The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Cζ, a mammalian egg-activating factor

Sperm-specific phospholipase C-zeta (PLC zeta) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLC zeta, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLC zeta mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374 - 381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg(376), Lys(377), Arg(378), Lys(379), or Lys(381) with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys(299) and/or Lys(301) in the C terminus of X domain, or Trp(13), Phe(14), or Val(18) in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. Ashort sequence 374 - 383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296 - 309 or 1 - 19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2 - 4 or C2 domain. Thus, PLC zeta is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLC delta 1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.