Abstract
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-delta1, but lacks the PH domain that anchors PLC-delta1 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-delta1. 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 5x10(3) 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.
Highlights
Phospholipase C- (PLC-) is a sperm-specific enzyme that initiates the Ca2؉ oscillations in mammalian eggs that activate embryo development
A peptide corresponding to the basic cluster within the linker region, phospholipase C (PLC)-(374 –385), binds to PC/PS vesicles with higher affinity than PLC-, but its binding is less sensitive to incorporating PIP2
Sequence alignment analysis of PLC isoforms indicates that PLC- has the greatest homology with PLC-␦1 (47% similarity, 3 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; PLC, phosphoinositide-specific phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; pleckstrin homology (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
Summary
Materials—We purchased 1-palmitoyl-2-oleoyl-sn-glycero3-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-glycero3-phosphatidylserine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)] (PG), and the triammonium salt of L-␣-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) from Avanti Polar Lipids (Alabaster, AL). [Inositol-2-3H]-L-␣phosphatidyl-D-myoinositol 4,5-bisphosphate ([3H]PIP2) and [ethyl-1, 2-3H]N-ethylmaleimide ([3H]NEM) were from PerkinElmer Life Sciences (Boston, MA). PIP2 Hydrolysis Assay—We used the centrifugation technique to measure the binding of catalytically active GSTPLC- to LUVs. We mixed ϳ5 nM GST-PLC- with sucroseloaded LUVs in a Ca2ϩ-free solution (100 mM KCl, 25 mM HEPES, 2 mM DTT, 100 M EGTA, pH 7.0) and collected the supernatant, which contained the unbound enzyme, following the procedure described above. 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), added recombinant GST-PLC- 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 used Super Signal West Dura (Pierce) to detect the HRP-coupled antibodies and a Bio-Rad Gel Doc system for image capture
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