Cross-linking of the cms-T maize mitochondrial pore-forming protein URF13 by N,N'-dicyclohexylcarbodiimide and its effect on URF13 sensitivity to fungal toxins.
URF13 is a membrane protein unique to mitochondria from maize having the Texas male-sterile cytoplasm (cms-T), which is capable of permeabilizing biological membranes in the presence of a family of pathotoxins (T-toxins) produced by certain fungi or the insecticide methomyl. The carboxylate-specific reagent dicyclohexylcarbodiimide has been shown previously to protect URF13-containing membranes against the permeabilizing effects of added T-toxin or methomyl. Dicyclohexylcarbodiimide was found to covalently cross-link URF13 into higher order oligomers, including dimers, trimers, and tetramers, in isolated cms-T mitochondria and Escherichia coli cells expressing URF13. In intact E. coli cells and isolated spheroplasts, the observed protection against the effects of methomyl was not associated with the appearance of dimers but was correlated with the appearance of cross-linked trimers and tetramers. Following treatment of E. coli cells expressing URF13 with dicyclohexylcarbodiimide, the specific binding of tritiated T-toxin was reduced by 50% and all binding cooperativity was lost. A similar decrease in the level of T-toxin binding and loss of binding cooperativity were observed with site-directed, T-toxin-insensitive URF13 mutants at aspartate 39, the residue known to undergo reaction with dicyclohexylcarbodiimide. When coupled with a postulated three membrane-spanning helical model of URF13, these results provide initial insights into the intermolecular interactions involved in URF13 oligomer formation.
Kaspi
C I
CI
Department of Botany, Duke University, Durham, North Carolina 27706.
Siedow
J N
JN
D001224:Aspartic Acid / Q000378:metabolism; D015153:Blotting, Western; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004926:Escherichia coli; D008724:Methomyl / Q000633:toxicity; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins; D009154:Mutation; D009183:Mycotoxins / Q000378:metabolism / Q000633:toxicity*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D003313:Zea mays
D003432:Cross-Linking Reagents; D024101:Mitochondrial Proteins; D009183:Mycotoxins; D010940:Plant Proteins; C071393:URF13 protein, Zea mays; D008724:Methomyl; D001224:Aspartic Acid; D004024:Dicyclohexylcarbodiimide