The calcium binding loops of the cytosolic phospholipase A2 C2 domain specify targeting to Golgi and ER in live cells.
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 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 cPLA2C2 on Syt1C2A and PKCalphaC2 domain backbones. In response to an intracellular calcium increase, PKCalphaC2 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 PKCalphaC2/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 PKCalphaC2 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.
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008961:Models, Structural; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium