Phospholipase Cζ binding to PtdIns(4,5)P2 requires the XY-linker region

Phospholipase C-zeta (PLC zeta) is a strong candidate for the mammalian sperm-derived factor that triggers the Ca2+ oscillations required for egg activation at fertilization. PLC zeta lacks a PH domain, which targets PLC delta 1 to the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) substrate in the plasma membrane. Previous studies failed to detect PLC zeta in the plasma membrane, hence the means of PLC zeta binding to PtdIns(4,5)P-2 is unclear. We find that the PLC zeta XY linker, but not the C2 domain, exhibits robust binding to PtdIns(4,5) P2 or to liposomes containing near-physiological levels of PtdIns(4,5)P-2. The role of positively charged residues within the XY linker was addressed by sequentially substituting alanines for three lysine residues, K374, K375 and K377. Microinjection of these mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLC zeta. The XY-linker mutant proteins were purified and the in vitro PtdIns(4,5)P-2 hydrolysis and binding properties were monitored. Successive reduction of net positive charge within the PLC zeta XY linker significantly affects both in vivo Ca2+-oscillation-inducing activity and in vitro PtdIns(4,5)P-2 interaction of mouse PLC zeta. Our data suggest that positively charged residues within the XY linker play an important role in the PLC. interaction with PtdIns(4,5)P-2, a crucial step in generating the Ca2+ activation signal that is essential for fertilization in mammals.