To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.

The machinery that inserts mitochondrially encoded proteins into the inner membrane and translocates their hydrophilic domains through the membrane is poorly understood. We have developed a genetic screen for Saccharomyces cerevisiae mutants defective in this export process. Here we show that mutations in the nuclear gene PNT1 block the translocation of mitochondrially encoded fusion proteins across the inner membrane. Pnt1p is a mitochondrial integral inner membrane protein that appears to have two hydrophilic domains in the matrix, flanking a central hydrophobic hairpin-like anchor. While an S. cerevisiae pnt1 deletion mutant was more sensitive to H 2 O 2 than the wild type was, it was respiration competent and able to export wild-type Cox2p. However, deletion of the PNT1 orthologue from Kluyveromyces lactis, KlPNT1, caused a clear nonrespiratory phenotype, absence of cytochrome oxidase activity, and a defect in the assembly of KlCox2p that appears to be due to a block of C-tail export. Since PNT1 was previously described as a gene affecting resistance to the antibiotic pentamidine, our data support a mitochondrial target for this drug.

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semisterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the 0RF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that 0RF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.The mitochondrion, as a cellular center for energy metabolism, serves essential functions in the development of eukaryotic organisms. In plants, one of the developmental transitions that appears to be particularly influenced by mitochondrial function is male reproductive (pollen) development. Mutations in the mitochondrial genome most commonly result in the inability of the plant to shed viable pollen. This phenomenon, known as cytoplasmic male sterility (CMS) and observed in more than 150 plant species, has presented the opportunity to investigate the special functions provided by mitochondria at this crucial developmental stage.The association of CMS with abnormal mitochondrial gene expression has been established in many plant species including maize (1, 2), petunia (3, 4), sunflower (5-8), and common bean (9, 10). In all cases, pollen disruption in CMS lines is accompanied by the expression of novel mitochondrial DNA sequences that contain protein-coding open reading frames. To date, there are two important issues regarding CMS yet to be resolved. (i) Definitive evidence that these aberrant mitochondrial genes actually cause pollen disruption is not yet available in any plant system, and (ii) the molecular mechanisms effecting the disruption of pollen development are still not understood.In CMS common bean, male-sterile plants contain a novel mitochondrial DNA sequence (designated pvs) of unknown origin that is associated with the pollen sterility phenotype by various genetic criteria (10, 11). The 3.7-kb pvs sequence contains at least two open reading frames, ocf239 and orf98.

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A DNA marker detection strategy that allows the rapid, efficient resolution of high levels of polymorphism among closely related lines of common wheat (Triticum aestivum) has been developed to circumvent the apparent lack of restriction fragment length polymorphism in many important self-pollinated crop species. The technique of randomly amplified polymorphic DNA (RAPD) was combined with a denaturing gradient gel electrophoresis system (DGGE) to explore DNA sequence polymorphisms among different genotypes of wheat. Of the 65 primer combinations used for the polymerase chain reaction (PCR) amplifications, over 38% of them produced readily detectable and reproducible DNA polymorphisms between a spring wheat line, SO852, and a winter wheat variety, 'Clark'. A high level of polymorphism was observed among a number of commercial varieties and breeding lines of wheat. This procedure was also used to detect polymorphisms in a recombinant inbred population to test the feasibility of its application in genome mapping. This DNA polymorphism detection system provides an opportunity for pedigree analysis and fingerprinting of developed wheat lines as well as construction of a high density genetic map of wheat. Without the need for (32)P and sophisticated DNA extraction procedures, this approach should make it feasible to utilize marker-based selection in a plant breeding program.