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.

URF13 is a mitochondrially encoded, integral membrane protein found only in maize carrying the cms-T cytoplasm. URF13 is associated with cytoplasmic male sterility, Texas type, and causes susceptibility to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 is predicted to contain three transmembrane alpha-helices and is a receptor for the pathotoxins (T-toxins) produced by B. maydis race T and P. maydis. Binding of T-toxin to URF13 leads to membrane permeability. Cross-linking of URF13 oligomers with N,N'-dicyclohexylcarbodiimide (DCCD) protects Escherichia coli cells expressing URF13 and cms-T mitochondria from the permeability caused by T-toxin or methomyl. Using mutated forms of URF13 expressed in E. coli cells, we determined the molecular mechanism of DCCD protection. We separately changed Lys-37 in helix II to isoleucine (K37I-URF13) and Lys-32 in the helix I/helix II loop region to alanine (K32A-URF13). DCCD treatment of K37I-URF13-expressing cells did not protect the cells from permeability caused by T-toxin or methomyl. DCCD cross-linking was greatly reduced in K37I-URF13 and in D39V-URF13-expressing cells, but it was unaffected in K32A-URF13-expressing cells. Binding of methomyl or T-toxin decreases DCCD cross-linking of URF13 oligomers expressed in either E. coli or cms-T mitochondria. We conclude that Asp-39 in helix II is cross-linked by DCCD to Lys-37 in helix II of an adjacent URF13 molecule and that this cross-linking protects against toxin-mediated permeabilization. Our results also indicate that helices II form a central core in URF13 oligomers.

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.

URF13, a maize mitochondrial pore-forming protein, is oligomeric and has a mixed orientation in Escherichia coli plasma membranes.

URF13, an inner mitochondrial membrane protein of the maize Texas male-sterile cytoplasm (cms-T), has one orientation in the inner membrane of maize mitochondria but two topological orientations in the plasma membrane when expressed in Escherichia coli. Antibodies specific for the carboxyl terminus of URF13 and for an amino-terminal tag fused to URF13 in E. coli were used to determine the location of each end of the protein following protease treatments of right-side-out and inside-out vesicles derived from cms-T mitochondria and the E. coli plasma membrane. Cross-linking studies indicate that a portion of the URF13 population in mitochondria and E. coli exists in membranes in an oligomeric state and, in combination with proteolysis studies, show that individual subunits within a given multimer have the same orientation. A three-membrane-spanning helical model for URF13 topology is presented.