INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Phosphatidylinositol transfer proteins (PITPs) are operationally defined by their ability to catalyze the transfer phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) monomers between membrane bilayers in vitro (Cleves et al., 1991a; Wirtz, 1991). Both yeast and metazoan PITPs contain one phospholipid-binding site that exhibits a dual headgroup specificity (Sha et al., 1998; Phillips et al., 1999; Yoder et al., 2001). Thus, PITPs accomodate two dissimilar phospholipid substrates in a mutually exclusive binding reaction.

From a biological perspective, little is known about the genuine physiological functions of PITPs. The best-studied case is in yeast where PITP function has been subjected to genetic analysis. Sec14p, the major yeast PITP, is essential for protein transport from the Golgi complex (Bankaitis et al., 1989, 1990). A dissection of how Sec14p translates PtdIns/PtdCho transfer activity into biological function is driven by analyses of mutations that relieve cells of the essential Sec14p requirement for Golgi function and cell viability (Cleves et al., 1989, 1991b; Fang et al., 1996; Kearns et al., 1997; Rivas et al., 1999; Li et al., 2000; Xie et al., 2001). Such "bypass Sec14p" mutants identify novel interfaces between Golgi function, lipid metabolism, and the actions of novel proteins whose functions are only now beginning to be understood. Precise execution points for individual PITPs in mammalian cells remain unknown, although reduced PITP function leads to specific neurodegenerative defects in flies and in the vibrator mouse (Hamilton et al., 1997; Milligan et al., 1997).

There are at least three soluble mammalian PITPs and these are designated PITP, PITP, and rdgB (Dickeson et al., 1989; Tanaka and Hosaka, 1994; Fullwood et al., 1999). The best characterized of these are PITP and PITP. These two isoforms share 77% sequence identity and are encoded by distinct genes. PITP is distinguished from PITP in that it catalyzes the in vitro transfer of PtdIns, PtdCho, and sphingomyelin (SM), whereas PITP is unable to catalyze SM transfer (van Tiel et al., 2000). PITP reconstitutes as a cytosolic factor required for 1) Ca²⁺-activated discharge of secretory granule cargo in permeabilized neuroendocrine cells (Hay and Martin, 1993), 2) budding of both constitutive secretory vesicles and immature secretory granules from the trans-Golgi network of neuroendocrine cells (Ohashi et al., 1995), 3) budding of constitutive secretory vesicles from hepatocyte trans-Golgi network (Jones et al., 1998), and 4) hydrolysis of phosphatidylinositol bisphosphate mediated by various G protein-coupled phospholipase C isoforms (Cunningham et al., 1996). The former two reconstitution systems implicate a role for mammalian PITP in regulation of vesicle trafficking, an activity analogous to Sec14p function in yeast.

In all of these in vitro systems, PITPs stimulate the synthesis of various phosphoinositides that play critical roles in facilitating the reactions that are being reconstituted. Moreover, PITP and yeast Sec14p can fully replace PITP in each of these reconstituted reactions. This is a rather remarkable result given that yeast Sec14p and mammalian PITPs share no primary sequence homology nor structural similarity at all (Skinner et al., 1993; Sha et al., 1998; Yoder et al., 2001). Yet, the interchangeability of PITPs in the reconstituted in vitro systems is significantly at odds with the results of biological experiments. Saccharomyces cerevisiae expresses five distinct Sec14p-like PITPs, but none of these PITPs shares perfect physiological redundancy with the others, and each regulates a distinct step in phospholipid metabolism (Li et al., 2000; Wu et al., 2000). Moreover, mammalian PITP exerts potent dominant-negative effects when expressed in lieu of a homologous Drosophila PITP that harbors the same biochemical properties as does PITP in vitro (Milligan et al., 1997). These data strongly suggest that individual mammalian PITPs also execute specific in vivo functions. Furthermore, these data indicate that the physiological specificity of mammalian PITPs is unavailable for experimental scrutiny in present in vitro systems.

In this report, we describe our use of genetic approaches to gain insight into the physiological functions of PITP and PITP in murine cells. Our data clearly indicate that disruption of both copies of the PITP structural gene in murine cells fails to compromise their growth or vigor, and that ablation of PITP function has no significant effect on bulk phosphoinositide or phospholipid metabolism. Moreover, PITP / cells are competent for protein trafficking through the constitutive secretory pathway, are normal with regard to endocytic pathway function, are fully competent for both biogenesis and agonist-induced exocytosis of dense core secretory granules (DSGs), are responsive to bulk growth factor stimulation, and retain their pluripotency. Thus, PITP does not uniquely execute an essential housekeeping function in murine embryonic stem (ES) cells. In contrast, we were unable to evict both PITP alleles from murine ES cells, and we demonstrate that PITP deficiency leads to early failure in murine embryonic development. We suggest that PITP is an essential housekeeping PITP in murine cells, whereas PITP plays a far more specialized function in mammals than what is indicated by in vitro systems that demonstrate PITP dependence.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Antibodies

R1 and AB1 ES cell lines have been described (McMahon and Bradley, 1990; Nagy et al., 1993) and were maintained in standard media consisting of DMEM (4.5 g/l glucose), 1 mM sodium pyruvate, 1× nonessential amino acids, 2 mM L-glutamine, a 50-µg/ml penicillin/streptomycin cocktail, 15% fetal bovine serum (Invitrogen, Carlsbad, CA), 2-mercaptoethanol, and leukemia inhibitory factor (LIF). Media used for differentiating ES cells to embryoid bodies were identical except LIF and 2-mercaptoethanol were omitted. ES cells were plated on feeder layers consisting of either mitomycin C-treated murine embryonic fibroblast feeder cells or gelatin-coated tissue culture dishes. When necessary, cells were dispersed with a 0.25% trypsin/0.1% EDTA solution.

Isolation of Genomic PITP and PITP Clones

A mouse 129SVJ genomic -phage library was screened by standard filter-lift plaque hybridization methods by using random primed ³²P-radiolabeled rat PITP or PITP cDNAs as probes. Hybridized filters were washed in 2× SSPE, 0.5% SDS at room temperature followed by two washes in 0.2× SSPE, 0.5% SDS at 65°C. From such screens, -phage clones containing 12- and 15-kb genomic insert fragments of PITP and PITP were recovered. Iterative cycles of physical mapping and nucleotide sequence analysis confirmed the identities of the genomic fragments, and identified the precise nature and physical positions of the exons contained in each genomic fragment.

Transfection and Selection of ES Cell Recombinants

All experiments were carried out with ES cells grown on embryonic murine fibroblast feeder cells. For electroporation, ES cells (80% confluence) were harvested by trypsinization, centrifuged through culture medium, and resuspended in fresh culture media. A 20-µg load of linearized targeting vector was electroporated into 2 × 10⁷ cells via a single discharge (300 V, 250 µF) delivered by a single cell electroporator (Bio-Rad, Hercules, CA). Immediately after electroporation the cells were distributed onto 100-mm embryonic mouse fibroblast feeder plates and allowed to recover for 24 h before challenge with 0.5 mg/ml G418 and 2 mM gangcyclovir. Selection media were exchanged every 3-4 d, for a total of 12 d, after which individual colonies were picked and expanded.

DNA Hybridization

DNA from each clonal ES cell line was prepared using the Wizard genomic DNA purification kit marketed by Promega (Madison, WI). Purified DNA (10 µg) was subsequently digested with either EcoRI or XbaI, electrophoresed through a 1% agarose gel, transferred to Hybond N nylon membrane (Amersham Biosciences, Piscataway, NJ), and hybridized with probe 1 (Figure 1A) under stringent nonaqueous conditions (Southern, 1975). Hybridized products were washed at 68°C in 1× SSC/0.1% SDS and 0.2× SSC/0.1% SDS.

View larger version (28K): [in this window] [in a new window]   Figure 1.   (A) Strategy for targeting of the PITP locus. An HSV-tk expression cassette from pPNT (Tybulewicz et al., 1991) was engineered so that it flanks a 12-kb mouse genomic PITP clone bracketed by SstI restriction sites. A 0.4-kb DNA fragment was released from the extreme 3' end of the genomic insert so that it is not represented in the targeting vector. This fragment was subsequently used as a diagnostic probe, designated probe 1, in Southern hybridization analyses. PITP exons 8-10 were exchanged for a selectable neo expression cassette derived by polymerase chain reaction from pMC1NEO (Stratagene, La Jolla, CA). As described in the text, this amplified cassette contains an uncharacterized mutation that attenuates, but does not abolish, activity of the gene product. (B) Identification of targeted ES cell clones by Southern hybridization. The PITP-targeting vector was linearized, introduced into ES cells by electroporation, and recombinants were selected on the basis of acquired resistance to G418 and gangcyclovir (see MATERIALS AND METHODS). Genomic DNA was prepared from +/+ controls and candidate targeted (/+) ES cells, digested to completion with either restriction endonuclease EcoRI or XbaI (as indicated at bottom), transferred to nitrocellulose, and hybridized with radiolabeled probe generated by random priming of probe 1 DNA. Diagnostic signatures of the wild-type (+) and targeted () PITP alleles are indicated at right. Approximately 5% of total R1- and AB1-derived recombinants exhibited physical signatures consistent with one wild-type and one targeted PITP allele as shown.

Quantitative Enzyme-linked Immunosorbent Assay (ELISA)

Total ES cell protein (20 µg) or 50 and 100 ng of PITP C-terminal peptide was affixed onto coated ELISA plates (Falcon Plastics, Oxnard, CA) by baking overnight at 60°C. Wells were incubated with 0.5% Tween 20 in phosphate-buffered saline (PBS) (30 min at 37°C), flushed three times with PBS, and blocked with 2% bovine serum albumin (BSA) for 90 min at 37°C. PITP-specific rabbit polyclonal antibody was added to wells and incubated at 37°C for 3 h. After extensive washing with PBS, secondary goat anti-chicken antibody was added to wells, incubated at 37°C for 3 h, and again washed with PBS. ELISA signal was developed with the OPD reagent system marketed by Sigma (St. Louis, MO). Signal was quantified as absorbance at 405 nm by using a Biospec Products plate reader.

Lipid Chromatography

ES cells were plated onto 100-mm tissue culture dishes at a density of 1 × 10⁶ cells/plate and labeled in standard media containing 10 µCi/ml of [³H]inositol and supplemented with serum (final concentration of 15%) dialyzed to remove all serum components with molecular mass of <1000 Da. After 72 h, cells were harvested by washing in cold PBS followed by scraping into glass tubes. Samples were then extracted with 1.88 ml of CHCl₃/methanol/HCl and incubated for 10 min on ice. Subsequently, 0.625 ml of CHCl₃ and 0.625 ml of 0.1 M HCl were sequentially added, and the phases separated by centrifugation for 10 min at 160 × g. The organic phase was collected and dried under a stream of nitrogen gas. Samples were resuspended in a small volume of CHCl₃ and loaded onto a dried silica gel 60 thin layer chromatography (TLC) plate and resolved in a CHCl₃/methanol/H₂O/concentrated ammonia (48:40:7:5) solvent system. The TLC plates were exposed to film, inositol lipid species were identified, and individual species were quantified by scraping from the TLC plate and scintillation counting.

SM, PtdCho, and water-soluble choline metabolite analyses were performed by plating and ES cells as described above with the exception that cells were radiolabeled with 1.5 µCi/ml of [¹⁴C]choline chloride. Cells were subsequently washed in ice-cold PBS and scraped off the plates into 1 ml of 5% trichloroacetic acid. Cells were pelleted, washed in PBS, resuspended in 1 ml of polar extraction solvent (15 ml of H₂O/15 ml of 95% ethanol/5 ml of diethylether/1 ml of pyridine/36 µl of NH₄OH/75 µl of 5% butylated hydroxytoluene in CHCl₃/MeOH [2:1]), and incubated at 60°C for 20 min. Subsequently, 0.5 ml of H₂O and 5 ml of CHCl₃/methanol/butylated hydroxytoluene (2:1:0.005) were added to the samples and the mixtures were incubated at 4°C for 60 min. Samples were centrifuged for 5 min at 160 × g to separate the aqueous (choline, phosphorylcholine, and cytidine-diphosphocholine-choline [CDP]-containing) and organic (PtdCho- and SM-containing) phases. These phases were individually collected and evaporated to dryness under nitrogen gas.

SM and PtdCho were further fractionated by deacylation of PtdCho upon addition of 0.1 N KOH to the lipid film, and incubation at 37°C for 1 h. After addition of CHCl₃/balanced salt solution/EDTA, the organic (SM-containing) and aqueous (PtdCho-derived, glycerophosphocholine-containing) phases were collected and dried. SM was resolved on silica gel TLC plates with a CHCl₃/methanol (1:1) solvent system. Water-soluble choline metabolites were separated on silica gel TLC plates by using a methanol/aqueous 0.5% NaCl/NH₄OH (100:100:4) solvent system. Individual choline-containing species were detected by autoradiography and quantified by scraping and scintillation counting.

Ratiometric Calcium Measurements

ES cells were grown on feeder layers in 100-mm dishes to 80% confluence in complete media, seeded onto gelatinized coverslips at a very low cell density, and allowed to grow for 14 h. Cells were incubated in serum-free media for 2 h before loading in saline solution with fura 2-acetoxymethylester (Teflabs, Austin, TX) for 40 min at a final concentration of 5 µM fura (Manning and Sontheimer, 1997). Cells were transferred to a Series 20 Microperfusion chamber on the stage of a Nikon Diaphot 200 inverted epifluorescence microscope and kept under constant perfusion with HEPES buffer supplemented with 2 mM Ca²⁺. Immediately before stimulation the chamber was flushed with Ca²⁺-free HEPES buffer and cells were stimulated with serum (3 or 10%) or LPA. Fura was alternately excited at 340 and 380 nm with a single-wavelength monochromator and fluorescence ratio obtained every 6 s. Emitted fluorescence >520 nm was captured with an intensified charge-coupled device camera, digitized, and analyzed using ImageMASTER software. The ratio of the two images (340/380 nm) was calculated and converted to absolute calcium concentrations (Grynkiewicz et al., 1985).

Transferrin Receptor (TfR) Recycling

Steady-state distribution of TfR at 37°C was performed by the method of Odorizzi et al. (1994). Cells were plated in triplicate wells (24-well plate) and 24 h later incubated first in serum-free media for 1 h then with 4-µg/ml ¹²⁵I-transferrin (Tf) in 0.1% BSA in PBS for 1 h at 37°C. The labeling media were removed, the cells rinsed three times in 0.1% BSA in PBS, and cells were then washed twice for 3 min with 0.5 ml of 0.2 M acetic acid, 0.5 M NaCl, pH 2.4, to remove surface-bound ¹²⁵I-Tf. Cells were lysed with 0.1 M NaOH to monitor intracellular ¹²⁵I-Tf. Radioactivity in the acid washes and the cell lysates was quantified and a ratio obtained.

Internalization assays used the IN/SUR method (Wiley and Cunningham, 1982; Kang et al. 1998). Externalization assays have been described (Jing et al., 1990).

Generation of Mast Cells

ES cells were cultured (10% CO₂ atmosphere) on gelatin-coated culture dishes for 48 h in Iscove's modified Dulbecco's medium (IMDM) with 15% fetal bovine serum (Invitrogen), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 100 µM monothioglycerol, and 10 ng/ml LIF. Cells were harvested using trypsin-EDTA and resuspended as single cells in IMDM.

Cells were seeded in methylcellulose-based medium (Stem Cell Technologies (Vancouver, British Columbia, Canada), supplemented as described above but without LIF and containing 40 ng/ml murine stem cell factor, at a density of 500 cells/ml (phase 1 medium) and cultured for up to 21 d. At various times after day 5, the embryoid bodies were disrupted using 0.5% collagenase (Sigma Chemical) and the cell suspension was passed through a 21-gauge needle three times. The hematopoietic progenitors were reseeded in methylcellulose medium supplemented as before but also including 1% BSA, 10 µg/ml insulin, 200 µg/ml transferrin, 30 ng/ml murine interleukin-3, and 30 ng/ml human interleukin-6 (phase 2 medium). Cultures were maintained for up to 40 d and were fed weekly with the same medium. At various times, cultures were scored for surface FcRI by using fluorescein isothiocyanate (FITC)-labeled mouse IgE and for morphology by electron microscopy.

IgE Labeling and Fluorescence-activated Cell Sorting (FACS) Analysis

Cells were harvested from methylcellulose cultures and treated with collagenase as described above to obtain a single cell suspension. Cells were "rested" for 12 h in IMDM supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 100 µM monothioglycerol. FITC-labeled IgE (gift of B. Wilson, University of New Mexico, Albuquerque, NM) was applied to cells (final concentration 5 nM). After 4 h, cells were collected and fixed. FACS analysis was performed on BD FACSCalibur and Coulter Epics Elite instruments.

Regulated Exocytosis Measurements

Permeabilized Cells. Calcium/EGTA buffers were prepared by mixing solutions, made up at identical concentrations adjusted to pH 6.8, of EGTA and endpoint-titrated Ca-EGTA (Gomperts and Tatham, 1993). Cells, suspended in an iso-osmotic salts buffer solution [137 mM NaCl, 2.47 mM KCl, 1 mM MgCl₂, 20 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.8] supplemented with 1 mg/ml BSA, were then incubated with metabolic inhibitors (0.6 mM 2-deoxyglucose and 10 µM antimycin A) for 5 min (37°C) and cooled on ice. Streptolysin O (SLO, final concentration 1.6 IU/ml in the same buffer) was then added. After 5 min, unbound SLO was washed away by dilution and centrifugation. Rundown was initiated by transferring cells to prewarmed (37°C) iso-osmotic buffer supplemented with 0.3 mM Ca-EGTA (to regulate pCa8) and 1 mM MgATP in wells of 96-microtiter plates. Cell load was ~10,000/well. After allowing predetermined times for rundown, the cells were stimulated to secrete by adding prewarmed solutions containing Ca-EGTA buffer (3 mM final) formulated to regulate pCa5 (or pCa7 as control) and guanosine-5'-O-(3-thio)triphosphate to a final concentration of 100 µM. After 10 min, the incubations were quenched by addition of an equal volume of ice-cold iso-osmotic buffer supplemented with 5 mM EGTA. The cells were sedimented by centrifugation and the supernatants sampled for secreted hexosaminidase as described (Gomperts and Tatham, 1993; Pinxteren et al., 1998). Secretion is expressed as percentage of total hexosaminidase released relative to total content release evoked by cell permeabilization with 0.2% Triton X-100.

Intact Cells. Cells were harvested from methylcellulose cultures, treated with collagenase, rested as described above, transferred to fresh IMDM (supplemented as described above) containing 1 or 10 µg/ml antidinitrophenol (DNP) IgE (Sigma Chemical), and incubated for 4 h under standard conditions (Buckley and Coleman, 1992). After three washes in PBS (with Ca²⁺ and Mg²⁺) 1% BSA, cells were transferred to 96-well microtiter plates. Human serum albumin conjugated to dinitrophenol (Sigma Chemical) was applied at 0, 1, 10, or 100 µg/ml. Cells were incubated (30 min, 37°C), pelleted, and sampled as described above.

Preparation of Mast Cells for Electron Microscopy

Mast cells were washed in PBS and placed in primary fixative consisting of 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at 4°C. Cells were rinsed three times in 0.1 M cacodylate buffer for 5 min and incubated in 1% osmium tetroxide for 1 h. After three rinses with 0.1 M cacodylate cells were stained in 0.5% tannic acid for 10 min.

Before embedding, cells were rinsed three times with 0.1 M cacodylate buffer, two times with water, and dehydrated by serial 5-min incubations in 50, 75, and 95% ethanol followed by three 5-min incubations in 100% ethanol. Samples were infiltrated overnight in a 1:1 mixture of ethanol/Spurr's resin followed by incubation in 100% Spurr's resin for 2 h. Samples were finally polymerized in fresh resin by overnight incubation at 60°C. Thin sections were prepared and stained with alcoholic uranyl acetate and Reynold's lead citrate before imaging by electron microscopy.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Targeting of PITP Mutation into Murine Embryonic Stem Cell Genome

Data from permeabilized cell systems suggest that PITP plays critical roles in a number of constitutive and regulated membrane trafficking events, and in the optimal activation of signaling pathways that involve G protein-coupled receptors (Cunningham et al., 1996). We therefore expected that PITP would be essential for the viability of eukaryotic cells. To test this prediction, we used homologous gene targeting methods to assess the cellular consequences of loss of PITP function. To this end, we recovered a 12-kb fragment of genomic DNA derived from a 129SVJ mouse strain (see MATERIALS AND METHODS). As diagrammed in Figure 1A, this genomic fragment harbors exons 7-10 of the PITP gene. These exons collectively encode amino acids 126-257 of the 278-residue PITP polypeptide. From this genomic DNA, we constructed a homologous recombination vector carrying a mutant PITP allele where exons 8-10 are replaced with a neo* cassette (Figure 1A). This mutation (PITP::neo*) deletes amino acid residues 162-257 from PITP, and we previously established that this region of the protein is critical for PITP activity (Alb et al., 1995). In the targeting vector, a total of 9 kb of PITP genomic flanking sequences is available for directing replacement of a wild-type PITP allele by PITP::neo* via homologous recombination.

The targeting vector was linearized by restriction in the vector backbone and introduced by electroporation into ES cell lines derived from R1 and AB1 mouse strains (see MATERIALS AND METHODS). Recombinants were selected using a positive-negative selection strategy of dual resistance to neomycin and gangcyclovir (Mansour et al., 1988). In these experiments, 120 and 76 clones derived from R1 and AB1 ES cells, respectively, survived the double selection and were screened for legitimate recombination events. The screening procedure used Southern hybridization to monitor the genomic arrangement of sequences flanking both ends of the integrated PITP::neo* allele. These analyses demonstrated that six and three clones derived from parental R1 and AB1 ES cell lines, respectively, exhibited the signatures of PITP::neo*/+ heterozygotes (Figure 1B). These data indicate a targeting efficiency at the PITP locus of ~5%.

PITP Is Nonessential for Viability of Murine Embryonic Stem Cells

The neo* cassette used to generate the PITP::neo* allele contains a missense mutation that reduces the activity of the neomycin acetyltransferase gene product such that a single genomic copy of the neo* cassette supports only low-level resistance to 400 µg/ml G418. Two or more genomic copies of the neo* cassette, however, endow cells with resistance to high concentrations of G418 (2 mg/ml). This property of neo* offers a facile method for generating PITP / ES cell lines provided cells remain viable when challenged with PITP deficiency.

To determine whether ES cells tolerate loss of PITP, three of the /+ R1 ES cell lines and two of the /+ AB1 ES cell lines were challenged with a high concentration of G418 (2 mg/ml) in attempts to convert PITP::neo* to a homozygous state. Surprisingly, we recovered survivors from the high G418 selection that bore the genetic signatures of / ES cells. Of the 98 and 72 /+ R1 and AB1 ES cell-derived cell lines that passed the 2 mg/ml G418 selection, 6 and 3 clones exhibit Southern hybridization profiles diagnostic of a homozygous PITP::neo* genotype (Figure 2A). The authenticity of these / clones was confirmed by immunoblotting by using an isoform-specific PITP antibody. No PITP antigen is detected in the candidate / ES cell lines (Figure 2B).

View larger version (27K): [in this window] [in a new window]   Figure 2.   (A) Genetic diagnosis of PITP / clones. R1- and AB-1-derived PITP /+ ES cells were challenged with 2 mg/ml G418 and surviving clones were isolated. Genomic DNA was prepared from /+ ES cell controls and high G418-resistant ES cell clones and analyzed as detailed in Figure 1B. Diagnostic signatures of the wild-type (+) and targeted () PITP alleles are indicated at right. Approximately 5% of total R1- and AB1-derived /+ ES cells that passed the high G418 selection exhibited physical signatures consistent with two targeted PITP alleles as shown. (B) Immunoblot analysis of candidate PITP / clones. ES cell strains were grown to confluence on gelatin-coated tissue culture dishes, harvested, and lysed by repeated trituration through a tuberculin syringe (Pierce Chemical). A sample load of 150 µg of total cell lysate was resolved by SDS-PAGE, and displayed proteins were transferred to nitrocellulose membranes. Membranes were decorated with PITP-specific antibodies and developed with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection (Amersham Biosciences). PITP genotypes are indicated at top. Chicken antibodies specific for PITP were raised against a peptide corresponding to the C-terminal 15 PITP amino acids. (C) Measurements of PITP levels. Top, an immunoblot experiment performed as described in A with 150 µg of total cell lysate per lane and PITP-specific antibodies as probe. The PITP-specific antibodies were rabbit polyclonal antibodies directed against the C-terminal 18 residues of this protein and were generously provided by Bruce Hamilton (University of California, San Diego, CA). PITP genotypes are indicated at top. Bottom, quantitative ELISA assay (see MATERIALS AND METHODS) was used to measure levels of PITP in isogenic PITP +/+, /+, and / ES cell lines (left, PITP genotype indicated at bottom). For comparison, the signals recorded for the indicated amounts of PITP C-terminal peptide against which the PITP-specific antibodies were raised is given (right).

Quantitative ELISA experiments indicated that PITP and PITP constitute 0.065 and 0.060% of total ES cell protein, respectively (our unpublished data). We therefore considered the possibility that expression of the closely related PITP may increase in PITP / ES cells as a compensatory mechanism for loss of PITP function. Immunoblotting experiments indicate that PITP expression is not increased in PITP / ES cells (Figure 2C), and quantitative ELISA experiments independently confirm that PITP levels are comparable in PITP +/+, /+, and / ES cells (Figure 2C). Thus, PITP expression is not increased in PITP / cells as part of some adaptive response to PITP deficiency. Finally, analysis of chromosome spreads demonstrated that the both the R1- and AB1-derived PITP / ES cell lines harbor the normal murine diploid count of 40 chromosomes (our unpublished data), indicating that aneuploidy is not associated with survival of PITP / ES cells. We conclude that PITP is nonessential for ES cell viability.

Choline Phospholipid Metabolism in PITP-deficient Cells

Functional analyses of yeast Sec14p reveal a role for this protein in regulating the interface between PtdCho metabolism and Golgi function (Cleves et al., 1991b; Skinner et al., 1995; Xie et al., 1998, 2001). A role for PITP in regulating metabolism of choline phospholipids in mammalian cells has also been proposed. Antisense approaches suggest that modest reductions in PITP levels result in significant alterations in PtdCho and SM metabolism in one breast cancer cell line (Monaco et al., 1998).

To test whether PITP regulates choline phospholipid metabolism, +/+ and / ES cells were radiolabeled to steady state with [¹⁴C]choline, lipids were extracted, and radiolabeled choline phospholipids were resolved by TLC. As shown in Figure 3A, there were no significant differences in steady-state levels of bulk SM or PtdCho between +/+ and / ES cells. Moreover, intracellular levels of soluble choline-containing compounds that serve as precursors of PtdCho synthesized via the CDP-choline pathway were unaffected by PITP deficiency (Figure 3B).

View larger version (24K): [in this window] [in a new window]   Figure 3.   (A) Bulk PtdCho and SM profiles of PITP +/+ and / ES cell lines. ES cells were labeled to steady state with [14C]choline, and choline-containing phospholipids (SM and PtdCho) were resolved from water-soluble choline-containing precursors of PtdCho in a two-phase organic/aqueous solvent system. (A) Choline-containing lipids SM and PtdCho were distinguished by deacylation of the lipid fraction and separation of the reaction products by organic/aqueous partitioning in a two-phase system. SM and deacylated PtdCho (glycerophosphocholine) were resolved from the organic and aqueous fractions by TLC, respectively (see MATERIALS AND METHODS). Data are from three independent experiments, and are presented as percentage of total choline lipid recovered as SM and PtdCho, respectively, in isogenic PITP +/+ (black bars) and PITP / ES cell lines (hatched bars). All ES cell lines exhibited similar incorporation of [14C]choline into phospholipid. (B) Choline-containing biosynthetic precursors of PtdCho in PITP +/+ and / ES cell lines were resolved by TLC. The data are given as the contribution of each indicated choline compound to total water-soluble radioactivity. Cho, choline; Cho-P, phosphorylcholine; CDP-Cho, cytidine-diphosphocholine. Values represent averages of three independent experiments, and the solid and hatched bars represent data from PITP +/+ and / ES cell lines, respectively.

Bulk Phosphoinositides in PITP-deficient Cells

Speculations regarding the physiological function of PITPs in mammalian cells have focused on the role of these proteins in regulating phosphoinositide synthesis. To determine the consequences of PITP deficiency on phosphoinositide levels in ES cells, isogenic PITP +/+ and / ES cells were radiolabeled to steady-state with [³H]inositol. The inositol lipids were then extracted and resolved by TLC.

The steady-state inositol lipid profiles of +/+ and / ES cells are essentially identical (Figure 4A). As expected, PtdIns was the major labeled lipid recovered from both +/+ and / ES cells (88.7 ± 1.1 and 88.4 ± 0.7% of inositol lipid, respectively). No differences were detected in the steady-state levels of monophosphorylated PtdIns species (i.e., combined levels of PtdIns-3-phosphate and PtdIns-4-phosphate with the latter representing by far the predominant species) in +/+ versus / ES cells (4.7 ± 0.8 and 5.4 ± 0.6% of inositol lipid, respectively). Similarly, bis-phosphorylated PtdIns derivatives (i.e., combined levels of PtdIns-4,5-P₂, PtdIns-3,4-P₂, and PtdIns-3,5-P₂ with PtdIns-4,5-P₂ representing by far the predominant species) were also indistinguishable in +/+ versus / cells (3.6 ± 1.1 and 3.7 ± 1.7% of inositol lipid, respectively). These results were verified by deacylating the inositol lipid fraction and resolving the glycerophosphoinositide derivatives by anion-exchange chromatography. No differences in bulk steady-state levels of PtdIns, PtdIns-3-phosphate, PtdIns-4-phosphate, or PtdIns-4,5-P₂ were observed (Figure 4B). Moreover, the deacylation data were in quantitative agreement with the TLC data.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Steady-state levels of inositol phospholipids. (A) ES cells were labeled to steady state with [3H]inositol, and phospholipids were extracted and resolved by TLC (see MATERIALS AND METHODS). A representative chromatogram is given (right) with identification of lipid species (left) and PITP genotype (top). At left are given the quantitative data obtained from the average of three independent experiments. (B) ES cells were labeled to steady state with [3H]inositol, phosphoinositides were extracted, deacylated in the presence of methylamine, and the corresponding glycerophospho-derivatives were resolved by anion exchange chromatography (Stolz et al., 1998). The left panel presents the deacylated PtdIns data for PITP +/+ (solid bars) and / cells (hatched bars), whereas the right panel shows data for deacylated PtdIns-3-P, PtdIns-4-P, and PtdIns-4,5-P2. All values represent averages of at least three independent experiments. (C) ES cells were pulse radiolabeled for 1 h with 100 µCi/ml of [3H]inositol. Lipids were extracted and resolved by TLC, identified by autoradiography, scraped, and quantified by scintillation counting. Data are expressed as percentage of total lipid [3H]inositol incorporated into each lipid species identified at bottom. Values from PITP +/+ (solid bars) and / cells (stippled bars) are given. PtdIns-P represents the sum of PtdIns-4-P and PtdIns-3-P values. Data represent the averages of three independent experiments.

To assess rates of phosphoinositide synthesis in PITP +/+ and / ES cells, cells were pulse-radiolabeled with [³H]inositol and radiolabeled inositol lipids were then extracted, resolved, and quantified. As shown in Figure 4C, the relative rates of PtdIns, PtdIns-phosphate (primarily PtdIns-4-phosphate), and PtdIns-4,5-P₂ were comparable in these isogenic ES cell lines. To compare efficiencies of phosphoinositide resynthesis in PITP +/+ and / ES cells after stimulation, cells were radiolabeled with [³H]inositol to steady state. Radiolabeled cells were then serum starved, stimulated with serum for various times, and labeled inositol phospholipids were extracted and quantified. The inositol phospholipid profiles of isogenic PITP +/+ and / cell lines as a function of time were again indistinguishable (our unpublished data). We conclude that there are no major reductions in rates of phosphoinositide resynthesis in PITP-deficient ES cells.

Because PITP is concentrated in the nucleus, PITP / cells might exhibit alterations in nuclear phosphoinositide levels. To address this issue, a detergent method was used to purify nuclei from isogenic +/+ and / ES cells labeled to steady state with [³H]inositol. Nuclear phosphoinositide levels were then analyzed. Again, we were unable to discern any significant differences in the identities of nuclear phosphoinositide species or in their steady-state levels between +/+ and / ES cells (our unpublished data). These data indicate that PITP deficiency does not manifest itself in discernible changes in steady-state levels of either bulk membrane or nuclear phosphoinositide pools.

Bulk Signaling in PITP-deficient Cells

Although the steady-state data suggest that PITP does not play a quantitatively obvious role in regulating bulk or nuclear phosphoinositide pools, these measurements are potentially insensitive to regulated fluctuations in specific pools. In that regard, PITP is proposed to be a critical regulator of the metabolism of PIP pools that sustain signaling via receptors localized at the plasma membrane (Kauffmann-Zeh et al., 1995). Thus, we used several independent assays to measure responsiveness of +/+ and / ES cells to serum stimulation. First, isogenic ES cells were labeled to steady state with [³H]inositol, cells were serum starved, and serum-deprived cells were stimulated with either undialyzed serum or lysophosphatidic acid. Inositol-1,4,5-trisphosphate was resolved and quantified from samples collected at various times poststimulation. These time course experiments failed to reveal obvious differences in bulk inositol-1,4,5-trisphosphate metabolism in / ES cells relative to isogenic +/+ cells (our unpublished data).

A second independent approach involved monitoring evoked Ca²⁺ mobilization in cells. We measured no significant difference between +/+ and / ES cells in the overall increase in cytosolic Ca²⁺ when serum-deprived cells were stimulated with 10% serum. Under these stimulation conditions, virtually all of the cells imaged responded to serum by rapidly increasing cytosolic Ca²⁺ to levels ~2.5-fold greater than those recorded for resting cells (Figure 5). Because stimulation with high serum might saturate cell surface receptors, and thereby obscure more subtle signaling defects in / cells, we repeated the experiments using a 3% serum stimulation. As seen in Figure 5, this low-serum condition represents a suboptimal stimulation as the amplitude of the response was reduced relative to that scored for the 10% serum condition. Moreover, only ~30% of the wild-type cells responded to the low-serum stimulus. Yet, even under these conditions, the latency of the Ca²⁺ response, the number of cells responding to the serum stimulus, and the response amplitude were all comparable for +/+ and / ES cells. Similar results were obtained when serum-deprived cells were stimulated with LPA alone (our unpublished data). The data indicate that PITP is dispensable for bulk cellular responses to serum stimulation, and that plasma membrane signaling through at least the LPA receptor is unaffected by PITP deficiency.

View larger version (27K): [in this window] [in a new window]   Figure 5.   Bulk signaling properties of PITP +/+ and / cells. ES cells were serum starved for 2 h, loaded with fura 2, and stimulated with serum (3 or 10%). Ratios of fura 2 excitation at 340 and 380 nm were determined and converted to absolute calcium concentrations. These concentrations are presented as a function of serum concentration used to stimulate cells (top) and PITP genotype (bottom). Values represent the averages and SDs of cytosolic Ca2+ concentrations measured for 69 PITP +/+ and 67 PITP / cells in the 10% serum stimulation condition, and 48 PITP +/+ and 45 PITP / cells in the 3% serum stimulation condition, respectively.

Constitutive Secretory Pathway Function in PITP-deficient Cells

The availability of isogenic +/+ and / cell lines allowed a survey of the involvement of PITP in defined cellular functions. Several lines of evidence obtained from biochemical reconstitution systems suggest that PITP regulates constitutive pathways for membrane trafficking through the Golgi complex (Paul et al., 1998), and from the trans-Golgi network to the cell surface (Jones et al., 1998). To assess constitutive secretory pathway function in PITP-deficient cells, we monitored the synchronized transport of the mutant vesicular stomatitis virus glycoprotein (VSV-G) tsO45 protein from the endoplasmic reticulum (ER) to the Golgi complex and the cell surface. Incubation of both +/+ and / cells at 39.5°C results in accumulation of core glycosylated VSV-G tsO45 in the ER (Aridor and Balch, 1996). Chase of VSV-G tsO45 from the ER was initiated by addition of cycloheximide to cells and shifting the infected cells to 32°C to permit proper folding of the mutant protein.

In both +/+ and / cells, ~35% of the VSV-G tsO45 was exported from the ER to the Golgi complex by 15 min of chase at 32°C as scored by the remodeling of VSV-G glycosyl chains to a form where these are resistant to endoglycosidase H (EndoH) treatment (Figure 6). By 45 and 60 min of chase, 50% of the VSV-G tsO45 had acquired EndoH resistance in the +/+ and the / cells (Figure 6). Longer chase times did not result in additional export of VSV-G tsO45 from the ER (our unpublished data). The 50% efficiency of VSV-G tsO45 export from the ER is in agreement with values reported by others (Bi et al., 1997). These data indicate that VSV-G tsO45 transport from the ER to the medial-Golgi (i.e., where EndoH resistance is acquired) occurs with similar kinetics and efficiencies in +/+ and / cells. Surface labeling methods with VSV-G antibodies revealed robust plasma membrane labeling in intact +/+ and / cells within 30 min of release of the 39.5°C block (our unpublished data). The surface-labeling data, although qualitative, suggest that the rates of VSV-G tsO45 transit from the ER to the cell surface are similar in PITP +/+ and / cells.

View larger version (45K): [in this window] [in a new window]   Figure 6.   Trafficking of VSV-G from the ER to medial-Golgi in PITP +/+ and / cells. The mutant VSV-G tsO45 protein was accumulated in the ER of infected cells (genotypes at left) at 39.5°C and chase was initiated by adding cycloheximide and reducing culture temperature to 32°C (t = 0 min). VSV-G tsO45 was chased from the ER for various times (top). Samples were collected and either challenged with endoglycosidase H (+) or not () as indicated at bottom. EndoH resistance signifies arrival in medial-Golgi, and the fraction of EndoH-resistant VSV-G tsO45 increases with chase. EndoH sensitivity is scored by increased migration of VSV-G tsO45 after EndoH challenge. Quantification of the fraction of VSV-G tsO45 that has acquired EndoH resistance at each time point is given at bottom. One set of quantifications is given because values were essentially identical for +/+ and / cells.

Constitutive Pathways for Endocytosis Operate Normally in PITP / ES Cells

Operation of the constitutive endocytic cycle is strongly perturbed by agents such as wortmannin that inhibit pathways for synthesis of 3-phosphorylated inositol lipids and inositides (Corvera et al., 1999). We therefore tested whether PITP deficiency compromises endocytic pathway activity. In these experiments, we used the constitutively recycling TfR as a sensitive reporter for function of the endocytic pathway in +/+ and / ES cells. Because TfR is a long-lived protein that recycles rapidly through the endosomal pathway, even subtle defects in TfR internalization from the cell surface, or in TfR recycling from endosomes back to the cell surface, exert large effects on steady-state TfR distribution. Using an ¹²⁵I-transferrin binding assay to register surface-exposed TfR, we found that the steady-state distributions of TfR are similar in +/+ and / cells irrespective of whether cells are incubated in 10 or 3% serum. In all cases, 60% of the TfR is present in intracellular compartments, whereas 40% of the TfR resides at the cell surface (Figure 7A). These steady-state distributions of TfR are in close agreement with those recorded for other cell types (Jing et al., 1990; Cotlin et al., 1999).

View larger version (12K): [in this window] [in a new window]   Figure 7.   (A) Steady-state distribution of TfR on the cell surface of PITP +/+ and / cells. Steady-state distribution of TfR was performed as described previously at 37°C (Odorizzi et al., 1994; see MATERIALS AND METHODS) with the modification that PITP +/+ and / ES cells were plated in triplicate in standard media containing either 10 or 3% serum. The percentage of total 125I-Tf (and therefore TfR) located at the cell surface (solid bars) and within the cell (hatched bars) is given for each ES cell PITP genotype (+/+ or /; bottom) and for each serum concentration tested (3 and 10%; top). (B) Kinetics of TfR internalization and recycling in PITP +/+ and / cells. For internalization assays, PITP +/+ (solid bars) and / cells (hatched bars) were preincubated in serum-free media at 37°C and prewarmed 125I-Tf was added for various times up to 10 min. At each time point, the ratio of internalized and surface 125I-Tf was distinguished by an acid strip of the cell surface, and Tf(internal)/Tf(surface) ratio was plotted as a function of time. The slope of each curve yields the TfR internalization constant kin, and the data represent the mean and SDs derived from at least three independent determinations. To measure the recycling constant kext, cells were incubated with 125I-Tf for 1 h at 37°C and chilled. Surface-bound 125I-labeled Tf was removed by chelation with deferosamine at 4°C, cells were warmed to 37°C to initiate chase of 125I-Tf from endocytic compartments to the cell surface, and measurements of cell-associated and -released radioactivity were taken at various times of chase. The Tf(secreted)/Tf(internal) ratio was plotted against time, and the slope of each curve yields kext. The data represent the mean and SE derived from at least three independent determinations.

The normal TfR distribution between the plasma membrane and intracellular pools in / ES cells suggests either that rates of TfR internalization and recycling are unadulterated in PITP-deficient cells, or that any alterations in internalization or recycling rates are compensated by alterations in the opposing arm of the TfR trafficking pathway. To measure TfR internalization rates (expressed as k_in), we used the IN/SUR method to monitor the rate at which surface ¹²⁵I-transferrin-TfR complexes acquired resistance to surface stripping with a low-pH wash. As shown in Figure 7B, +/+ cells exhibit a k_in = 0.123 ± 0.075 when incubated in 10% serum and 0.125 ± 0.064 when the assay is performed in cells cultured in 3% serum. Similarly, TfR k_in values are 0.129 ± 0.078 and 0.133 ± 0.042 in / cells incubated in 10 and 3% serum, respectively. Thus, PITP deficiency does not impose a defect on rate of TfR internalization into cells.

Finally, the data show that the rate of TfR recycling from intracellular compartments to the cell surface is also refractory to PITP dysfunction. By monitoring the rate at which internalized ¹²⁵I-transferrin-TfR complexes reach the cell exterior, rates of TfR recycling (k_ext) in +/+ and / cells were calculated. The recycling rates are essentially the same in +/+ and / cell lines irrespective of whether cells are incubated at 3% serum (k_ext = 0.085 ± 0.007 vs. 0.085 ± 0.007 min¹) or 10% serum (k_ext = 0.075 ± 0.021 vs. 0.080 ± 0.028 min¹; Figure 7B). These collective data indicate that the constitutive endocytic cycle operates normally in PITP-deficient cells.

Regulated Exocytosis in PITP-deficient Mast Cells

Because PITP has been implicated in catalyzing formation and priming of secretory granules for agonist-induced exocytosis (Hay and Martin, 1993; Ohashi et al., 1995; Fensome et al., 1996; Pinxteren et al., 1998, 2001), we investigated the consequence of PITP deficiency to the activity of the regulated exocytotic pathway in ES cell-derived mast cells. Initially, we tested whether / ES cells could be induced to become functional mast cells. We subjected isogenic +/+ and / ES cell lines to conditions that induce ES cells to differentiate into the mast cell lineage (see MATERIALS AND METHODS). At various times during a 40-d period of maturation in phase 2 medium, the differentiating cell cultures were harvested and subjected to two tests for acquisition of mast cell properties. First, FACS was used monitor expression of FcRI, a mast cell marker, on the cell surface. Second, we used to electron microscopy to visualize formation of mast cell-like DSGs in the differentiating cell population. Mature cells (>30 d in phase 2) were then used in established assays that measure secretory capacity directly.

As shown in Figure 8A, both +/+ and / ES cells express the high-affinity receptor for IgE (FcRI) equally well and the efficiency of differentiation to mast cells is dependent on the length of culture incubation in phase 1 medium. The optimum incubation was 9 d, with 75-80% of the cells expressing FcRI after 35 d in phase 2 (Figure 8A, bottom). Shorter or longer periods in phase 1 medium gave a lower yield of mast cells, e.g., 50% (5 d) and 58% (21 d). No further increase in the expression of FcRI was observed after 35 d in phase 2 (our unpublished data).

View larger version (79K): [in this window] [in a new window]   Figure 8.   (A) Expression of IgE binding sites on the surface of +/+ and isogenic PITP / ES cells during phase 2 culture. Cells were treated with FITC-labeled IgE (5 nM) and surface IgE-FITC was quantified by flow cytometry. Shaded histograms represent populations from phase 2 cultures with ages in days indicated at right. Results from / cells are in the right-hand panels. Control cells (open histograms) represent passage-matched cells maintained for the same period exclusively in phase 1 medium. Results are representative of three independent experiments. (B) Mast cells from isogenic PITP +/+ and / ES cells (genotype at top) were visualized by thin-section electron microscopy. Magnifications for each panel are given. PITP +/+ and / ES cells yielded mast cells packed with morphologically correct dense core secretory granules.

Visualization of differentiated cell populations by electron microscopy confirmed that both +/+ and / ES cells efficiently manufacture DSGs, and that these granules are morphologically indistinguishable from those present in authentic mast cells. In cells derived from both +/+ and / lineages, the mast-like cells were packed with DSGs (Figure 8B). Classification of these granules as DSGs is further supported by the demonstration that the granule-bearing cells express both the mast cell-specific marker tryptase and histamine. By these criteria, ~80% of the +/+ and / ES cells adopted the morphological characteristics of mast cells. We conclude that both DSG biogenesis and mast cell differentiation proceed efficiently in the face of PITP deficiency.

To characterize the secretory competence of these differentiated cells, we stimulated intact cells to exocytose. Both +/+ and / ES cell-derived mast cells are perfectly able to secrete granule contents after loading of the cell surface with anti-DNP IgE and stimulating the loaded cells with a DNP-conjugated human serum albumin agonist (Table 1). These data demonstrate that the physiologically relevant regulated exocytotic machinery is in place in PITP-deficient cells.

View this table: [in this window] [in a new window]   Table 1.  IgE-dependent secretion from peritoneal mouse mast cells and ES cell-derived mast cells   The values (%) express the secretion (± SEM, n = 4 separate determinations) elicited by applying human serum albumin-DNP to anti-DNP IgE-loaded cells at the indicated concentrations and under the conditions as described in MATERIALS AND METHODS. Genotypes of the cells are indicated at left. Authentic peritoneal mast cells (PMCs) were us as positive controls and were isolated by peritoneal lavage of 2-10-wk-old male C57 Bl/6J mice. Mast cells were purified (>95%) by centrifugation through a Percoll cushion, essentially as previously described (Pinxteren et al., 1998).

Rundown in Permeabilized PITP-deficient Mast Cells

Agonist-induced exocytotic events have been reconstituted in permeabilized mast cells, and this reaction exhibits "rundown." Rundown is the progressive decrease in exocytotic efficiency that is recorded as the time interval between cell permeabilization and addition of secretory stimulus increases, and reflects the progressive destruction of phosphoinositides in the permeabilized cell ghosts (Pinxteren et al., 1998, 2001). Addition of either cytosol or purified PITP to the permeabilized cells strongly diminishes rundown rate, suggesting that PITP may represent the key cytosolic factor that stimulates the reconstituted exocytotic event (Pinxteren et al., 1998, 2001).

If PITP is indeed a major factor in synthesis of phosphoinositide pools required for agonist-induced exocytosis, significant differences in the rates of rundown in permeabilized PITP +/+ versus PITP / mast cells should be apparent. As shown in Table 2, application of 100 µM guanosine-5'-O-(3-thio)triphosphate at pCa5 to SLO-permeabilized peritoneal mast cells or to +/+ and / ES cell-derived mast cells elicited the massive degranulation normally recorded upon such treatment, and the corresponding values recorded for each cell type were set at 100%. As reported previously (Pinxteren et al., 1998, 2001), the exocytotic efficiency recorded for peritoneal mast cells was inversely proportional to time of preincubation of permeabilized cells before stimulation. This rate of peritoneal mast cell rundown closely resembles that measured for permeabilized PITP +/+ ES cell-derived mast cells. These data further demonstrate that ES cell-derived mast cells exhibit the properties of genuine mast cells. Rundown in the PITP-deficient system was not accelerated relative to the peritoneal mast cell and PITP +/+ ES cell-derived mast cell controls (Table 2). These data indicate that PITP is not a major factor in the resynthesis of phosphoinositide pools whose integrity is scored in the reconstituted reaction. It remains possible that PITP is the physiological PITP isoform that is dedicated to this reaction. Quantitative ELISA experiments indicated that PITP is more abundant in mast cells than is PITP (our unpublished data).