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 colt. Antibodies specific for the carboxyl terminus of URF13 and for an amino-terminal tag fused to URF13 in E. colU were used to determine the location of each end of the protein following protease treatments of right-sideout 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. coil 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.URF13 is a mitochondrially encoded 13-kDa protein uniquely associated with the inner mitochondrial membrane of maize carrying the Texas male-sterile cytoplasm (cms-T) (1-3). The DNA encoding URF13 (T-urfl3) arose by multiple recombinational events and contains nucleotide sequences derived from four disparate origins (4). The open reading frame is made up of sequences originating from coding and flanking regions of the mitochondrial 26S rRNA gene and a small region ofunknown origin. cms-Tmaize fails to produce viable pollen and is particularly susceptible to two fungal pathogens, Bipolaris maydis race T and Phyllosticta maydis. These pathogens caused widespread disease in the United States maize crop in 1969 and 1970 and effectively stopped the use of cms-T maize for the production of hybrid seed. Maize carrying normal cytoplasm is not seriously affected by these pathogens (reviewed in ref. 5). Isolated cms-T maize mitochondria exposed to specific toxins (T toxins) produced by these fungal pathogens exhibit swelling, inhibition of malatestimulated respiration, uncoupling of oxidative phosphorylation, and leakage of small molecules and ions (NAD+ and Ca2+). Identical effects are seen when cms-T mitochondria are exposed to methomyl, an insecticide structurally unrelated to T toxins (reviewed in ref. 6). The T toxin/URF13 interaction results in pore formation in the cms-T inner mitochondrial membrane (5).Escherichia coli expressing the cloned T-urfl3 gene product respond to T toxin or methomyl like cms-T mitochondria (7,8). This observation provides direct evidence that URF13 is responsible for susceptibility of cms-T maize to the fungal toxins. The analogous responses of cms-T maize mitochondria and E. coli to T toxins or methomyl suggest that URF13 has comparable structural and topographical properties in both membrane systems. P. maydis toxin has been shown to cooperatively bind to URF13 produced in E. coli. The binding is reversible, and T toxins and methomyl compete for the same or overlapping binding sites (9). A possible explanation for the cooperative binding is that URF13 exists in the membrane as a multimeric co...