The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells

Translocation of cytosolic phospholipase A(2) (cPLA,) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA(2)C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (SytIC2A) and protein kinase Calpha C2 (PKCalphaC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA(2)C2 on Syt1C2A and PKCalphaC2 domain backbones. In response to an intracellular calcium increase, PKCaC2 targeted plasma membrane regions rich in phosphatidylinositol4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the SytlC2A/cPLA(2)C2 and PKCalphaC2/cPLA(2)C2 hybrids targeted Golgi/ER and colocalized with cPLA(2)C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA(2)C2, but not PKCalphaC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA(2)C2 and the hybrid domains.