Identification of a 50-kDa systemin-binding protein in tomato plasma membranes having Kex2p-like properties.
A protein of 50-kDa (SBP50) was identified in plasma membranes of tomato leaves which resembles proteases of the family of Kex2p-like prohormone convertases. To our knowledge, proteases of this class have not been reported in plants previously. A biotinylated derivative of systemin, the 18-aa polypeptide inducer of proteinase inhibitors in tomato and potato leaves, was bound by SBP50 with high specificity. When a systemin derivative was labeled with biotin at residue 8 and with [35S]methionine at position 15, the biotin moiety but not the radioactive label was bound by SBP50. At least 4 aa from the C terminus that included [35S]methionine were missing, indicating that proteolytic cleavage had occurred. Whereas residues in systemin most important for binding SBP50 appear to be located in the N-terminal half of the molecule, amino acids crucial for proteinase inhibitor induction are located within the C terminus. The residues important for binding include a cleavage site for furin, a member of the family of Kex2p-like prohormone-processing enzymes. Processing of systemin at the predicted furin cleavage site was confirmed in vitro. An antiserum against a Kex2p-like protease from Drosophila inhibited binding of biotinylsystemin to SBP50 and recognized a protein of about 60 kDa in Western blot analyses of tomato plasma membrane proteins. The data suggest a possible role for a membrane bound, furin-like protease in the mechanism of defense gene signaling by systemin.
Schaller
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Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Ryan
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CA
D000595:Amino Acid Sequence; D001710:Biotin; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018551:Solanum lycopersicum / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D043484:Proprotein Convertases; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; C073704:systemin; D001710:Biotin; D008715:Methionine; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae