Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.
Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).
Heinze
Michael
M
Department of Molecular Cell Biology, Institute of Pharmaceutical Biology and Pharmacology, Martin-Luther-University, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Steighardt
Jörg
J
Gesell
Andreas
A
Schwartze
Wieland
W
Roos
Werner
W
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D031675:Eschscholzia / Q000235:genetics / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D054467:Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D044385:GTP-Binding Protein alpha Subunits; D010940:Plant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D054467:Phospholipases A2