Membrane targeting of C2 domains of phospholipase C-δ isoforms

The C2 domain is a Ca2+-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca2+-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca2+, Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca2+-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca2+-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca2+-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca2+-binding loops and the substitution of Ser for the Ca2+-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique prelocalization to the plasma membrane prior to Ca2+ import and non-selective Ca2+-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca2+-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.