Abstract
The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.
Highlights
The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria
Transport was saturable with an apparent Km of 1 M for GTP and 5 M for GDP. It was strongly inhibited by pyridoxal 5-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate
Mitochondrial protein synthesis is stimulated by the addition of external GTP [6], and second, on incubating yeast mitochondria with labeled GTP, the amount of radioactivity associated with mitochondria is time-dependent, inhibited by GDP and insensitive to carboxyatractyloside [7]
Summary
The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. In Saccharomyces cerevisiae, succinyl-CoA ligase produces ATP instead of GTP [4], and the mitochondrial nucleoside diphosphate kinase is localized in the intermembrane space and absent in the matrix [5] These observations imply that in S. cerevisiae GTP has to be imported into the mitochondria probably via a carrier system embedded in the inner mitochondrial membrane. The inner membranes of mitochondria contain a family of proteins that transport various substrates and products into and out of the matrix (for a review see Ref. 8) These proteins are characterized by three tandem sequence repeats, each being approximately 100 amino acids in length and folded into two transmembrane ␣-helices joined by an extensive hydrophilic loop. We report the identification and functional characterization of Ggc1p This protein has been overexpressed in Escherichia coli, reconstituted into phospholipid vesicles, and identified from its transport properties as a carrier for GTP and GDP. This report presents the first information on the molecular properties of the mitochondrial GTP/GDP carrier and a definitive identification of its gene in S. cerevisiae
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