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

Abstract

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...

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

“…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. This pore-forming model is consistent with early observations on mitochondria] electrolyte leakage, uncoupling of oxidative phosphorylation, and chan-nel formation in the presence of T-and PM-toxins (Matthews et Bervil-16 et al, 1984;Holden et al, 1985).…”

The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes

“…[4][5][6] A few CMS-causing proteins were effectively shown to moderately impair respiratory chain activity or to form large pores within the mitochondrial inner membrane, but how this correlates with pollen abortion remains elusive. [7][8][9][10][11] Male fertility can be restored by the expression of nuclearencoded genes called restorer of fertility (Rf ), which re-establish partial to normal pollen production to plants carrying a corresponding sterility-inducing cytoplasm. Fertility restoration is generally associated with a strong reduction in the production of mitochondrial CMS-inducing proteins.…”

Plant Mitochondrial Genome Evolution and Cytoplasmic Male Sterility

“…Novel and chimeric mitochondrial sequences are a frequent result of this recombination (Wise et al, 1987;Kennell and Pring, 1989;Wen and Chase, 1999;Gallagher et al, 2002;Okazaki et al, 2013;Yamagishi and Bhat, 2014;Tang et al, 2017), in which recombination sometimes leads to the creation of transcripts that interfere with normal male gametophyte development (Kitazaki and Kubo, 2010) via the generation of toxic and/or disruptive transmembrane proteins (Korth et al, 1991;Kim et al, 2007;Wan et al, 2007;Zhang et al, 2007;Yang et al, 2009;Gulyas et al, 2010;Jing et al, 2012;Flores-Renteria et al, 2013;Ji et al, 2013;Luo et al, 2013;Okazaki et al, 2013;Park et al, 2013;Hu et al, 2014). Surprisingly, such genes are not only abundant in many fertile plants, such as Arabidopsis thaliana Unseld et al, 1997), Beta vulgaris (Kubo et al, 2000), Oryza sativa (Notsu et al, 2002), Brassica napus (Handa, 2003), Zea mays (Clifton et al, 2004), Triticum aestivum (Ogihara et al, 2005), and Nicotiana tabacum (Sugiyama et al, 2005), but are also constitutively expressed.…”

“…Maladaptive retrograde signaling has also been implicated in mitochondrial interference with the rice nuclear gene COX11, resulting in the premature programmed cell death of tapetal cells and subsequent pollen abortion (Luo et al, 2013). Still, other species have the features and function of cytotoxic proteins (Dewey et al, 1987;Korth et al, 1991;Levings, 1993;Korth and Levings, 1993;Nakai et al, 1995;Duroc et al, 2005;Wang et al, 2006;Jing et al, 2012).…”

“…These proteins can directly interfere with energy production (Jing et al, 2012;Wang et al, 2013c;Wu et al, 2013), induce the release of cytochrome C via accumulation of unusually large numbers of reactive oxygen species (ROS) (Yan et al, 2014), and stimulate premature programmed cell death in male reproductive tissues (Luo et al, 2013;Horn et al, 2014). Several CMS proteins have demonstrated toxicity, such as URF13 in CMS-T maize (Korth et al, 1991), ORFH79 in HL-CMS rice , Orf507 in CMS pepper , and ROS homeostasis associated protein in cotton . Restoration of fertility can occur at the translational or post-translational level.…”