Affiliation:
1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703
Abstract
ABSTRACT
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 Arg8
m
p is exported from the matrix 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, 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 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.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
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