Cross-linking and disulfide bond formation of introduced cysteine residues suggest a modified model for the tertiary structure of URF13 in the pore-forming oligomers.

URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea mays L.) carrying the cms-T cytoplasm. This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogens Bipolaris maydis race T and Phyllosticta maydis. URF13 contains three transmembrane alpha-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers in cms-T maize mitochondria and when expressed in Escherichia coli cells. To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking. We introduced Cys residues individually into consecutive positions 78-82, believed to be in helix III. We expressed these proteins in E. coli cells and tested for cross-linking through disulfide bond formation or by using Cys-Cys cross-linkers. URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys-Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules. Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers. Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers. Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys-Cys cross-linker) and N,N'-dicyclohexylcarbodiimide (Lys-Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers.

Functional analysis of a recently originating, atypical presequence: mitochondrial import and processing of GUS fusion proteins in transgenic tobacco and yeast.

A gene family of at least five members encodes the tobacco mitochondrial Rieske Fe-S protein (RISP). To determine whether all five RISPs are translocated to mitochondria, fusion proteins containing the putative presequences of tobacco RISPs and Escherichia coli beta-glucuronidase (GUS) were expressed in transgenic tobacco, and the resultant GUS proteins were localized by cell fractionation. The amino-terminal 75 and 71 residues of RISP2 and RISP3, respectively, directed GUS import into mitochondria, where fusion protein processing occurred. The amino-terminal sequence of RISP4, which contains an atypical mitochondrial presequence, can translocate the GUS protein specifically into tobacco mitochondria with apparently low efficiency. Consistent with the proposal of a conserved mechanism for protein import in plants and fungi, the tobacco RISP3 and RISP4 presequences can direct import and processing of a GUS fusion protein in yeast mitochondria. Plant presequences, however, direct mitochondrial import in yeast less efficiently than the yeast presequence, indicating subtle differences between the plant and yeast mitochondrial import machineries. Our studies show that import of RISP4 may not require positively charged amino acid residues and an amphipathic secondary structure; however, these structural properties may improve the efficiency of mitochondrial import.

URF13, a ligand-gated, pore-forming receptor for T-toxin in the inner membrane of cms-T mitochondria.

URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis. Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation. Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E. coli cells. The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria.

The relationship between the mitochondrial gene T-urf13 and fungal pathotoxin sensitivity in maize.

Mitochondria isolated from maize containing cms-T cytoplasm are specifically sensitive to pathotoxins (T-toxins) produced by the fungi Bipolaris maydis race T and Phyllosticta maydis. T-toxins interact with a 13 kDa membrane-bound toxin receptor protein, URF13, to produce hydrophillic pores in the membrane. Expression of URF13 in Escherichia coli produces bacterial cells that form hydrophillic pores in the plasma membrane when exposed to T-toxin or methomyl. Topological studies have established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and may contribute to pore formation. URF13 specifically binds T-toxin in a cooperative manner. Oligonucleotide-directed mutagenesis of URF13 led to the isolation of methomyl/T-toxin-resistant mutations at 39 separate positions throughout the URF13 primary sequence. Chemical cross-linking of URF13 demonstrated the presence of URF13 oligomers and established that the pore-forming species is oligomeric. The ability of the carboxylate-specific reagent, dicyclohexycarbodiimide to cross-link URF13 has been used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle that may undergo a conformational change after T-toxin or methomyl binding. Experimental evidence indicates that URF13 acts as a ligand-gated, pore-forming T-toxin receptor.

Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration.

The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility. We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species. The coding sequence of T-urf13 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Plants expressing high levels of URF13 were methomyl sensitive. Subcellular analysis indicated that URF13 is mainly associated with the mitochondria. Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity. Most control plants, which expressed the T-urf13c construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction. Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria. No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria. Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria. There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.

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.

Flower-enhanced expression of a nuclear-encoded mitochondrial respiratory protein is associated with changes in mitochondrion number.

The mitochondrial Rieske iron-sulfur protein is an obligatory component of the respiratory electron transport chain that is encoded by a single-copy gene in mammals and fungi. In contrast, this protein is encoded by a small gene family in dicotyledonous tobacco and monocotyledonous maize. We cloned four cDNAs from tobacco that encode the mitochondrial Rieske iron-sulfur protein. These clones, along with a previously isolated cDNA, represent five independent members of the gene family that can be divided into three subfamilies. All of these genes were derived from the two progenitor species and were expressed in amphidiploid tobacco. The proteins encoded by these five genes are probably functional because they all contain the universally conserved hexyl peptides necessary for the 2Fe-2S cluster formation. The expression of the Rieske protein gene family is differentially regulated; a 6- to 11-fold higher level of steady state transcripts was found in flowers than in leaves, stems, and roots. Members of at least two subfamilies were preferentially expressed in flowers, indicating that they share a common cis-regulatory element(s), which can respond to a flower-specific signal(s). Although approximately 10 times more transcripts occurred in flowers than in leaves, flower and leaf mitochondria contained a similar amount of the Rieske protein. Flowers, however, contained seven times more Rieske proteins than leaves. These results indicated an increase in mitochondrion number in flowers. High-energy demands during anther development might bring about an increase in mitochondrion numbers in flowers and the flower-enhanced expression of the Rieske protein gene family. Our results suggested that nuclear genes encoding mitochondrial respiratory proteins could sense and respond to changes in energy metabolism and/or changes in mitochondrion numbers.

The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein.

We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide. The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria. A mitochondrial polypeptide with an apparent molecular weight of 21,000 was detected with a polyclonal antibody raised against an ORF221 fusion protein. In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria. The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.

Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.

RNA editing of a chimeric maize mitochondrial gene transcript is sequence specific.

RNA editing was analysed in the mitochondrial ATPase complex subunit 6 gene (atp6) transcripts of the C male-sterile cytoplasm (cms-C) of maize. The only copy of atp6 in cms-C, designated C-atp6, is a triple gene fusion product comprised of DNA sequences derived from atp9, atp6, and an unknown origin. Sequences of cDNAs revealed 19 C to U alterations resulting in 16 amino acid residue changes compared to the genomic sequence. The only C to U edit in the 39-nucleotide sequence similar to atp9 was comparable to a change in the complete atp9 mRNAs of Petunia, Oenothera, wheat, and sorghum. The 442 nucleotides of unknown origin were not edited. The 18 editing events within the atp6 homologous region were similar to those in the atp6 transcripts of sorghum. RNA editing in maize C-atp6 transcripts introduces a translational stop codon at the same position where it is created by editing in sorghum and Oenothera atp6 mRNAs and is already present in atp6 open reading frames of most other plant and non-plant organisms. Our results, along with other reports on editing in chimeric transcripts, indicate that RNA editing is not influenced by rearrangements but instead is sequence specific.

Functional analysis in yeast of cDNA coding for the mitochondrial Rieske iron-sulfur protein of higher plants.

cDNA clones coding for the nuclear-encoded mitochondrial Rieske iron-sulfur protein (RISP) have been isolated from maize and tobacco. Complementation analysis of hybrid proteins consisting of different domains of plant and yeast RISPs showed that the carboxyl two-thirds of the plant protein is functionally equivalent to that of the yeast protein. The amino terminus of the RISP, however, seems to be species specific because this region is not interchangeable between plant and yeast proteins. Complementation analysis of hybrid proteins also identified a structurally conserved domain probably essential for the function of bc1 complex RISPs. A specific domain from the plant RISP was found to cause temperature-sensitive respiratory growth in yeast. We have demonstrated that yeast can serve as a model system to study the structural and functional relationships of plant gene products that are enzymatic components of the mitochondrial respiratory chain.

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.

Chimeric mitochondrial genes expressed in the C male-sterile cytoplasm of maize.

Aberrant recombinations involving the mitochondrial atp9, atp6 and coxII genes have created unique chimeric sequences in the C male-sterile cytoplasm (cms-C) of maize. An apparent consequence of the rearrangements is the interchanging of transcriptional and/or translational regulatory signals for these genes, and alterations in the reading frames encoding the atp6 and coxII genes in the C cytoplasm. Particularly unusual is the organization of the atp6 gene in cms-C mitochondria, designated atp6-C. The atp6-C sequence is a triple gene fusion product comprised of DNAs derived from atp9, atp6 and an open reading frame of unknown origin. Although there is no direct evidence indicating that these chimeric genes are responsible for the cytoplasmic male sterility (cms) trait, their novel arrangements and the strong correlation between these genes and the C type of male sterility suggest such a role.

The protein-encoding gene T-urf13 is not edited in maize mitochondria.

RNA editing of T-urf13, a gene specific to the mitochondria of cytoplasmic male-sterile, type-T (cms-T) maize, and an adjacent, cotranscribed gene orf221, have been studied by cDNA sequencing. No editing was detected in 22 cDNA clones. This is the only report of a polypeptide-encoding gene in higher-plant mitochondria that is not edited. T-urf13 may not be edited because it is derived largely from the coding and flanking regions, which are rarely edited, of a ribosomal RNA gene. orf221 is edited; however, the similarity between the predicted amino acid sequences of orf221 in cms-T and normal cytoplasms is not increased.

The Texas cytoplasm of maize: cytoplasmic male sterility and disease susceptibility.

The Texas cytoplasm of maize carries two cytoplasmically inherited traits, male sterility and disease susceptibility, which have been of great interest both for basic research and plant breeding. The two traits are inseparable and are associated with an unusual mitochondrial gene, T-urf13, which encodes a 13-kilodalton polypeptide (URF13). An interaction between fungal toxins and URF13, which results in permeabilization of the inner mitochondrial membrane, accounts for the specific susceptibility to the fungal pathogens.