Mutations affecting a yeast mitochondrial inner membrane protein, Pnt1p, block export of a mitochondrially synthesized fusion protein from the matrix

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 ce revisiae mutants defective in this export process. The screen is based on t he fact that the hydrophilic polypeptide Arg8(m)p is exported from the matr ix if it is synthesized within mitochondria as a bifunctional Cox2p-Arg8(m) p fusion protein. Since export of Arg8(m)p causes an Arg(-) phenotype, defe ctive mutants can be selected as Arg(+). Here we show that mutations in the nuclear gene PNT1 block the translocation of mitochondrially encoded fusio n proteins across the inner membrane. Pnt1p is a mitochondrial integral inn er membrane protein that appears to have two hydrophilic domains in the mat rix, flanking a central hydrophobic hairpin-like anchor. While an S. cerevi siae pnt1 deletion mutant was more sensitive to H2O2 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, a nd a defect in the assembly of KICox2p that appears to be due to a block of C-tail export, Since PNT1 was previously described as a gene affecting res istance to the antibiotic pentamidine, our data support a mitochondrial tar get for this drug.