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...

No abstract

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 Phylosticta maydis. URF13 is predicted to contain three transmembrane a-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'-dicyclohexylcarbodlimide (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-linkin 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.Maize plants (Zea mays L.) that exhibit Texas cytoplasmic male sterility (cms-T) are unable to produce pollen and are specifically susceptible to the fungal pathogens Bipolaris maydis race T and Phyllosticta maydis (1, 2). Early experiments established that mitochondria isolated from cms-T maize plants and exposed to the host-specific toxins (Ttoxins) produced by B. maydis race T or P. maydis (BmTtoxin and Pm-toxin, respectively) exhibit rapid swelling, uncoupling of oxidative phosphorylation, inhibition of malate-driven respiration, and leakage of NAD+ and other ions (1-5).A 13-kDa protein, URF13, is the product of the mitochondrial gene T-urfl3, which is specific to cms-T maize (6). URF13 is located in the inner mitochondrial membrane of cms-T maize plants (7,8) and is predicted to contain three transmembrane a-helices (Fig. 1), two of which (helices II and III) are amphipathic (1,2). 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). Radiolabeled Pm-toxin binds specifically to cms-T mitochondria and to E...

No abstract