The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes.
Cytosolic phospholipase A2 (cPLA2) catalyzes release of arachidonic acid from membranes following translocation to Golgi and endoplasmic reticulum. In response to an intracellular calcium concentration ([Ca2+]i) increase, the C2 domain binds Ca2+ and brings the catalytic domain into proximity with its phospholipid substrate. Because membrane residence is important in the regulation of cPLA2 activity, we explored the contributions of the C2 and catalytic domains in mediating membrane residence using an imaging approach in live cells with fluorescent protein chimeras of cPLA2. The isolated cPLA2 C2 domain associated with Golgi membranes rapidly in proportion to the [Ca2+]i, allowing for its use as a [Ca2+]i indicator. cPLA2 association with Golgi was slower than the isolated C2 domain in response to a [Ca2+]i increase. After [Ca2+]i decrease, cPLA2 remained associated with membrane in a Ca(2+)-independent fashion whereas C2 domain rapidly dissociated. Ca(2+)-independent membrane association was greatly reduced by mutation of Trp464, located at the membrane-exposed face of the catalytic domain, to Gly or Ala. Mutation of Trp464 to Phe supported Ca(2+)-independent association similar to wild type. These results demonstrate a role for the cPLA2 catalytic domain in regulating membrane association and membrane residence time.
Evans
John H
JH
Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Leslie
Christina C
CC
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002122:Calcium Chloride / Q000737:chemistry; D020134:Catalytic Domain; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009991:Oscillometry; D018408:Patch-Clamp Techniques; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry
D014364:Tryptophan; D010741:Phospholipases A; D054467:Phospholipases A2; D002122:Calcium Chloride; D002118:Calcium