LeRALF, a plant peptide that regulates root growth and development, specifically binds to 25 and 120 kDa cell surface membrane proteins of Lycopersicon peruvianum.
A photoaffinity analog of tomato leaf RALF peptide (LeRALF), (125)I-azido-LeRALF, bound saturably to tomato suspension cultured cells in the dark in a classical receptor binding assay. Classical kinetic analyses revealed that the analog interacted with a single binding site on the surface of the cells with a KD of 0.8x10(-9) M, typical of known peptide hormone-receptor interactions in both plants and animals. The (125)I-azido-LeRALF, when exposed to UVB light in the presence of the cells, strongly labeled only two proteins of 25 kDa and 120 kDa, with the 25 kDa protein being more strongly labeled than the 120 kDa protein. The cell-surface localization of the interaction was indicated, as suramin, a known inhibitor of peptide-receptor interactions, and native LeRALF peptide competed with (125)I-azido-LeRALF labeling of both proteins. Two biologically inactive LeRALF analogs were not competitors. Incubation of (125)I-azido-LeRALF with suspension cultured cells in the dark, where it was fully active, could subsequently be totally dissociated from cells by acid washes, indicating that it was interacting at the cell surface and was not internalized. The (125)I-azido-LeRALF-labeled 25 kDa and 120 kDa proteins could not be solubilized from cell membranes by methods that release peripheral proteins, indicating that they are integral membrane components. The cumulative kinetic and biochemical evidence strongly indicates that the two proteins may be components of a LeRALF receptor complex.
Scheer
Justin M
JM
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Pearce
Gregory
G
Ryan
Clarence A
CA
D002473:Cell Wall / Q000502:physiology; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D018551:Solanum lycopersicum / Q000502:physiology*; D000455:Medicago sativa; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D010940:Plant Proteins / Q000378:metabolism / Q000502:physiology*; D018517:Plant Roots / Q000254:growth & development*; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000502:physiology; D015398:Signal Transduction
D008565:Membrane Proteins; D010940:Plant Proteins; D011956:Receptors, Cell Surface