title: Signal transfer in the plant plasma membrane: phospholipase A(2) is regulated via an inhibitory Gα protein and a cyclophilin.
abstract: The plasma membrane of the California poppy is known to harbour a PLA2 (phospholipase A2) that is associated with the Gα protein which facilitates its activation by a yeast glycoprotein, thereby eliciting the biosynthesis of phytoalexins. To understand the functional architecture of the protein complex, we titrated purified plasma membranes with the Gα protein (native or recombinant) and found that critical amounts of this subunit keep PLA2 in a low-activity state from which it is released either by elicitor plus GTP or by raising the Gα concentration, which probably causes oligomerization of Gα, as supported by FRET (fluorescence resonance energy transfer)-orientated fluorescence imaging and a semiquantitative split-ubiquitin assay. All effects of Gα were blocked by specific antibodies. A low-Gα mutant showed elevated PLA2 activity and lacked the GTP-dependent stimulation by elicitor, but regained this capability after pre-incubation with Gα. The inhibition by Gα and the GTP-dependent stimulation of PLA2 were diminished by inhibitors of peptidylprolyl cis-trans isomerases. A cyclophilin was identified by sequence in the plasma membrane and in immunoprecipitates with anti-Gα antibodies. We conclude that soluble and target-associated Gα interact at the plasma membrane to build complexes of varying architecture and signal amplification. Protein-folding activity is probably required to convey conformational transitions from Gα to its target PLA2.
authors.affiliation: Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, Department of Molecular Cell Biology, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Heinze
authors.forename: Madeleine
authors.initials: M
authors.lastname: Herre
authors.forename: Carolin
authors.initials: C
authors.lastname: Massalski
authors.forename: Isabella
authors.initials: I
authors.lastname: Hermann
authors.forename: Udo
authors.initials: U
authors.lastname: Conrad
authors.forename: Werner
authors.initials: W
authors.lastname: Roos
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation; D031675:Eschscholzia / Q000201:enzymology / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D014018:Tissue Distribution
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D054467:Phospholipases A2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D021983:Cyclophilins