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Originally published In Press as doi:10.1074/jbc.M701072200 on April 12, 2007

J. Biol. Chem., Vol. 282, Issue 22, 16644-16653, June 1, 2007
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Binding of Phosphoinositide-specific Phospholipase C-{zeta} (PLC-{zeta}) to Phospholipid Membranes

POTENTIAL ROLE OF AN UNSTRUCTURED CLUSTER OF BASIC RESIDUES*Formula

Michail Nomikos{ddagger}1, Anna Mulgrew-Nesbitt§1, Payal Pallavi, Gyongyi Mihalyne, Irina Zaitseva, Karl Swann||, F. Anthony Lai{ddagger}, Diana Murray§, and Stuart McLaughlin2

From the {ddagger}Cell Signaling Laboratory, Wales Heart Research Institute and the ||Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom, the §Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, and the Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794

Received for publication, February 5, 2007 , and in revised form, April 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phospholipase C-{zeta} (PLC-{zeta}) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-{delta}1, but lacks the PH domain that anchors PLC-{delta}1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-{zeta} interacts with membranes. The linker region between the X and Y catalytic domains of PLC-{zeta}, however, contains a cluster of basic residues not present in PLC-{delta}1. Application of electrostatic theory to a homology model of PLC-{zeta} suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-{zeta} to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5 x 103 M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-{zeta} X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge =+7) within the linker region, PLC-{zeta}-(374-385), binds to PC/PS vesicles with higher affinity than PLC-{zeta}, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-{zeta} may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian eggs, fertilizing sperm trigger a series of oscillations in the intracellular free calcium concentration, [Ca2+]i, because of release of Ca2+ from the endoplasmic reticulum; the oscillations initiate the major events of egg activation (1-4). Inositol 1,4,5-trisphosphate (IP3)3 appears to mediate the release of Ca2+ at fertilization (5, 6), but it is not established exactly how the sperm triggers the IP3 and Ca2+ changes. There is substantial evidence that mammalian sperm introduce a protein factor into the egg after fusion (1, 2, 7); this sperm factor exhibits phospholipase C (PLC) activity, but is not a conventional somatic PLC isoform (8-12). A genomic search for sperm- and testis-specific PLCs identified a novel sperm-specific isoform, PLC-{zeta}, that has been characterized in mouse (13), human (14), and other species (15). Several lines of evidence indicate PLC-{zeta} fulfills the main criteria for the sperm factor: injection of either recombinant PLC-{zeta} or its RNA produces Ca2+ oscillations similar to those seen after sperm extract injection, and immunoprecipitation to remove PLC-{zeta} from sperm extracts abolishes their Ca2+-releasing activity (13, 14, 16). Mass spectrometry shows PLC-{zeta} is the factor responsible for activating the egg after intracytoplasmic sperm injection (17).

The four well characterized somatic isoforms of PLC (beta, {gamma}, {delta}, and {epsilon}) use different mechanisms to interact with plasma membranes (18, 19). PLCs beta, {gamma}, and {epsilon} (20, 21) bind to specific plasma membrane proteins, while PLC-{delta}1 has a pleckstrin homology (PH) domain that binds with high affinity and specificity to the lipid PIP2 (22-26). Membrane interactions are important because when PLCs bind to the plasma membrane, they experience a ~1000-fold higher local concentration of their PIP2 substrate, which increases the hydrolysis rate significantly (18, 19, 27, 28).

Sequence alignment analysis of PLC isoforms indicates that PLC-{zeta} has the greatest homology with PLC-{delta}1 (47% similarity, 33% identity) (13), but lacks the PH domain that mediates PLC-{delta}1 interactions with the plasma membrane. How then is PLC-{zeta} targeted to membranes? Does it use protein-protein interactions like the beta, {gamma}, and {epsilon} isoforms, or protein-lipid interactions like the {delta}1 isoform? Alignment analysis (13) also reveals differences in the apparently unstructured linker region between the conserved X and Y catalytic domains of PLC-{zeta} and PLC-{delta}1: the PLC-{zeta} linker region proximal to the Y domain contains a cluster of basic residues (15) not found in the homologous region of PLC-{delta}1 (29). Scheme 1 below compares these sequences of the X-Y linker region of mouse PLC-{zeta} and rat PLC-{delta}1; basic residues are underlined and acidic residues are italicized.Formula

Unstructured clusters of basic residues similar to the one in PLC-{zeta} help anchor proteins such as Src (30) and K-Ras4B (31, 32) to membranes via electrostatic interactions with acidic lipids. These clusters may target proteins to the plasma membrane for two reasons. First, it has more monovalent acidic lipids (and thus a more negative electrostatic surface potential) than internal membranes (32-34). Second, it has a higher fraction of polyphosphoinositides, such as PIP2 (35). Although there is good evidence the basic cluster in the X-Y linker region of PLC-{zeta} acts as a nuclear localization sequence (36, 37), we hypothesized it could also interact with acidic lipids to help anchor the enzyme to biological membranes. We tested this hypothesis by measuring the binding of (a) catalytically active PLC-{zeta} to phospholipid vesicles, (b) a glutathione S-transferase (GST) fusion construct containing the PLC-{zeta} X-Y linker region to phosphoinositide-impregnated nitrocellulose filters, and (c) a peptide corresponding to the basic cluster within the X-Y linker, PLC-{zeta}-(374-385), to phospholipid vesicles. We also constructed homology models of PLC-{zeta} and PLC-{zeta}-(374-385) and used them to calculate whether the basic cluster can interact electrostatically with acidic lipids. Finally, we used fluorescence resonance energy transfer (FRET) measurements to examine the lateral interaction of membrane-bound PLC-{zeta}-(374-385) with PIP2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—We purchased 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (PG), and the triammonium salt of L-{alpha}-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) from Avanti%20Polar%20Lipids">Avanti Polar Lipids (Alabaster, AL). [Inositol-2-3H]-L-{alpha}-phosphatidyl-D-myoinositol 4,5-bisphosphate ([3H]PIP2) and [ethyl-1, 2-3H]N-ethylmaleimide ([3H]NEM) were from PerkinElmer Life Sciences (Boston, MA). Non-radioactive NEM was from Sigma. Bodipy-TMR-PIP2 (see Ref. 38 for structure) and PI(3,5)P2 were from Echelon (Salt Lake City, UT), and Texas Red was from Molecular Probes (Eugene, OR).

Peptides—Peptides, purchased from American Peptide Co., Inc. (Sunnyvale, CA), were blocked with an acetyl group at the N terminus and an amide at the C terminus. An N-terminal cysteine was added to facilitate covalent attachment of either a radioactive ([3H]NEM) or a fluorescent (Texas Red) label. The designations and sequences of the peptides are: PLC-{zeta}-(374-385), acetyl-CKKRKRKMKIAMA-amide; PLC-{delta}-(480-491), acetyl-CKPKEDKLKLVPE-amide; Asp3Lys13Asp3, acetyl-CDDDKKKKKKKKKKKKKDDD-amide.

Peptide Labeling—We labeled peptides with radioactive NEM by placing 250 µCi of [3H]NEM in pentane on top of 20 µl of dimethyl formamide (DMF), evaporating the pentane with argon gas, then mixing the [3H]NEM in DMF with 1 ml of a ~0.1 mM peptide solution. We then added an excess of unlabeled NEM (molar ratio of 1.5:1 NEM:peptide) to block unlabeled cysteines. We labeled peptides with Texas Red, a thiol-reactive fluorescent probe, using a modified version of the protocol supplied by the manufacturer. Briefly, we mixed 1 ml of a ~0.1 mM peptide solution (10 mM K2HPO4/KH2PO4, pH 7.0) with the appropriate volume of probe solution (N,N'-dimethylformamide) to give a molar ratio of 1:1 probe:peptide and let the reaction proceed for 1 h at room temperature. We purified the labeled peptides using HPLC and determined the final purity (>95%) by MALDI-TOF mass spectroscopy.

Expression Constructs of PLC-{zeta} and PLC-{delta}1—Mouse PLC-{zeta} and its corresponding X-Y linker region were amplified by polymerase chain reaction (PCR) from the original clone cDNA (GenBankTM accession no. AF435950 [GenBank] ; (13)) using Pfu polymerase and the appropriate primers to incorporate an EcoRI site at the 5'-end and a SalI site at the 3'-end. The primer pairs used for PLC-{zeta}, and the corresponding XY linker were, respectively, 5'-AATCGAATTCTCATGGAAAGCCAACTTCATGAG-3' (forward) and 5'-ATGG TCGACATGCGTCACTCTCTGAAGTA-3' (reverse); and 5'-TAGAATTCGGAAAGTGGGAACCTTATCTGAAAC-3' (forward) and 5'-AATGGTCGACAAGGCCATGGCTATTTTCATCT-3' (reverse). The PCR products were then cloned into pGEX-5X2 (Amersham Biosciences).

The rat PLC-{delta}1 clone (GenBankTM accession no. M20637 [GenBank] ) was kindly provided by Matilda Katan (Institute for Cancer Research, London, UK) in plasmid vector pGEX-1{lambda}t. Appropriate primers were designed to incorporate a SalI site at the 5'-end and a NotI site at the 3'-end of PLC{delta}1. PLC{delta}1 and the XY linker of PLC-{delta}1 were amplified by PCR and the amplicon was cloned into pGEX-5X2 vector using these SalI and NotI restriction sites. The primers used for PLC{delta}1 and for the corresponding XY linker, respectively, were 5'-CTTCGTCGACCATGGACTCGGGTAGGGAC-3' (forward) and 5'-CACCGCGGCCGCTTAGTCCTGGATGGAGATCTTC-3' (reverse), and 5'-TAGAATTCGGAAGAAGCTGGGAGGGCTGCTGCCT-3' (forward) and 5'-TGGTCGACTCCGGCACCAGCTTTAGTTTATCC-3' (reverse).

Protein Expression and Purification—For bacterial expression of the GST-tagged fusion proteins, Escherichia coli (BL21 strain, Novagen) was transformed with the appropriate construct cloned into the bacterial expression vector pGEX-5X2 (39). Cultures were incubated at 30 °C until the A600 reached 0.5, expression was induced at 25 °C with 0.5 mM isopropyl-beta-D-thiogalactopyranoside, Promega) for 4 h, and bacteria were harvested by centrifugation for 15 min at 10,000 x g at 4 °C. Bacterial pellets were resuspended in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 x 7H2O, 1.4 mM KH2PO4, pH 7.4) containing 20% glycerol, 2 mM dithiothreitol (DTT), and protease inhibitor mixture (Roche Applied Science). The suspension was sonicated 3 times for 20 s on ice and centrifuged for 15 min at 15,000 x g at 4 °C to pellet unlysed cells and cellular debris. GST-tagged recombinant proteins were purified from the soluble lysate using glutathione-SepharoseTM 4B (GS4B, Amersham Biosciences), 2-ml bed volume/liter starting culture. The GS4B was washed three times with 10 ml of PBS, mixed with lysate, and incubated at 4 °C for 2 h, and then centrifuged at 5000 rpm (5 min at 4 °C). The supernatant was discarded, and the GS4B was washed four times with 25 ml of PBS at 4 °C. Proteins were eluted with 10 mM reduced glutathione (Sigma) in 50 mM Tris-HCl, pH 8. Eluates were placed in snake-skin dialysis tubing (10,000 dalton cut off; Pierce) and dialyzed against 4 liters of PBS overnight at 4 °C. Dialyzed GST fusion proteins were concentrated and stored at -80 °C, in buffer containing 40% glycerol, 2 mM DTT, and protease inhibitors.

Vesicle Preparations—We used 100 nm sucrose-loaded large unilamellar vesicles (LUVs) for centrifugation experiments. As described previously (40, 41), we added solutions of PC, PS, and PIP2 dissolved in 1-2 ml chloroform to a 50-ml round-bottomed flask, which was then immersed into a 30-35 °C water bath and attached to a rotary evaporator. After ~5 min rotation to allow thermal equilibration of the flask and lipid solution, we applied the maximum vacuum that does not boil the chloroform and rapidly evaporated most of the solvent to produce a uniform thin dry film of the lipid mixture; we then maintained the flask under full vacuum for ~30 min. Adding a buffer solution containing 176 mM sucrose, 1 mM MOPS at pH 7.0 produces a multilamellar vesicle solution, which we then froze and thawed five times. We formed 100-nm diameter LUVs by extruding the solution through polycarbonate filters, then resuspended the sucrose-loaded vesicles in a 100 mM KCl, 1 mM MOPS, pH 7.0 buffer.

Centrifugation Binding Experiments—We used the centrifugation technique described in detail elsewhere (40-42) to measure the binding of 3H-labeled peptides to sucrose-loaded PC/PS, PC/PIP2, and PC/PS/PIP2 LUVs. In brief, we mixed LUVs with trace concentrations (2-10 nM) of radiolabeled peptide and centrifuged the mixture at 100,000 x g for 1 h (we added sonicated unilamellar PC vesicles to the lipid-peptide mixtures, which reduces loss of peptide on the walls of the centrifuge tubes). We compared the radioactivity counts of the supernatant (free peptide) and pellet (peptide bound to sucrose-loaded vesicles) to determine the percentage of peptide bound. We describe the binding using the molar partition coefficient K, given by Equation 1,

Formula(Eq.1)

where [Pm] is the molar concentration of peptide partitioned onto the membrane; [Ptotal] is the total concentration of peptide in solution and [L] is the accessible lipid concentration, which is one-half the total lipid concentration because we add radioactive peptide to preformed vesicles. We applied Equation 1 to the experimental data and obtained a value for K. It is apparent from Equation 1 that K is the reciprocal of the accessible lipid concentration required to bind 50% of the peptide. The derivation of Equation 1 makes no assumption about the mechanism by which the peptide binds to the membrane (43). An identical equation is obtained if one assumes (incorrectly) that the unstructured basic peptide forms a 1:1 stoichiometric complex with a lipid in the outer leaflet of the vesicle. Thus, 1/K may be considered an effective dissociation constant of the peptide with a lipid.

PIP2 Hydrolysis Assay—We also used the centrifugation technique to measure the binding of catalytically active GST-PLC-{zeta} to LUVs. We mixed ~5 nM GST-PLC-{zeta} with sucrose-loaded LUVs in a Ca2+-free solution (100 mM KCl, 25 mM HEPES, 2 mM DTT, 100 µM EGTA, pH 7.0) and collected the supernatant, which contained the unbound enzyme, following the procedure described above. We used a PIP2 hydrolysis assay to determine the concentration of PLC-{zeta} in the supernatant, adding aliquots of the supernatant to 33:33:33:1 PC/PS/PE/PIP2 100 nm LUVs containing a trace amount of [3H]PIP2, then initiating hydrolysis by adding calcium chloride (total [CaCl2] 110 µM; free [Ca2+] ~ 10 µM). We removed 75-µl samples at different times and terminated the reaction by adding 375 µl of ice-cold 10% trichloroacetic acid and 50 µl of 10% Triton X-100, then incubated the samples on ice until a white precipitate formed. Following centrifugation at 14,000 x g for 5 min, we removed the supernatant and mixed it with 1 ml of 2:1 chloroform/methanol. We used scintillation counting of the upper phase of this mixture to determine the concentration of methanol-soluble [3H]IP3 products.

We used different known concentrations of PLC-{zeta} to determine calibration curves: the initial slopes of curves illustrating %[3H]PIP2 hydrolysis versus time (as in Fig. 1A for binding data) are directly proportional to the [PLC-{zeta}] over the concentration range we used. We measured the initial slopes of the experimental curves (e.g. Fig. 1A), and compared them to the calibration curves to determine the concentration of active PLC-{zeta} in the supernatants. The experimental points in Fig. 1A are fit with a hyperbola. Fig. 1B plots % bound enzyme against the accessible lipid concentration; the curve is the best fit of Equation 1 to the data. We measured hydrolysis by PLC-{delta}1 in control experiments and observed no significant binding to PC/PS vesicles, consistent with previous measurements (44). GST-PLC-{delta}1, however, bound with a Kd of ~2 µM to vesicles containing PIP2 (not shown), a value similar to that reported for PLC-{delta}1 and the PH domain of PLC-{delta}1 (23, 25, 26).

Electrostatic Calculations and Potentials—We used a modified version of the DelPhi program (45) to solve the nonlinear Poisson Boltzman (PB) equation for the model peptide/membrane systems considered in this study (46). DelPhi produces finite difference solutions to the PB equation for a system where the solvent is described in terms of bulk dielectric constant and concentrations of mobile ions, whereas solutes (peptides and phospholipid membranes) are described in terms of coordinates of individual atoms as well as atomic radii and partial charges. Our calculations assume PIP2 has a valence of -4 and basic peptides are in their minimum free energy orientation. We followed the protocol outlined in Ref. 47 to calculate the interaction of the peptides with membranes containing PIP2. We used the Modeler 8v1 (48) program to construct the homology models for mouse PLC-{zeta} (GenBankTM accession number: Q3KPE5) based on the crystal structure of rat PLC-{delta}1 (pdb_id: 1djx:b) (29). The PLC-{delta}1 structure did not include coordinates for a portion of the catalytic linker region spanning residues 446-483, which correspond to the mouse PLC-{zeta} segment 313-377. We modeled the catalytic linker residues HKPKE (479-483 in PLC-{delta}1) and VKKRK (373-377 in PLC-{zeta}) de novo into the structure and homology model, respectively, using the program Loopy (49) so that the regions corresponding to the experimental peptides were included in both the available structure and the model. However, we did not include other residues in the linker region of PLC-{delta}1 not visible in the structure, i.e. residues 446-478 for the PLC-{delta}1 structure and residues 313-372 for the PLC-{zeta} model are not shown. Hence, the electrostatic maps in panels B and C of Fig. 2 do not reflect these missing linker regions. Electrostatic maps adjacent to full models of human PLC-{delta}1 and PLC-{zeta} are in supplemental materials. The electrostatic potentials were calculated by solving the PB equation and visualized using GRASP (50).

FRET Experiments—We used an SLM-AMINCO spectrofluorometer to measure FRET between a Bodipy-TMR-PIP2 and a Texas red label on the membrane-adsorbed basic peptides. We prepared PC/PS/Bodipy-TMR-PIP2 (70:30:0.1) LUVs as described previously (38), excited the Bodipy-TMR donor fluorophore at 547 nm, and collected emission spectra from 560 to 660 nm after addition of peptide. We used a lipid concentration (0.75 mM) sufficient to bind >90% of the peptide to LUVs. We deconvoluted the raw data by performing a four-parameter two-peak Lorentzian fit, keeping the peak wavelengths fixed at 571 nm for Bodipy-TMR-PIP2 and 607 nm for Texas-Red PLC-{zeta}-(374-385). We calculated fluorescence intensity using the peak amplitudes and full width at half-maximum amplitude for each peak. The energy transfer efficiency, % transfer, was analyzed by calculating the quenching of the fluorescence intensity of the donor (Equation 2) or by calculating the fluorescence intensity of the acceptor (Equation 3).

Formula(Eq.2)

Ida is the donor fluorescence intensity at the given wavelength {lambda}, in the presence of the acceptor, and Id is the corresponding intensity in the absence of the acceptor.

Formula(Eq.3)

{epsilon}ad is the absorbance of the acceptor when donor is present; {epsilon}da is the absorbance of the donor when acceptor is present; {lambda}1 is the peak absorbance wavelength of the donor; Iad is the intensity at the acceptor wavelength, {lambda}2, in the presence of the donor; and Ia is the corresponding intensity in the absence of the donor (38, 51). Texas Red-labeled peptides permeate vesicles, so the peptide quenches the Bodipy-TMR-PIP2 on both the inner and outer leaflets (38).

X-Y Linker Binding to Inositol Phosphate Lipids—We first preblocked PIP strips (Molecular Probes) for 2 h with binding buffer (TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.4) containing 3% bovine serum albumin, lipid-free), then added recombinant GST-PLC-{zeta} X-Y fusion protein (100 pmol) in 5 ml of TBS-T and incubated for 4 h at room temperature. After washing three times in TBS-T, we visualized GST fusion protein interaction with the inositol phosphate lipids by first incubating the PIP strips with rabbit anti-GST polyclonal antibody (T103, 1:5000 dilution in 5 ml of binding buffer) overnight at 4 °C, followed by three 15-min washes. We then incubated the strips with a horseradish peroxidase (HRP)-conjugated anti-rabbit antibody in the same binding buffer for 1 h at room temperature, followed by 3 x 15-min washes with TBS-T. We used Super Signal West Dura (Pierce) to detect the HRP-coupled antibodies and a Bio-Rad Gel Doc system for image capture.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Mouse PLC-{zeta} Binds More Avidly to Phospholipid Vesicles That Include PIP2—As described under "Experimental Procedures," the centrifugation assay we used monitors only binding of the active enzyme, determining the concentration of unbound PLC-{zeta} by assaying its activity. Fig. 1B compares the binding of GST-PLC-{zeta} to LUVs formed from 2:1 PC/PS or 66:33:1 PC/PS/PIP2; the enzyme binds ~10-fold more strongly to the latter. Fig. 1A illustrates the data we used to calculate the % enzyme bound in Fig. 1B (% [3H]PIP2 hydrolyzed versus time at 3 different lipid concentrations); calibration experiments ("Experimental Procedures") showed the initial slopes of these curves are proportional to the [PLC-{zeta}]. For example, the initial slope of the 0.3 mM PC/PS/PIP2 curve in Fig. 1A is about one-half the initial slope of the 0 mM lipid curve, indicating ~50% of the active enzyme had bound to the LUVs; this corresponds to the 0.3 mM PC/PS/PIP2 point in Fig. 1B. The curves in Fig. 1B show the fit of Equation 1 to the data, which allows us to determine the molar partition coefficient K, the reciprocal of the lipid concentration that binds 50% of the PLC-{zeta}.(K may be considered as equivalent to the 1:1 association constant of a protein with any lipid in the membrane). Averaging the values obtained in three independent experiments, we deduced K = 4 ± 0.5 x 102 M-1 for 2:1 PC/PS and 6 ± 4 x 103 M-1 for 66:33:1 PC/PS/PIP2 vesicles.

Is this binding sufficiently strong to be biologically important? The effective concentration of lipids in a spherical cell of radius 10 µmis ~10-3 M (assuming the lipids from the inner leaflet of the plasma membrane are dissolved uniformly in the cytoplasm); thus, PLC-{zeta} should have a K ≥ 103 M-1 to anchor a significant fraction of the enzyme to the plasma and/or internal membranes of a mammalian egg. The results in Fig. 1 suggest PLC-{zeta} can bind significantly to the inner leaflet of a plasma membrane that contains a typical physiological mol fraction (1%) of PIP2. Curiously, the binding affinities of PLC-{zeta} and PLC-{delta}1 for PC/PS/PIP2 vesicles are similar, even though the former lacks a PIP2-targeting PH domain. Specifically, if we assume (incorrectly) that PLC-{zeta} forms a 1:1 complex with PIP2, the apparent 1:1 association constant, 6 x 105 (Kd ~ 2 µM), is similar to the binding constant of the PH domain for PIP2 in a membrane (18, 25). Substituting PtdIns(3,5)P2 for PtdIns(4,5)P2 in PC/PS/PIP2 vesicles did not affect GST-PLC-{zeta} binding, but ablated GST-PLC-{delta}1 binding (data not shown). The latter result is consistent with previous work showing the PLC-{delta}1 PH domain binds to PtdIns(4,5)P2 with high specificity (25, 26). Thus, PLC-{zeta} selects polyvalent phosphoinositides strongly over monovalent acidic lipids, but lacks the specificity of PLC-{delta}1 for the 4,5-versus 3,5-phosphoinositide head group.


Figure 1
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  		FIGURE 1.
Binding of PLC-{zeta} to PC/PS and PC/PS/PIP2 LUVs. A, plot of the % hydrolysis of radioactive PIP2 in LUVs versus time after addition of supernatant from a centrifugation experiment containing unbound PLC-{zeta}. In the centrifugation experiment, ~5 nM PLC-{zeta} was mixed with 66:33:1 PC/PS/PIP2 LUVs present at an accessible lipid concentration of 0 mM ({circ}), 0.3 mM ({square}), or 2 mM ({blacksquare}); following centrifugation to remove the PLC-{zeta} bound to the LUVs, the assays measured the activity of the unbound PLC-{zeta} in the supernatants. The initial slopes are proportional to the [PLC-{zeta}]. B, plot of % PLC-{zeta} bound to 2:1 PC/PS (bullet) and to 66:33:1 PC/PS/PIP2 ({circ}) vesicles versus accessible lipid concentration. The curves show the best fits of Equation 1 to the data. The molar partition coefficients (K) for binding of the enzyme to 2:1 PC/PS and 66:33:1 PC/PS/PIP2 vesicles are 3.5 x 102 M-1 and 5 x 103 M-1, respectively. The data in B are the results from A and additional data from that experiment. The average and S.D. for three similar experiments are: (4 ± 0.5) x 102 M-1 (n = 3) for 2:1 PC/PS and (6 ± 4) x 103 M-1 (n = 3) for 66:33:1 PC/PS/PIP2 vesicles.

 


Figure 2
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  		FIGURE 2.
Homology model of PLC-{zeta} and comparison of the electrostatic properties of the mouse PLC-{zeta} model versus the rat PLC-{delta} structure. A, C{alpha} trace of a comparative model of PLC-{zeta}, based on the crystal structure of PLC-{delta}1 (29). Catalytic X in green, catalytic Y in olive, the calcium ion in the catalytic site shown as a yellow sphere, X-Y linker region in dark gray (with basic residues shown in blue in atomic detail), EF hand 1 in orange, EF hand 2 in gold, and the C2 domain in light gray. The dashed line indicates a putative position of the membrane. B and C, electrostatic potential profiles calculated using the Poisson Boltzmann equation (ionic strength = 0.1 M) and displayed using GRASP. The ±25 mV equipotential profiles are shown as blue/red mesh surfaces, and the C{alpha} traces of the PLC-{zeta} model (B) and the PLC-{delta} (C) structures are colored gray. Basic and acidic residues in the linker regions of both enzymes are shown in atomic detail and colored blue and red, respectively. The orientations are the same as in A. Note residues 446-478 for the PLC-{delta}1 structure and residues 313-372 for the PLC-{zeta} model are not depicted.

 
Homology Model and Potential Profiles—We constructed the homology model of mouse PLC-{zeta} shown in Fig. 2A using the programs Modeler 8v1 (48) and Loopy (49) and the published structure of rat PLC-{delta}1 (29, 52). Potential profiles predict that the cluster of basic residues in the linker region (shown in dark gray with basic residues highlighted in blue in Fig. 2A) produces a local positive electrostatic potential (blue mesh surface in Fig. 2B). PLC-{delta}1 lacks this basic cluster and the consequent strongly positive electrostatic potential at this location (Fig. 2C). (See supplemental materials for electrostatic potentials adjacent to models of human PLC-{delta} and human PLC-{zeta}.) The model and potential calculations suggest the cluster of basic residues in the X-Y linker region could play a role in anchoring the enzyme to phosphoinositides in a membrane.


Figure 3
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  		FIGURE 3.
Binding of recombinant PLC-{zeta} XY linker region to various lipids. A, analysis of purified X-Y linker regions of PLC-{zeta} and PLC-{delta}1. Purified recombinant GST-tagged proteins were separated using 8% SDS-PAGE and subjected to Coomassie Brilliant Blue staining (left panel) and immunoblot analysis using T103 (anti-GST) polyclonal antibody (right panel). B, binding of recombinant X-Y linker regions and GST to phosphoinositides and other lipids spotted onto a nitrocellulose membrane (PIP strips).

 
PLC-{zeta} X-Y Linker Region Binds More Strongly to Polyvalent than Monovalent Phosphoinositides—We tested whether the X-Y linker of PLC-{zeta}, absent the remainder of the protein domains, could itself bind specifically to inositol phosphoinositides by monitoring binding of a recombinant protein fragment to phosphoinositides and other lipids spotted onto nitrocellulose membranes (PIP strips). We compared binding of the GST-X-Y linker regions from PLC-{delta}1, PLC-{zeta}, and GST alone (Fig. 3A). Fig. 3B shows neither the PLC-{delta}1 X-Y linker nor GST alone binds detectably to any of the phosphoinositides on the PIP strip. In contrast, the PLC-{zeta} X-Y linker binds significantly to all multivalent phosphoinositides tested; among the diphosphoinositides, it apparently binds more strongly to PI(3,5)P2 than to PI(4,5)P2. We did not observe this specificity, however, in the more physiologically relevant measurements of binding of PLC-{zeta} to phospholipid vesicles (see above); the PIP strip assay is not quantitative and often gives both false positive and false negative readings (53).

PLC-{zeta}-(374-385) Binds to PC/PS and PC/PS/PIP2 LUVs—We next investigated how PLC-{zeta}-(374-385), a synthetic peptide that corresponds to the basic cluster in the mouse PLC-{zeta} X-Y linker region, binds to membranes containing acidic lipids. The peptide has 7 basic and no acidic residues (see Scheme 1 above). Fig. 4A shows the percent PLC-{zeta}-(374-385) bound to vesicles with increasing mol fractions of the monovalent acidic lipid PS; the peptide does not bind significantly to electrically neutral PC vesicles, while 50% of the peptide is bound at 5 x 10-5 M accessible lipid, or K = 2 x 104 M-1 for 2:1 PC/PS vesicles. Fig. 4B shows the molar partition coefficient K increases exponentially with the mol fraction of PS. (Equivalently, the binding energy increases linearly with the mol fraction of PS.) This is expected theoretically if the peptide binds to the vesicle via nonspecific electrostatic interactions (46, 54). As discussed in the supplemental materials, experiments at a different ionic strength and with a different monovalent acidic lipid also suggest the binding is caused by electrostatics.

Comparison of the data in Figs. 1B (filled circles) and 4A (triangles) shows that the native enzyme binds to 2:1 PC/PS vesicles ~100-fold less strongly than does the peptide. Moreover, comparison of the binding of PLC-{zeta} and PLC-{zeta}-(374-385) to LUVs with 1% PIP2 shows binding of the former is enhanced more significantly. Specifically, for PLC-{zeta}-(374-385), K = 5 ± 2 x 104 M-1 (n = 4, data not shown) with 66:33:1 PC/PS/PIP2 vesicles versus K = 2 x 104 M-1 with 2:1 PC/PS vesicles (Fig. 4A); thus adding 1% PIP2 increases K by 15-fold for PLC-{zeta} (Fig. 1B) versus 2-fold for the peptide. Our electrostatic calculations with computer-modeled PLC-{zeta}-(374-385) predicted this 2-fold effect; calculations with the full-length enzyme, for which we do not know the structure, are beyond the scope of the current work. Fig. 2B shows there are acidic residues adjacent to the basic cluster in the X-Y linker region of PLC-{zeta} that were not included in the peptide, and we hypothesized these might explain the differences in the binding behavior of the molecules. We tested this hypothesis by studying the binding of a simple model peptide comprising a core of 13 basic residues flanked by 3 acidic residues at each end; note that this peptide, Asp3Lys13Asp3, has the same net charge (+7) as PLC-{zeta}-(374-385).


Figure 4
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  		FIGURE 4.
Binding of PLC-{zeta}-(374-385) to PC/PS LUVs. A, binding of trace concentrations (~25 nM)of[3H]PLC-{zeta}-(374-385) to PC ({circ}) and PC/PS (5:1bullet, 3:1 {blacksquare}, and 2:1 {blacktriangledown}) LUVs. The molar partition coefficients, K, are calculated from the best fit of Equation 1 to the data (curves). Each symbol on the plot represents an average of ≥ three independent experiments (±S.D. if larger than symbol). B, plot of the molar partition coefficient K versus mol % of PS in lipid vesicles. As predicted theoretically for electrostatic interactions, the value of K increases exponentially as the mol fraction of PS increases. Control experiments with PLC-{delta}1-(480-491), a peptide corresponding to a similar portion of the X-Y linker region of this enzyme (see Scheme 1), confirm that this peptide binds very weakly (K < 102 M-1) to 2:1 PC/PS vesicles (result not shown).

 
Binding of Asp3Lys13Asp3 to Lipid Vesicles—We used the centrifugation assay to measure the molar partition coefficient, K, of Asp3Lys13Asp3 for LUVs. Table 1 shows the data: K = 5 ± 0.5 x 102 M-1 with 5:1 PC/PS vesicles, which is about 100-fold lower than the value with Lys13 (K = 7 x 104 M-1; (41)). Electrostatic theory suggests the flanking Asp residues on the Asp3Lys13Asp3 peptide have, on average, an oblique orientation with respect to the membrane surface, and our calculations predict that this peptide should bind ~50-fold less strongly to 5:1 PC/PS membranes than Lys13. Thus when a peptide has negative residues flanking a basic cluster, it binds considerably less strongly to PC/PS vesicles because the interaction depends on the net charge of the peptide. The equally charged Asp3Lys13Asp3 and PLC-{zeta}-(374-385) peptides bind equally well to 5:1 PC/PS vesicles (Table 1); this is also predicted by electrostatic theory. Next, we tested whether incorporating PIP2 in the LUVs increases the binding affinity of Asp3Lys13Asp3. Table 1 shows that when 5:1 PC/PS LUVs include 1% PIP2, K increases 20-fold for Asp3Lys13Asp3 compared with just 6-fold for PLC-{zeta}-(374-385). Hence, the binding of Asp3Lys13Asp3 is ~3-fold stronger than the binding of PLC-{zeta}-(374-385) when PIP2 is incorporated into the vesicles. Electrostatic theory predicts that this difference should be 10-fold; an overprediction, but one that captures the essence of the phenomenon.


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  		TABLE 1
Binding of two peptides with a net charge of +7 to PC/PS and PC/PS/PIP2 LUVs

The molar partition coefficients, K, were obtained from centrifugation measurements similar to those illustrated in Fig. 4. We obtained paired results with the two peptides on the same sets of vesicles. Incorporating 1% PIP2 into the vesicles increased K 6-fold for PLC-{zeta}-(374-385) and 20-fold for Asp3Lys13Asp3; the difference is statistically significant. The solutions contained 100 mM KCl, 1 mM MOPS, pH 7.

 
These results agree well with complementary calculations based on the nonlinear Poisson-Boltzmann equation, an equation of classical electrostatics, using atomistic models of the peptides and membranes. This agreement suggests simple electrostatic interactions can explain our observations both that PLC-{zeta} binds to PC/PS vesicles with lower affinity than the PLC-{zeta}-(374-385) peptide, and that incorporating PIP2 into the vesicles produces a more significant effect on binding of the protein than the peptide. Electrostatic theory predicts the binding of Asp3Lys13Asp3 to PC/PS vesicles depends on the net charge of the peptide because the electrostatic potential at a given distance from the surface of a PC/PS vesicle is relatively constant, as shown in Fig. 5c of reference (55). In contrast, binding of PLC-{zeta}-(374-385) to ≥1 PIP2 in a membrane depends on the distribution of basic residues in its central region because this binding involves more local electrostatic interactions with the field produced by the polyvalent acidic lipid (see Fig. 5b of Ref. 55). Because the acidic residues are >1 Debye length (~1 nm under physiological conditions) from the center of the peptide interacting electrostatically with PIP2, they exert a minimal effect.

The ability of a membrane-bound cluster of basic residues to sequester PIP2 electrostatically in a membrane that also contains an excess of monovalent acidic lipid is distinct from the ability of PIP2 to enhance the binding of the basic peptide, as we discuss in detail elsewhere (38). Thus, we used FRET to examine directly whether a membrane-bound PLC-{zeta}-(374-385) can sequester PIP2 in PC/PS/PIP2 membranes.

PLC-{zeta}-(374-385) Sequesters PIP2 in Model Membranes—Previous work showed that peptides corresponding to basic clusters within several proteins produce a local positive electrostatic potential when they adsorb to a membrane, and that this potential enables the adsorbed peptides to sequester multivalent acidic lipids like PIP2 (28, 41, 47, 55). We examined whether the basic cluster in the PLC-{zeta} X-Y linker region can also laterally sequester PIP2, concentrating this substrate in the region of the catalytic domain. We used Bodipy-TMR-PIP2 and Texas Red-PLC-{zeta}-(374-385) as the donor and acceptor fluorphores, respectively, and made FRET measurements on LUVs. Fig. 5A illustrates that there is strong FRET between the probes even when the vesicles contain the monovalent acidic lipid PS in 300-fold excess over multivalent PIP2. The deconvoluted spectra show that stepwise increases in peptide concentration from 0 to 600 nM produce progressive quenching of the Bodipy-TMR-PIP2 fluorescence (Fig. 5B); conversely, the Texas Red-PLC-{zeta}-(374-385) fluorescence signal increases (Fig. 5C), indicating a FRET between the labeled PIP2 and peptide. Fig. 5D plots the % energy transfer as a function of peptide concentration: 50% energy transfer occurs at ~450 nM ([PIP2] = 750 nM). If we assume that 50% transfer corresponds to 50% sequestration of PIP2, each peptide sequesters approximately one PIP2. This is also predicted by our electrostatic calculations, as shown previously for Lys7 (47).


Figure 5
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  		FIGURE 5.
FRET from Bodipy-TMR-PIP2 to membrane-bound Texas Red PLC- {zeta}-(374-385). Adding Texas Red-labeled PLC-{zeta}-(374-385) to PC/PS/Bodipy-TMR-PIP2 (70:30:0.1) LUVs quenches the fluorescence of the Bodipy-TMR label and enhances the fluorescence of the Texas Red label. The lipid concentration (0.75 mM) is sufficiently high to bind most of the peptide to the membrane. A, representative corrected emission spectra for the peptide concentrations indicated. B and C show the deconvoluted curves for Bodipy-TMR-PIP2 and Texas Red-PLC-{zeta}-(374-385), respectively. As peptide concentration increases from 0 to 600 nM, the Bodipy-TMR-PIP2 fluorescence signal decreases (B) and the Texas Red signal increases (C). D, % energy transfer from Bodipy-TMR-PIP2 to Texas Red-PLC-{zeta}-(374-385) as the peptide concentration increases from 0 to 600 nM. We calculated the % energy transfer from B by using Equation 2. Each point on the graph is an average of ≥ five independent experiments (±S.D.). The solutions contained 100 mM KCl, 1 mM MOPS, pH 7.0. Bodipy-TMR-PIP2 was excited at 547 nm.

 
If a membrane-bound peptide sequesters PIP2 mainly due to electrostatics, increasing the salt concentration in the solution should screen the charges on the membrane, reducing sequestration, and hence energy transfer (38). FRET experiments show that increasing the salt from 100 to 200 mM KCl reduces energy transfer from 60 to 30% (data not shown). Our FRET results with PLC-{zeta}-(374-385) thus support the theoretical prediction that clusters of basic residues on proteins can act as basins of attraction for multivalent acidic lipids such as PIP2, enhancing their local concentration through nonspecific electrostatic interactions (28, 47, 55, 56). If the catalytic site is within a Debye length (~1 nm in a physiological salt solution; see Ref. 55) of the basic region in the intact protein, this electrostatic concentration effect could increase the local concentration of PIP2 significantly, which could in turn enhance the catalytic activity of the enzyme.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Membrane-anchoring of PLCs is biologically important because it enhances enzyme catalytic activity by exposing them to a higher local concentration of their PIP2 substrate (18, 19, 27). The well characterized PLC isoforms (PLC-beta,-{gamma},-{delta},-{epsilon}) interact with specific plasma membrane proteins or lipids. We investigated whether PLC-{zeta} binds strongly enough to lipids to anchor it to the bilayer component of the plasma (or possibly internal) membranes of eggs. The cytoplasmic leaflet of a typical mammalian plasma membrane comprises 20-30% monovalent acidic phospholipids, mainly PS. Native mouse PLC-{zeta} does not bind to 2:1 PC/PS membranes with sufficient affinity to anchor a significant fraction of enzyme to the bilayer component of biological membranes (Fig. 1B). Incorporating a physiological mol fraction of PIP2 (1%) into 2:1 PC/PS vesicles; however, enhances binding of the enzyme ~10-fold (Fig. 1B). The resulting affinity is strong enough to be biologically significant.

This enhancement in PLC-{zeta} binding upon incorporation of PIP2 is reminiscent of PLC-{delta}1, which has a PH domain that binds with high affinity and specificity to PI(4,5)P2 to form a stoichiometric 1:1 complex of known structure (25, 26). PLC-{zeta}, however, lacks a PH domain and our experiments show it does not distinguish between the 4,5 and 3,5 isoforms of PIP2 incorporated into lipid vesicles. This result is consistent with our working hypothesis that an unstructured cluster of basic residues in the X-Y linker region of PLC-{zeta} (Fig. 2B) helps anchor the protein to membranes through nonspecific electrostatic interactions. PLC-{delta}1 lacks this basic cluster in the X-Y linker region: as expected from our hypothesis, a PLC-{delta}1 fragment that lacks an intact PH domain (Fig. 2C) does not bind significantly to PC/PIP2 vesicles (57). Calculations of the electrostatic potential adjacent to homology models of mouse (Fig. 2) and human (supplemental materials) PLC-{zeta} reveal that the cluster of basic residues in the X-Y linker region is the most prominent positively charged region within PLC-{zeta}. PIP-strip assay binding measurements with a construct corresponding to the entire XY linker region of PLC-{zeta} (Fig. 3) demonstrated the apparent affinity of this fragment for polyvalent phosphoinositides over monovalent lipids, consistent with the results obtained with PLC-{zeta} (Fig. 1B). This supports the notion that this region of PLC-{zeta}, which is markedly distinct from PLC-{delta}1 in sequence alignment comparisons, may mediate specific protein-lipid interactions of this novel PLC. Importantly, Meyer and co-workers (35) recently reported that many Ras, Rab, Arf, and Rho proteins also use unstructured clusters of basic residues to anchor themselves to phosphoinositides in the plasma membrane.

We also studied the membrane binding of a peptide corresponding to the cluster of basic residues in the PLC-{zeta} X-Y linker region, PLC-{zeta}-(374-385), measuring its binding to LUVs composed of zwitterionic PC and the monovalent acidic lipids PS or PG (Fig. 4). Our results agree well with key predictions of electrostatic theory, as explained under supplemental materials. Comparing the binding of PLC-{zeta} and PLC-{zeta}-(374-385) reveals two notable differences: the enzyme binds less strongly to PC/PS vesicles than the peptide, and its binding affinity increases more strongly when the vesicles include PIP2.We postulated that both these effects could be due to the presence of acidic residues adjacent to the basic cluster in the enzyme (Fig. 2B), and our results with Asp3Lys13Asp3, a peptide comprising a basic core flanked by acidic residues, support this postulate.

We have not yet established whether the X-Y linker region is the main membrane anchor for intact PLC-{zeta}, and this region is likely to have several other functions; e.g., it produces a local positive electrostatic potential that may attract PIP2, laterally sequestering it in the region of the PLC-{zeta} catalytic site. Our FRET experiments (Fig. 5) show this is possible and suggest additional experimental investigations of this phenomenon may be fruitful. Additional experiments (supplemental material) demonstrate calcium/calmodulin can bind to a peptide corresponding to the basic/hydrophobic cluster in the X-Y linker region of human PLC-{zeta} sufficiently strongly (Kd = 10 nM) to remove the peptide from a membrane. The biological significance of this observation is not clear. Finally, we note that it is well established the basic cluster is also important for nuclear targeting and overlaps with a nuclear localization signal sequence, NLS (37, 58, 59). When injected into unfertilized mouse eggs, PLC-{zeta} was observed to undergo nuclear localization upon formation of the pronucleus; changing a single lysine or arginine residue to glutamate in the NLS (residues 376-381 of mouse PLC-{zeta}) disrupts translocation to the nucleus (36, 37, 58). These data suggest that the basic cluster in PLC-{zeta} plays an important role in its nuclear translocation after the egg enters interphase. Nuclear targeting sequences are important for controlling the activity of other enzymes that affect the level of PIP2 in cells; e.g. the yeast PI4,5P2 kinase, Mss4, becomes inactive upon nuclear translocation (60) and PLC-{delta}1 translocates into the nucleus in a Ca2+-dependent manner (61, 62). There is precedence for our suggestion that the basic cluster in PLC-{zeta} is a dual function motif that mediates both nuclear import and membrane binding. The Ste5 scaffolding protein also has a basic cluster that mediates both nuclear import and membrane binding (63).

Many important questions about the membrane binding of PLC-{zeta} remain unanswered. (i) Do the enzyme EF hand and C2 domains contribute to membrane binding? These domains help maintain the enzyme's high Ca2+ sensitivity (37, 39, 64), which is critical for its biological function, but their role in membrane binding is not well understood, despite extensive mutational studies (37). (ii) Does intracellular Ca2+ affect membrane binding (see the discussion of calcium/calmodulin binding under supplemental materials)? (iii) Is binding to lipids the main determinant of membrane anchoring, or do protein-protein interactions also contribute to the binding in cells? (iv) Where are the multivalent phosphoinositides (to which PLC-{zeta} may bind) located in mammalian eggs? Phosphoinositides are not distributed uniformly in the membranes of most eukaryotic cells: PI(3)P is concentrated on early endosomal membranes, PI(3,5)P2 is probably concentrated on late endosomal and lysosomal membranes, and PI(4)P is concentrated on the Golgi complex membranes (65). Analysis of many cell types, including mammalian eggs (59, 66), suggests PI(4,5)P2 is mainly on the plasma membrane and within the nucleus (67). These studies do not, of course, rule out the possibility that mammalian eggs have low but significant mol fractions of PI(4,5)P2 in other membranes (10). Because PLC-{zeta} interacts nonspecifically with polyvalent phosphoinositides, it could be anchored to both the plasma and internal membranes. (v) Perhaps most importantly, where does PLC-{zeta} reside within the egg prior to its translocation to the pronucleus? Fluorescently tagged PLC-{zeta} appears to be distributed uniformly throughout the cytoplasm of mouse eggs (37, 59); PLC-{delta}1, in contrast, localizes preferentially on the plasma membrane, presumably because it binds specifically to PI(4,5)P2 (59). These experiments, however, are unlikely to detect a minor fraction of PLC-{zeta} (e.g. 10%) bound to the plasma membrane. A PLC bound to the plasma membrane experiences ~1000-fold greater local concentration of PIP2 due to the ratio between the volume of the surface phase and the cell. (Specifically, if the thickness of the surface phase, d, is about the dimension of the PLC molecule, its volume = 4{pi}r2d where r is the radius of the spherical egg cell. The volume of the cell = (4/3){pi}r3; the ratio of these two volumes is r/(3d), which is >1000 if r > 10 µm and d = 3 nm.) Thus if 10% of the PLC-{zeta} is adsorbed to the membrane, it could hydrolyze PIP2 ~100-fold more efficiently than the 90% of the PLC-{zeta} in the cytoplasm. This suggests experiments to determine more accurately the membrane localization of PLC-{zeta} in eggs, and to investigate the factors that affect this membrane binding could provide important new information about how PLC-{zeta} functions in eggs.


   FOOTNOTES
 
* This work was supported in part by Grant GM24971 from the National Institutes of Health (to S. M.) and Grants MCB0212362 and MCB030028 for supercomputing resources (to D. M.) and an NSF Graduate Research Fellowship (to A. M.-N.) from the National Science Foundation, a grant from the ELWa Knowledge Exploitation Fund and Cardiff Partnership Fund (to A. L.), a grant from the Wellcome Trust (to K. S.), and a Cardiff School of Medicine PhD studentship (to M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Eq. S1. Back

1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661. Tel.: 631-444-3615; Fax: 631-444-3432; E-mail: stuart.mclaughlin{at}stonybrook.edu.

3 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PLC, phosphoinositide-specific phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; PH, plextrin homology; Ac, Acetyl; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; LUVs, large unilamellar vesicles; MLVs, multilamellar vesicles; SUVs, small unilamellar vesicles; PB, Poisson Boltzmann; FRET, fluorescence resonance energy transfer; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; GRASP, graphical representation and analysis of structural properties; PS, phosphatidylserine; PG, phosphatidylglycerol; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; DTT, dithiothreitol; DMF, dimethyl formamide; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Matilda Katan for the gift of the PLC-{delta}1 plasmid.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Stricker, S. A. (1999) Dev. Biol. 211, 157-176[CrossRef][Medline] [Order article via Infotrieve]
  2. Swann, K., Larman, M. G., Saunders, C. M., and Lai, F. A. (2004) Reproduction 127, 431-439[Abstract/Free Full Text]
  3. Kurokawa, M., Sato, K., and Fissore, R. A. (2004) Biol. Cell 96, 37-45[CrossRef][Medline] [Order article via Infotrieve]
  4. Malcuit, C., Kurokawa, M., and Fissore, R. A. (2006) J. Cell Physiol. 206, 565-573[CrossRef][Medline] [Order article via Infotrieve]
  5. Miyazaki, S., Shirakawa, H., Nakada, K., and Honda, Y. (1993) Dev. Biol. 158, 62-78[CrossRef][Medline] [Order article via Infotrieve]
  6. Brind, S., Swann, K., and Carroll, J. (2000) Dev. Biol. 223, 251-265[CrossRef][Medline] [Order article via Infotrieve]
  7. Runft, L. L., Jaffe, L. A., and Mehlmann, L. M. (2002) Dev. Biol. 245, 237-254[CrossRef][Medline] [Order article via Infotrieve]
  8. Jones, K. T., Cruttwell, C., Parrington, J., and Swann, K. (1998) FEBS Lett. 437, 297-300[CrossRef][Medline] [Order article via Infotrieve]
  9. Jones, K. T., Matsuda, M., Parrington, J., Katan, M., and Swann, K. (2000) Biochem. J. 346, 743-749[CrossRef][Medline] [Order article via Infotrieve]
  10. Rice, A., Parrington, J., Jones, K. T., and Swann, K. (2000) Dev. Biol. 228, 125-135[CrossRef][Medline] [Order article via Infotrieve]
  11. Wu, H., Smyth, J., Luzzi, V., Fukami, K., Takenawa, T., Black, S. L., Allbritton, N. L., and Fissore, R. A. (2001) Biol. Reprod. 64, 1338-1349[Abstract/Free Full Text]
  12. Parrington, J., Jones, M. L., Tunwell, R., Devader, C., Katan, M., and Swann, K. (2002) Reproduction 123, 31-39[Abstract]
  13. Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544[Abstract/Free Full Text]
  14. Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623[Abstract]
  15. Saunders, C. M., Swann, K., and Lai, F. A. (2007) Biochem. Soc. Symp. 23-36
  16. Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and Miyazaki, S. (2004) J. Biol. Chem. 279, 10408-10412[Abstract/Free Full Text]
  17. Fujimoto, S., Yoshida, N., Fukui, T., Amanai, M., Isobe, T., Itagaki, C., Izumi, T., and Perry, A. C. (2004) Dev. Biol. 274, 370-383[CrossRef][Medline] [Order article via Infotrieve]
  18. Rebecchi, M. J., and Pentyala, S. N. (2000) Physiol. Rev. 80, 1291-1335[Abstract/Free Full Text]
  19. Rhee, S. G. (2001) Annu. Rev. Biochem. 70, 281-312[CrossRef][Medline] [Order article via Infotrieve]
  20. Kelley, G. G., Reks, S. E., and Smrcka, A. V. (2004) Biochem. J. 378, 129-139[CrossRef][Medline] [Order article via Infotrieve]
  21. Kelley, G. G., Kaproth-Joslin, K. A., Reks, S. E., Smrcka, A. V., and Wojcikiewicz, R. J. (2006) J. Biol. Chem. 281, 2639-2648[Abstract/Free Full Text]
  22. Ferguson, K. M., Lemmon, M. A., Schlessinger, J., and Sigler, P. B. (1995) Cell 83, 1037-1046[CrossRef][Medline] [Order article via Infotrieve]
  23. Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10472-10476[Abstract/Free Full Text]
  24. Garcia, P., Gupta, R., Shah, S., Morris, A. J., Rudge, S. A., Scarlata, S., Petrova, V., McLaughlin, S., and Rebecchi, M. J. (1995) Biochemistry 34, 16228-16234[CrossRef][Medline] [Order article via Infotrieve]
  25. Lemmon, M. A., and Ferguson, K. M. (2000) Biochem. J. 350, 1-18[CrossRef][Medline] [Order article via Infotrieve]
  26. Lemmon, M. A. (2003) Traffic 4, 201-213[Medline] [Order article via Infotrieve]
  27. Kholodenko, B. N., Hoek, J. B., and Westerhoff, H. V. (2000) Trends Cell Biol. 10, 173-178[CrossRef][Medline] [Order article via Infotrieve]
  28. McLaughlin, S., Wang, J., Gambhir, A., and Murray, D. (2002) Annu. Rev. Biophys. Biomol. Struct. 31, 151-175[CrossRef][Medline] [Order article via Infotrieve]
  29. Essen, L. O., Perisic, O., Cheung, R., Katan, M., and Williams, R. L. (1996) Nature 380, 595-602[CrossRef][Medline] [Order article via Infotrieve]
  30. Murray, D., Hermida-Matsumoto, L., Buser, C. A., Tsang, J., Sigal, C. T., Ben-Tal, N., Honig, B., Resh, M. D., and McLaughlin, S. (1998) Biochemistry 37, 2145-2159[CrossRef][Medline] [Order article via Infotrieve]
  31. Hancock, J. F., Cadwallader, K., Paterson, H., and Marshall, C. J. (1991) EMBO J. 10, 4033-4039[Medline] [Order article via Infotrieve]
  32. Okeley, N. M., and Gelb, M. H. (2004) J. Biol. Chem. 279, 21833-21840[Abstract/Free Full Text]
  33. Cho, W., and Stahelin, R. V. (2005) Annu. Rev. Biophys. Biomol. Struct. 34, 119-151[CrossRef][Medline] [Order article via Infotrieve]
  34. Yeung, T., Terebiznik, M., Yu, L., Silvius, J., Abidi, W. M., Philips, M., Levine, T., Kapus, A., and Grinstein, S. (2006) Science 313, 347-351[Abstract/Free Full Text]
  35. Heo, W. D., Inoue, T., Park, W. S., Kim, M. L., Park, B. O., Wandless, T. J., and Meyer, T. (2006) Science 314, 1458-1461[Abstract/Free Full Text]
  36. Swann, K., Saunders, C. M., Rogers, N. T., and Lai, F. A. (2006) Semin. Cell Dev. Biol. 17, 264-273[CrossRef][Medline] [Order article via Infotrieve]
  37. Kuroda, K., Ito, M., Shikano, T., Awaji, T., Yoda, A., Takeuchi, H., Kinoshita, K., and Miyazaki, S. (2006) J. Biol. Chem. 281, 27794-27805[Abstract/Free Full Text]
  38. Gambhir, A., Hangyas-Mihalyne, G., Zaitseva, I., Cafiso, D. S., Wang, J., Murray, D., Pentyala, S. N., Smith, S. O., and McLaughlin, S. (2004) Biophys. J. 86, 2188-2207[Medline] [Order article via Infotrieve]
  39. Nomikos, M., Blayney, L. M., Larman, M. G., Campbell, K., Rossbach, A., Saunders, C. M., Swann, K., and Lai, F. A. (2005) J. Biol. Chem. 280, 31011-31018[Abstract/Free Full Text]
  40. Buser, C. A., and McLaughlin, S. (1998) Methods Mol. Biol. 84, 267-281[Medline] [Order article via Infotrieve]
  41. Wang, J., Gambhir, A., Hangyas-Mihalyne, G., Murray, D., Golebiewska, U., and McLaughlin, S. (2002) J. Biol. Chem. 277, 34401-34412[Abstract/Free Full Text]
  42. Wang, J., Arbuzova, A., Hangyas-Mihalyne, G., and McLaughlin, S. (2001) J. Biol. Chem. 276, 5012-5019[Abstract/Free Full Text]
  43. Peitzsch, R. M., and McLaughlin, S. (1993) Biochemistry 32, 10436-10443[CrossRef][Medline] [Order article via Infotrieve]
  44. Rebecchi, M., Peterson, A., and McLaughlin, S. (1992) Biochemistry 31, 12742-12747[CrossRef][Medline] [Order article via Infotrieve]
  45. Gallagher, K., and Sharp, K. (1998) Biophys J. 75, 769-776[Medline] [Order article via Infotrieve]
  46. Ben-Tal, N., Honig, B., Peitzsch, R. M., Denisov, G., and McLaughlin, S. (1996) Biophys. J. 71, 561-575[Medline] [Order article via Infotrieve]
  47. Wang, J., Gambhir, A., McLaughlin, S., and Murray, D. (2004) Biophys. J. 86, 1969-1986[Medline] [Order article via Infotrieve]
  48. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779-815[CrossRef][Medline] [Order article via Infotrieve]
  49. Petrey, D., Xiang, Z., Tang, C. L., Xie, L., Gimpelev, M., Mitros, T., Soto, C. S., Goldsmith-Fischman, S., Kernytsky, A., Schlessinger, A., Koh, I. Y., Alexov, E., and Honig, B. (2003) Proteins 53, Suppl. 6, 430-435[CrossRef][Medline] [Order article via Infotrieve]
  50. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281-296[CrossRef][Medline] [Order article via Infotrieve]
  51. Berney, C., and Danuser, G. (2003) Biophys. J. 84, 3992-4010[Medline] [Order article via Infotrieve]
  52. Grobler, J. A., and Hurley, J. H. (1996) Protein Sci. 5, 680-686[Medline] [Order article via Infotrieve]
  53. Downes, C. P., Gray, A., and Lucocq, J. M. (2005) Trends Cell Biol. 15, 259-268[CrossRef][Medline] [Order article via Infotrieve]
  54. Arbuzova, A., Wang, L., Wang, J., Hangyas-Mihalyne, G., Murray, D., Honig, B., and McLaughlin, S. (2000) Biochemistry 39, 10330-10339[CrossRef][Medline] [Order article via Infotrieve]
  55. McLaughlin, S., and Murray, D. (2005) Nature 438, 605-611[CrossRef][Medline] [Order article via Infotrieve]
  56. Murray, D., Arbuzova, A., Honig, B., and McLaughlin, S. (2002) in Current Topics in Membranes: Peptide-Lipid Interactions (Simon, S. A., and McIntosh, T. J., eds) pp. 277-307, Academic Press, San Diego, CA
  57. Cifuentes, M. E., Honkanen, L., and Rebecchi, M. J. (1993) J. Biol. Chem. 268, 11586-11593[Abstract/Free Full Text]
  58. Larman, M. G., Saunders, C. M., Carroll, J., Lai, F. A., and Swann, K. (2004) J. Cell Sci. 117, 2513-2521[Abstract/Free Full Text]
  59. Yoda, A., Oda, S., Shikano, T., Kouchi, Z., Awaji, T., Shirakawa, H., Kinoshita, K., and Miyazaki, S. (2004) Dev. Biol. 268, 245-257[CrossRef][Medline] [Order article via Infotrieve]
  60. Audhya, A., and Emr, S. D. (2003) EMBO J. 22, 4223-4236[CrossRef][Medline] [Order article via Infotrieve]
  61. Okada, M., Ishimoto, T., Naito, Y., Hirata, H., and Yagisawa, H. (2005) FEBS Lett. 579, 4949-4954[CrossRef][Medline] [Order article via Infotrieve]
  62. Stallings, J. D., Tall, E. G., Pentyala, S., and Rebecchi, M. J. (2005) J. Biol. Chem. 280, 22060-22069[Abstract/Free Full Text]
  63. Winters, M. J., Lamson, R. E., Nakanishi, H., Neiman, A. M., and Pryciak, P. M. (2005) Mol. Cell 20, 21-32[CrossRef][Medline] [Order article via Infotrieve]
  64. Kouchi, Z., Shikano, T., Nakamura, Y., Shirakawa, H., Fukami, K., and Miyazaki, S. (2005) J. Biol. Chem. 280, 21015-21021[Abstract/Free Full Text]
  65. Behnia, R., and Munro, S. (2005) Nature 438, 597-604[CrossRef][Medline] [Order article via Infotrieve]
  66. Halet, G., Tunwell, R., Balla, T., Swann, K., and Carroll, J. (2002) J. Cell Sci. 115, 2139-2149[Abstract/Free Full Text]
  67. Irvine, R. F. (2003) Nat. Rev. Mol. Cell. Biol. 4, 349-360[CrossRef][Medline] [Order article via Infotrieve]

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