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Vol. 15, Issue 1, 371-383, January 2004

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

John H. Evans ^*, Stefan H. Gerber , Diana Murray , and Christina C. Leslie ^* 

^* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, and Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262; Department of Cardiology, University of Heidelberg, 69115 Heidelberg, Germany; and Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021

Submitted May 28, 2003; Revised August 6, 2003; Accepted August 26, 2003
Monitoring Editor: Vivek Malhotra

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 C C2 (PKCC2) 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 PKCC2 domain backbones. In response to an intracellular calcium increase, PKCC2 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 PKCC2/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 PKCC2 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.

Abbreviations used: BFA, brefeldin A; cPLA₂, cytosolic phospholipase A₂; FP, fluorescent protein; GT, 1,4 galactosyltransferase; IONO, ionomycin; PC, phosphatidylcholine; PH, pleckstrin homology; PIP₂, phosphatidylinositol-4,5-bisphosphate; PS, phosphatidylserine; Syt1C2A, synaptotagmin I C2A.

Online version of this article contains videos for some figures. Online version is available at www.molbiolcell.org.

Corresponding author. E-mail address: lesliec{at}njc.org.

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