The role of X/Y linker region and N-terminal EF-hand domain in nuclear translocation and Ca2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor.
Sperm-specific phospholipase C-zeta (PLCzeta) causes intracellular Ca(2+) oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCzeta, 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 PLCzeta 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 Ca(2+) 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. Ca(2+) oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short 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, PLCzeta 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 PLCdelta1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.
Kuroda
Keiji
K
Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo 162-8666, Japan.
Ito
Masahiko
M
Shikano
Tomohide
T
Awaji
Takeo
T
Yoda
Ayako
A
Takeuchi
Hiroyuki
H
Kinoshita
Katsuyuki
K
Miyazaki
Shunichi
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D020013:Calcium Signaling; D020134:Catalytic Domain; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D051379:Mice; D008958:Models, Molecular; D019913:Nuclear Localization Signals; D010063:Ovum / Q000502:physiology; D054801:Phosphoinositide Phospholipase C; D021381:Protein Transport; D010738:Type C Phospholipases / Q000737:chemistry* / Q000502:physiology
D019913:Nuclear Localization Signals; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C499234:Plcz1 protein, mouse