The Calcium Binding Loops of the Cytosolic Phospholipase A2 C2 Domain Specify Targeting to Golgi and ER in Live Cells

摘要

Translocation of cytosolic phospholipase A₂ (cPLA₂) 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 cPLA₂C2 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 (Syt1C2A) and protein kinase Cα C2 (PKCαC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA₂C2 on Syt1C2A and PKCαC2 domain backbones. In response to an intracellular calcium increase, PKCαC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-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 Syt1C2A/cPLA₂C2 and PKCαC2/cPLA₂C2 hybrids targeted Golgi/ER and colocalized with cPLA₂C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA₂C2, but not PKCαC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA₂C2 and the hybrid domains.