in which 5, 10, and 20 amino acids were deleted from the C terminus. Delta 5- and Delta 10-PITP had reduced PI and PC transfer activities compared with wild-type PITP, with the effect on PI transfer being more marked than that on PC transfer. Delta 20-PITP was inactive at all concentrations tested. All three truncated mutants were unable to restore G-protein-mediated phospholipase Cbeta stimulation in HL-60 cells. Delta 5- and Delta 10-PITP, but not Delta 20-PITP, inhibited the signaling function of wild-type protein without any effect on lipid transfer in vitro. We conclude that (a) the carboxyl terminus of PITP plays a critical role in phospholipase C signaling; (b) the transfer activity is not the only determining factor that dictates the restorative function of PITP in inositol lipid signaling; and (c) the dominant inhibitory effects of Delta 5- and Delta 10-PITP on wild-type PITP in phospholipase C signaling suggest the existence of a receptor for PITP.


INTRODUCTION

Phosphatidylinositol transfer protein (PITP)1 is a ubiquitous and abundant cytosolic protein that was originally identified because of its ability to transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membrane bilayers in vitro (1, 2). Two isoforms of PITP (alpha  and beta ) that show different lipid binding properties have been identified in mammalian cells. PITPalpha has a single lipid-binding site that can reversibly bind PI and PC, with a 16-fold higher affinity for PI than for PC (3, 4). In vitro, PITPalpha can transfer PI, PC, and PG to a lesser extent (5), whereas PITPbeta can transfer sphingomyelin in addition (6). In cells, both PITPalpha and PITPbeta participate in phospholipase C (PLC)-mediated signaling (7-9) and in vesicular traffic (10-12).

PITPalpha was identified as the major reconstituting factor that allowed restoration of PLCbeta signaling in HL-60 cells (7). A requirement for PITP has also been identified for inositol 1,4,5-trisphosphate production by receptors that activate PLCgamma 1. When activated by the appropriate agonist, both the epidermal growth factor and IgE receptors are dependent on PITP for PLCgamma 1 signaling in A431 and RBL-2H3 cells, respectively (8, 9). The mechanism of PITP function in lipid signaling has been attributed to the lipid binding/transfer properties of PITP, and the delivery of PI to a signaling complex composed of PI 4-kinase, phosphatidylinositol-phosphate 5-kinase, and the receptor has been proposed as its physiological function (7-9, 13).

A separate role for PITP in exocytosis has also been identified (10, 12, 14). PITP was purified as a reconstituting factor together with phosphatidylinositol-phosphate 5-kinase for restoration of ATP-dependent priming of secretory vesicles for fusion with the plasma membrane in PC12 cells (10, 14). Cells of the myeloid lineage including neutrophils and HL-60 cells secrete lysosomal enzymes when activated with Ca2+ and guanine nucleotides. HL-60 cells depleted of cytosolic proteins become refractory to stimulation, and PITP was found to be required to restore secretory competence for Ca2+- and guanine nucleotide-mediated exocytosis (12). The addition of PITP led to increased synthesis of PI 4,5-bisphosphate, and this function of PITP provides the most likely explanation of how PITP participates both in exocytosis and in PLC-mediating signaling. In PLC signaling, PI 4,5-bisphosphate functions as a substrate, whereas its function in exocytosis is probably due to the recruitment of specific protein(s) required for the exocytotic machinery.

Secretory vesicle formation is also dependent on cytosolic proteins, and PITPalpha and PITPbeta were identified as the active components (11). This function of PITP in mammalian cells is analogous to the requirement of yeast PITP (SEC14p) for export of secretory proteins from the Golgi complex (15). Evidence has been presented that suggests that it functions as a lipid sensor that controls the PC content of yeast Golgi membranes (16). Although yeast PITP (SEC14p) shares no primary sequence homology with mammalian PITP, SEC14p can restore the function of mammalian PITPs in PLC signaling, exocytosis, and vesicle formation (9-12). The function of yeast PITP is not conserved as mutations in SEC14p in the dimorphic yeast Yarrowia lipolytica do not lead to impaired secretion. Instead, it is required for differentiation from the yeast to the mycelial form (17).

In this report, we show that the carboxyl terminus of the PITP molecule plays a critical role in the restoration of PLC signaling. Deletion of 5 amino acids is sufficient to inactivate the protein with regard to PLC-mediated signaling, although lipid transfer is only reduced. In addition, the Delta 5- and Delta 10-PITP deletion mutants were found to inhibit restoration of PLC signaling by wild-type PITP, although they did not interfere with the lipid transfer function of PITP in vitro. We conclude that (a) the carboxyl terminus of PITP plays a critical role in PLC signaling; (b) the transfer activity is not the only determining factor that dictates the restorative function of PITP in inositol lipid signaling; and (c) the dominant inhibitory effects of Delta 5- and Delta 10-PITP on wild-type PITP in PLC signaling suggest the existence of a receptor for PITP.


MATERIALS AND METHODS

Preparation of Recombinant PITPalpha Proteins

Recombinant wild-type and mutant PITPalpha proteins were cloned by the polymerase chain reaction using human PITPalpha cDNA as template and the following primers: 5'-GAACTTCTAGATCCCATATGGTGCTGCTCA-3' and 5'-CTCGGGATCCTAGTCATCTGCTGTCATTCC-3' for wild-type PITP, 5'-CATCTGGATCCTATCCTTTCACTGGGTC-3' for Delta 5-PITP, 5'-CCTTTGGATCCCTACTTTTGTCTCATTTCA-3' for Delta 10-PITP, 5'-CCAGGGATCCCTACGTCTCTTCTTCCATCC-3' for Delta 20-PITP, 5'-GCCGGATCCTAGTCATCTGCTGTCATTCCTATCACTGG-3' for K264I, 5'-GCCGGATCCTAGTCATCTGCTGCCATTCCT-3' for T267A, and 5'-GCCGGATCCTAGTCATCTGCTACCATTCCT-3' for T267V. The polymerase chain reaction products were cloned into the NdeI-BamHI restriction site of the pET14b expression vector (QIAGEN Inc.) and transformed into XL1-Blue cells (Stratagene). Positive clones were sequenced, compared with published human PITPalpha cDNA sequence, and transformed into DE3 (pLysS) cells (Promega). Expression of recombinant PITPalpha was induced with isopropyl-beta -D-thiogalactopyranoside (0.1 mM) for 4 h at room temperature, and bacterial cells were collected by centrifugation. The pellet was resuspended in buffer containing 50 mM sodium phosphate and 300 mM NaCl (pH 8.0). After freeze-thaw, the samples were centrifuged at 100,000 × g for 30 min at 4 °C. Recombinant proteins were purified from the supernatants using Ni2+/nitrilotriacetic acid-agarose resin (QIAGEN Inc.) as described previously (9).

Assay for PI and PC Transfer

PI transfer activity was assayed as described previously (7). This assay measures the transfer of [3H]PI from rat liver microsomes to unlabeled liposomes in the presence of transfer protein. Briefly, protein samples were added to tubes containing [3H]PI-labeled microsomes (62.5 µg of microsome protein), liposomes (50 nmol of phospholipid; 98 mol % PC:2 mol % PI), and SET buffer (0.25 M sucrose, 1 mM EDTA, and 5 mM Tris-HCl (pH 7.4)) in a final volume of 125 µl. After incubation at 27 °C for 30 min, microsomes were precipitated by the addition of 25 µl of ice-cold 0.2 M sodium acetate (pH 5.0) and removed by centrifugation (12,000 × g for 15 min). A 100-µl aliquot of the supernatant was measured for radioactivity.

Assay for PC transfer activity measures the transfer of radioactivity from [3H]PC-labeled liposomes to rat liver mitochondria. The liposomes consisted of 2 mmol of egg yolk PC/ml containing 1 µCi of [3H]PC in SET buffer and were sonicated on ice prior to use. [3H]PC-labeled liposomes (40 nmol) were incubated with transfer protein and rat liver mitochondria (2 mg of protein) in a final volume of 0.2 ml of SET buffer for 30 min at 37 °C. The reaction was halted by placing samples on ice, and mitochondria were sedimented by centrifugation at 12,000 × g for 10 min. The sedimented mitochondria were resuspended in 0.5 ml of SET buffer and sedimented by centrifugation at 12,000 × g for 10 min through 0.5 ml of 14.3% sucrose. The pellet was resuspended in 50 µl of 10% SDS and boiled for 5 min, and this solution was counted for radioactivity.

Determination of the Lipid Bound to Recombinant PITPalpha Proteins Purified from Escherichia coli

After overnight culture of E. coli cells expressing PITPalpha proteins in the presence of [3H]acetate (20 ml, 15 MBq), expression of PITPalpha proteins was induced with isopropyl-beta -D-thiogalactopyranoside for 4 h at room temperature, and recombinant proteins were prepared as described above. Lipids were extracted from purified proteins and separated by TLC (solvent: chloroform/methanol/acetic acid/water (75:45:3:0.2, v/v)). Unlabeled standards were added to the samples to aid recovery and for identification on the TLC plate. The TLC plate was stained with iodine to locate the lipids, and the radioactivity was measured after excision of the spots for cardiolipin, PG, and PE. The TLC plate was also analyzed by imaging the radioactivity on a PhosphorImager.

Exchange of the Endogenous Lipids with PI and PC

50 µg of purified PITPalpha proteins (45 µl) were incubated with 5 µl of [3H]PC/phosphatidic acid (70:30 mol %; 120 µM and 0.1 µCi/µl final concentrations) or [3H]PI (120 µM and 0.0135 µCi/µl final concentrations) in 20 mM Tris buffer containing 5 mM MgCl2 and 60 mM NaCl (pH 7.4). After the solution was incubated at room temperature for 1 h, PITPalpha proteins were repurified with nitrilotriacetic acid spin (QIAGEN Inc.), and the radioactivity was counted.

For exchange of endogenous bacterial lipids for PI or PC prior to use in PLC assays, purified wild-type PITP and Delta 5-PITP were incubated with the appropriate lipid vesicles at a ratio of 1 mg of protein to 2.4 mg of lipid (1:100 molar ratio) and incubated at 4 °C overnight. The lipid vesicles were removed by the addition of DE52 in 20 mM Tris buffer containing 5 mM MgCl2 and 60 mM NaCl (pH 7.4) as described previously (7).

Reconstitution of G-protein-regulated Phospholipase C Activity in Cytosol-depleted HL-60 Cells with PITPalpha Proteins

HL-60 cells were grown and labeled with [3H]inositol as described previously (13). Labeled HL-60 cells (5 × 107) were permeabilized with 0.6 IU/ml streptolysin O in PIPES (supplemented with 1 mM Mg2+·ATP at pCa 7) for 10 min to deplete the cells of cytosolic proteins including PITP. The permeabilized cells were washed and finally resuspended in PIPES supplemented with 4 mM Mg2+·ATP, 20 mM LiCl, and 4 mM MgCl2 at pCa 6. Permeabilized cells (20 µl) were incubated with recombinant proteins in the presence of 10 µM GTPgamma S in a final volume of 45 µl at 37 °C for 20 min. The reaction was quenched by the addition of chloroform/methanol, and inositol phosphates were extracted, separated by passage through Dowex 1-X8 anion-exchange resin, and counted for radioactivity.


RESULTS

C-terminal Truncation of PITP Reduces Its Ability to Transfer Phospholipids

We have constructed three C-terminally truncated forms of PITPalpha (Delta 5, Delta 10, and Delta 20) in which 5, 10, and 20 amino acids residues are deleted, respectively (Fig. 1A). The proteins were expressed in E. coli and purified, and the in vitro transfer activities of these recombinant proteins were compared with those of wild-type PITP (Figs. 1B and 2 (A and B)). In addition to these three mutants, we constructed Delta 80-PITP, but this mutant could not be recovered from the soluble fraction of E. coli.


Fig. 1. A, linear display of the mutants used in this study; B, SDS gel electrophoresis illustrating recombinant PITPalpha proteins (10 µg of protein/lane) and staining with Coomassie Blue. Lane 1, molecular mass markers (205, 116, 97, 66, 45, 29, 20.1, and 18.4 kDa); lane 2, wild-type PITP; lane 3, Delta 5-PITP; lane 4, Delta 10-PITP; lane 5, Delta 20-PITP. a.a, amino acids.
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Fig. 2. Comparison of in vitro transfer of PI and PC of wild-type PITP with C-terminally truncated mutants and with site-directed mutants. Recombinant PITP (rPITP) proteins were examined in a PI (A) or PC (B) in vitro transfer assay. The results are expressed as % of maximal transfer observed with wild-type PITP. bullet , wild-type PITP; black-square, Delta 5-PITP; black-triangle, Delta 10-PITP; black-down-triangle , Delta 20-PITP. Also shown is the PI transfer of two site-directed mutant PITP with wild-type PITP (C). bullet , wild-type PITP; black-square, T267V; black-triangle, K264I. Data are means of duplicate measurements that did not vary by >5% and are representative of three independent experiments.
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Transfer activity was monitored using a donor membrane that contained radiolabeled PI or PC and an acceptor membrane compartment. The acceptor compartment was reisolated and monitored for the transferred radiolabeled lipid. Deletion of 5 amino acids was sufficient to reduce the transfer activity for both PI and PC (Fig. 2, A and B). 1 µg/ml wild-type PITP was maximal in the PI transfer assay, and corresponding concentrations of the mutant proteins were inactive. The Delta 5- and Delta 10-PITP mutants displayed substantial PI transfer activity when concentrations as high as 200 µg/ml were tested (Fig. 2A, panel b). In comparison, near normal transfer of PC could be observed at high concentrations of the Delta 5- and Delta 10-PITP mutant proteins. Although Delta 5- and Delta 10-PITP retained some ability to transfer PI and PC, Delta 20-PITP could not transfer PI or PC (Fig. 2, A and B).

To carry out phospholipid transport, PITP has to interact with a phospholipid membrane surface and exchange the endogenous lipid for another. When PITP is purified from mammalian tissues, the two major lipids that are bound to the protein are PI and PC at a ratio of 65:35 (3, 4). The recombinant proteins were expressed in E. coli, an organism devoid of both PI and PC. When expressed in E. coli cells, PITP is exposed to PE (84.4%), PG (12.5%), and cardiolipin (2.6%), the main phospholipids present in these cells. We first established which of the endogenous E. coli lipids were associated with purified PITPs.

E. coli cells were grown in the presence of [3H]acetate overnight prior to induction to label the endogenous E. coli lipids. PITP was purified, and the lipids associated with the protein were extracted and analyzed by TLC (Fig. 3A). The lipid incorporated into wild-type PITP was predominantly PG, with some PE (8:2 PG/PE) (Fig. 3B). In comparison to wild-type PITP, the Delta 5- and Delta 10-PITP mutant proteins had a higher proportion of PE, shifting the ratio to 6:4 PG/PE. This change in binding properties was more marked in Delta 20-PITP, where the ratio was nearly 5:5. In addition, the amount of lipid bound to Delta 20-PITP was decreased by >60%. The results in Fig. 3B indicate that Delta 5- and Delta 10-PITP have a similar occupancy compared with wild-type PITP, but only 35% of Delta 20-PITP has a lipid bound to the protein.


Fig. 3. A, PG and PE are associated with wild-type PITP purified from E. coli. Wild-type PITP was purified from E. coli cells grown in the presence of [3H]acetate overnight prior to induction, and the lipids associated with the purified protein were extracted and separated by TLC. The radiolabel associated with the lipids was analyzed using a PhosphorImager. The positions of the lipid standards (PE, PG, and cardiolipin (CL)) are indicated. B, shown is the quantitation of the lipids associated with wild-type PITP and with Delta 5-, Delta 10-, and Delta 20-PITP. Wild-type and truncated forms of PITP were purified from E. coli cells grown in the presence of [3H]acetate overnight prior to induction, and the lipids associated with the purified protein were extracted and separated by TLC. The radioactivity associated with the lipids was determined and is expressed as dpm/mg of protein. For each lipid, the dpm is also expressed as a percentage of the total radioactivity found in PE + PG + cardiolipin. The data from three independent experiments were pooled to calculate the percentage of the total radioactivity (±S.E., n = 3). C, shown is the binding of radiolabeled PI or PC to the recombinant proteins. Purified PITPalpha proteins were incubated with [3H]PC/phosphatidic acid or [3H]PI. After the solution was incubated at room temperature for 1 h, PITPalpha proteins were repurified, and the radioactivity was counted. The results are expressed as % of binding observed with wild-type PITP.
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The exchange of the endogenous lipids with radiolabeled PI and PC was also examined. The proteins were incubated with PC and PI lipid vesicles and reisolated using the His tag, and the radioactivity associated with the proteins was compared with that of wild-type PITP. The data in Fig. 3C illustrate that although Delta 5-PITP can exchange the endogenous lipids with PI and PC as well as wild-type PITP, the abilities of the Delta 10- and Delta 20-PITP mutants to exchange the lipids are significantly decreased.

Deletion Mutants Have No Ability to Restore Inositol Signaling in Cytosol-depleted HL-60 Cells

Delta 5- and Delta 10-PITP had reduced PI transfer activity, but near normal PC transfer activity at high concentrations of protein. If lipid transfer was the sole determining factor that was responsible for PITP function in PLC signaling, it would be expected that these proteins may retain some residual capacity to restore signaling in cells when used at high concentrations. We utilized the G-protein-driven PLCbeta signaling in HL-60 cells for this purpose. HL-60 cells were depleted of endogenous PITP, and the washed cells were incubated with wild-type PITP and Delta 5-, Delta 10-, and Delta 20-PITP in the presence of GTPgamma S (Fig. 4). Near maximal restoration of PLC signaling occurred around 100 µg of PITP/ml. None of the truncated proteins were able to restore PLC-mediated signaling. The maximal concentration that was practical to test in this assay was 1.5 mg/ml, and despite the addition of such a huge concentration, no reconstitution was observed. Instead, at these high concentrations, a slight inhibition of the GTPgamma S response was evident.


Fig. 4. A, restoration of PLC signaling with PITP and Delta 5-, Delta 10-, and Delta 20-PITP. HL-60 cells were permeabilized for 10 min and washed. The cytosol-depleted cells were stimulated with GTPgamma S in the presence of recombinant PITPalpha proteins for 20 min. bullet , wild-type PITP; black-square, Delta 5-PITP; black-triangle, Delta 10-PITP; black-down-triangle , Delta 20-PITP. B, restoration of PLC signaling after exchange of bacterial lipids with PI or PC. Wild-type PITP and Delta 5-PITP were incubated with PI or PC vesicles prior to use in the permeabilized HL-60 cells. The cytosol-depleted cells were stimulated with GTPgamma S in the presence of the recombinant PITPs loaded with PI or PC (200 µg/ml) for 20 min. Data are averaged from four independent experiments.
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To exclude the possibility that the bacterial lipids could in any way interfere with the restoration of PLC signaling when added to permeabilized cells, the bacterial lipids were exchanged for PI or PC. Wild-type PITP and Delta 5-PITP were converted into the PC and PI forms and compared with the protein loaded with the bacterial lipid PG. Fig. 4B illustrates that the ability of PITP to function in permeabilized cells in PLC signaling is independent of the nature of the loaded lipid.

Site-directed Mutational Analysis

To identify which specific residues may be important for the lipid binding/transfer properties of PITP, we mutated 2 candidate residues. There are clusters of basic amino acids in the C-terminal region in both PITPalpha and PITPbeta , and it was reported that basic amino acid residues are very important for PI 4,5-bisphosphate binding (18). PITPalpha contains a lysine residue at position 264, whereas PITPbeta has a similar basic amino acid, arginine, in this position. In addition to basic amino acids, Alb et al. (19) have reported that replacement of threonine 59 with several different amino acids abolished the PI transfer activity of PITP without any effect on PC transfer activity. Threonine 267 in PITPalpha is a serine in PITPbeta , again a conservative change. Although we replaced lysine 264 with isoleucine and threonine 267 with valine or alanine, the site-directed mutants had normal lipid transfer activity (Fig. 2C) and exhibited normal activity in the PLC reconstitution assay (data not shown).

Delta 5-PITP Inhibits the Function of Wild-type PITP in PLC Signaling, but Not in Lipid Transfer in Vitro

The truncated mutants were examined for their ability to inhibit the restorative effects of wild-type PITP (Fig. 5A). Delta 5-PITP inhibited wild-type PITP when added simultaneously to permeabilized HL-60 cells. The concentration of Delta 5-PITP required for inhibition was much lower than the amount of wild-type protein added. Although Delta 10-PITP acted as well as Delta 5-PITP, Delta 20-PITP did not affect the activity of wild-type PITP to restore PLC signaling. The inhibitory effects of Delta 5- and Delta 10-PITP were not observed when lipid transfer was examined in vitro (Fig. 5B).


Fig. 5. A, Delta 5-PITP inhibits restoration of PLC signaling with full-length PITP. The cytosol-depleted cells were stimulated with GTPgamma S in the presence of 0.3 mg/ml full-length PITP and the indicated concentrations of recombinant PITPalpha (rPITP) proteins for 20 min. bullet , Delta 5-PITP; black-square, Delta 10-PITP; black-triangle, Delta 20-PITP. B, Delta 5-PITP does not inhibit the transfer activity of full-length PITP. PI transfer activity was measured in the presence of 5 µg/ml full-length PITP and the indicated concentrations of recombinant PITPalpha proteins for 20 min. bullet , Delta 5-PITP; black-square, Delta 10-PITP; black-triangle, Delta 20-PITP.
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DISCUSSION

PITP has diverse effects on several cell functions ranging from PLC signaling to membrane traffic. In all cases reported to date, mammalian PITPalpha and PITPbeta can be used interchangeably with SEC14p, the yeast form of PITP, despite the lack of sequence homology with mammalian PITP. Since the PI binding/transfer activity is the common feature shared by the two mammalian forms of PITP and SEC14p, it must be the relevant activity that determines their abilities to restore PLC signaling and membrane traffic. Rescue of SEC14 defects in yeast with mammalian PITP has also been observed (20). However, PITPalpha only rescues the temperature-sensitive mutations, but not the null mutation. This result clearly indicates that SEC14p and mammalian PITP have distinct as well as overlapping functions.

To identify the structural requirements of PITP, we have systematically deleted the carboxyl terminus of PITPalpha and have compared the lipid binding/transfer characteristics in vitro with the ability of the truncated proteins to restore inositol lipid signaling in HL-60 cells. Deletion of just 5 amino acid residues was sufficient to impair the function of PITP both in lipid transfer and in PLC signaling. In addition, the Delta 5-truncated form of PITP was inhibitory to wild-type PITP and potentially functions as a dominant-negative mutant.

Deletion of 5 residues at the C terminus decreased the lipid transfer activity of both PI and PC. PI transfer activity was affected more than PC transfer activity. For example, Delta 5-PITP retained 30% of PI transfer activity when examined at 10 times the concentration of wild-type PITP, whereas PC transfer activity was comparable to that of wild-type PITP except that three to four times more protein was required. Despite the residual transfer activity obtained with Delta 5- and Delta 10-PITP, both these deletion mutants had no activity when examined for restoration of G-protein-mediated PLC signaling in HL-60 cells even when 15-fold higher concentrations over wild-type protein were used. The uncoupling of transfer function with reconstitution supports the emerging view that the function of PITP is not just to passively transfer PI from intracellular membranes where it is synthesized to sites of PLC signaling, but to directly participate in the synthesis of PI 4,5-bisphosphate (8, 12, 13).

It is possible that the deletion of the 5 amino acids from the C terminus affects the lipid binding properties of PITP significantly and hence the loss in transfer function. This was not found to be the case. We first examined which lipids were associated with the full-length recombinant protein purified from E. coli and found that PITP was loaded with PG predominantly (80%) and the remainder with PE. A previous study reported that PITP purified from E. coli had only PG bound to it (21). Their failure to detect PE probably resulted from the use of different techniques to monitor the lipid content. In the study reported here, we radiolabeled E. coli prior to induction so that the protein expressed would incorporate radiolabeled lipid. Analysis by TLC clearly revealed that both PG and PE could associate with PITP. The other study extracted the lipid from PITP and examined the chemical mass of the lipid (21). It is very possible that the low levels of PE were not detectable by mass determination.

Deletion mutants of PITP (Delta 5 and Delta 10) were still capable of binding the endogenous E. coli lipid, but had slightly altered ratios of anionic lipids (PG) to zwitterionic lipids (PE). Deletion of 20 amino acids had a more dramatic effect in that the amount of lipid associated was decreased by 60%, and this implies that a substantial amount of PITP does not have a lipid bound to it. In all three deletion mutants, a detectable amount of label in cardiolipin was also observed, suggesting that PITP mutants are less discriminatory than wild-type PITP (Fig. 3B).

Truncation also affected the ability to exchange the endogenous lipids with PI or PC in vitro. But the effects were small, and Delta 5-PITP could exchange PI or PC as well as wild-type PITP. However, their ability to transfer PI or PC was significantly impaired. Delta 20-PITP was completely inactive when examined for PI or PC transfer. Loss of PI transfer with Delta 5-PITP was reduced by 30%, whereas restoration of PLC signaling was completely disrupted taking into account the 10-fold higher concentrations of Delta 5-PITP used for both assays. This uncoupling of transfer with PLC signaling indicates that in addition to transfer, the PITP molecule has additional properties that are required for PLC signaling.

Delta 5-PITP inhibits PITP function in cells, but not in the in vitro PI transfer assay. Inhibition by Delta 5-PITP was not of a competitive nature. This suggests that in cells Delta 5-PITP interacts with the "putative" receptor for PITP more strongly and effectively inhibits full-length PITP from exerting its effect. In the in vitro transfer assay, lipid transfer is a passive event and is not dependent on interaction with another protein. The identification of the binding partners for PITP will provide additional insights into the mechanism of PITP function. It should be recognized that in the visual transduction system in Drosophila, PITP is part of a larger entity that is an integral membrane protein (22).

In summary, we have identified the carboxyl terminus of PITP as being critical in PLC-mediated signaling. Deletion of the carboxyl terminus does not lead to complete loss of transfer activity, but does lead to complete loss of signaling in cells. Thus, the transfer activity is not the only determining factor that dictates the restorative function of PITP in inositol lipid signaling. It has been recently reported that limited proteolysis of PITP by trypsin cleaves the carboxyl terminus at Arg253 and Arg259, and this leads to a decrease in PC transfer activity (23). These data are in accord with our results. In addition, our results suggest that in cells PITP interacts with a putative receptor. The identification of the binding partners for PITP will be greatly facilitated by taking advantage of Delta 5-PITP. The ability of the mutant proteins to block signaling will also provide valuable tools in assessing the function of PITP in living cells.


FOOTNOTES

*   This work was supported in part by a grant from the Wellcome Trust.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    Recipient of a Japan-Europe scientist exchange program for the promotion of sciences from the Ciba-Geigy Foundation (Japan).
§   To whom correspondence should be addressed: Dept. of Physiology, Rockefeller Bldg., University College London, University St., London WC1E 6JJ, United Kingdom. Tel.: 44-171-209-6084; Fax: 44-171-387-6368; E-mail: ucgbsxc{at}ucl.ac.uk.
1   The abbreviations used are: PITP, phosphatidylinositol transfer protein; PI, phosphatidylinositol; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PIPES, 1,4-piperazinediethanesulfonic acid; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate).

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