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PMID: 13679516 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Molecular biology of the cell ·Vol. 15 ·No. 1 ·2004-01-00 ·Pages 371-83

Evans JH, Gerber SH, Murray D, Leslie CC

Abstract

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.

MeSH Terms
Amino Acid Sequence Animals Calcium/metabolism Calcium-Binding Proteins Cells, Cultured Dogs Endoplasmic Reticulum/metabolism Golgi Apparatus/metabolism Membrane Glycoproteins/metabolism Microscopy, Fluorescence Models, Structural Molecular Sequence Data Nerve Tissue Proteins/metabolism Phosphatidylinositol 4,5-Diphosphate/metabolism Phospholipases A/metabolism Phospholipases A2 Protein Binding Protein Kinase C/metabolism Protein Kinase C-alpha Protein Structure, Tertiary/physiology Protein Transport/physiology Recombinant Fusion Proteins/metabolism Sequence Analysis, Protein Synaptotagmin I Synaptotagmins
Chemicals
Calcium-Binding Proteins Membrane Glycoproteins Nerve Tissue Proteins Phosphatidylinositol 4,5-Diphosphate Recombinant Fusion Proteins Synaptotagmin I Synaptotagmins Protein Kinase C Protein Kinase C-alpha Phospholipases A Phospholipases A2 Calcium
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Evans John H
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Gerber Stefan H
Murray Diana
Leslie Christina C
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e03-05-0338
Published
2004-01-00
Epub
2003-00-17
Pages
371-83
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC307554
Subset
IM
Grants
NIGMS NIH HHS · R01 GM066147 · United States
NHLBI NIH HHS · P01 HL034303 · United States
NHLBI NIH HHS · HL-61378 · United States
NIGMS NIH HHS · GM-66147 · United States
NHLBI NIH HHS · F32 HL010507 · United States
NHLBI NIH HHS · HL-34303 · United States
NHLBI NIH HHS · R01 HL061378 · United States
External Links
PubMed source DOI: 10.1091/mbc.e03-05-0338 PMC Full Text View journal details
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