Fungal Toxins Bind to the URF13 Protein in Maize Mitochondria and Escherichia coli

Abstract

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13.

The Genetics, Pathology, and Molecular Biology of T-Cytoplasm Male Sterility in Maize

“…[3H]PM-toxin binds to wild-type URF13 in E. coli, but not to the 82-amino-acid truncated URF13 (Braun et al, 1990). This is consistent with the toxin sensitivity phenotypes in cms-T maize and insensitivity in the T-4 mutant.…”

“…There is evidence that restoration can reduce the sensitivity of T-cytoplasm mitochondria to T-toxin (Watrud et al, 1975a;Barratt and Flavell, 1975). This is likely due to the ability of Rfl to reduce URF13 levels in the mitochondria and thus reduce the amount of substrate available for toxin binding (Braun et al, 1990;Hack et al, 1991). However, this reduction in mitochondrial sensitivity to the toxins does not provide significant resistance to the fungi (as evidenced by the 1970 epidemic on restored T-cytoplasm maize).…”

“…Based on this finding, it was proposed that amino acids 2 through 11 are unnecessary for toxin binding, but essential for the toxin-URFl3 interaction that leads to membrane permeabilization. Hence, the binding to URF13 in E. coli cells is cooperative, suggesting that URF13 exists as oligomers in E. coli membranes (Braun et al, 1990). Based on the additional studies of Korth et al (1991) and Rhoades et al (1994), it has now been postulated that URF13 monomers containing three trans-membrane a-helices interact with T-toxin (or methomyl) and are assembled as tetrameric pore-forming structures spanning the inner mitochondria1 membrane.…”

N,N'-dicyclohexylcarbodiimide cross-linking suggests a central core of helices II in oligomers of URF13, the pore-forming T-toxin receptor of cms-T maize mitochondria.

“…Protease accessibility studies support this postulated topography of URF13 in the membrane (9). Escherichia coli cells that express URF13 are also sensitive to T-toxin, and the effects of the toxin are similar to those observed when T-toxin interacts with isolated cms-T mitochondria (10)(11)(12). When methomyl, a compound structurally unrelated to T-toxin, is added to cms-T mitochondria or E. coli cells expressing URF13, the effects are the same as when T-toxin is added (12).…”

“…When methomyl, a compound structurally unrelated to T-toxin, is added to cms-T mitochondria or E. coli cells expressing URF13, the effects are the same as when T-toxin is added (12). Radiolabeled Pm-toxin binds specifically to cms-T mitochondria and to E. coli cells expressing URF13 (11). The binding to URF13 in E. coli cells is cooperative, suggesting that URF13 exists as oligomers in E. coli membranes (11).…”