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Molecular and Cellular Biology, October 1999, p. 6598-6607, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutations Affecting a Yeast Mitochondrial Inner Membrane Protein, Pnt1p, Block Export of a Mitochondrially Synthesized Fusion Protein from the Matrix

Shichuan Hedagger and Thomas D. Fox*

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703

Received 15 April 1999/Returned for modification 13 May 1999/Accepted 13 July 1999

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. The screen is based on the fact that the hydrophilic polypeptide Arg8mp is exported from the matrix if it is synthesized within mitochondria as a bifunctional Cox2p-Arg8mp fusion protein. Since export of Arg8mp causes an Arg- phenotype, defective mutants can be selected as Arg+. 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 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, 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.

* Corresponding author. Mailing address: Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853-2703. Phone: (607) 254-4835. Fax: (607) 255-6249. E-mail: tdf1{at}cornell.edu.

dagger Present address: Monsanto Life Sciences Co., St. Louis, MO 63167.

Molecular and Cellular Biology, October 1999, p. 6598-6607, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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