Identification of the Mitochondrial GTP/GDP Transporter in Saccharomyces cerevisiae

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 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. 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.In the mitochondrial matrix, GTP is required as an energy source for protein synthesis; as a substrate for the synthesis of tRNA, mRNA, rRNA, and RNA primers; and as a phosphate group donor for the activity of GTP-AMP phosphotransferase (1) and G proteins (2, 3). In several organisms, GTP is synthesized in the mitochondria by succinyl-CoA ligase, which catalyzes the conversion of succinyl-CoA to succinate with the generation of GTP, and by nucleoside diphosphate kinase, which catalyzes the transfer of the ␥ phosphate from ATP to a nucleoside diphosphate to yield nucleotide triphosphates. In Saccharomyces cerevisiae, however, 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.Despite the importance of GTP in mitochondrial metabolism, the transport of guanine nucleotides has not been characterized in yeast mitochondria, nor has any mitochondrial protein responsible for this transport been identified. There are only two indirect observations that suggest that GTP is transported across the inner mitochondrial membrane of S. cerevisiae. First, mitochondri...

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

“…The GTP/ GDP carrier (Fig. S9 in SI Appendix) is predicted to be a net importer in agreement with its biological function, because the net influx of nucleotides is required for nucleic acid synthesis, yet no uniport activity has been observed (44). If the matrix network is weak and the cytoplasmic network is strong, net export would be anticipated when the substrate is absent from the intermembrane space.…”

A Mitochondrial-Vacuolar Signaling Pathway in Yeast That Affects Iron and Copper Metabolism

“…Deletion of these genes leads to marked changes in mitochondrial metal homeostasis. For example, deletion of the mitochondrial carrier YHM1 leads to mitochondrial iron accumulation (24), yet Yhm1p appears to be a GTP/GDP exchanger (25). It is not clear how defects in mitochondrial GTP levels would lead to increased mitochondrial iron accumulation.…”

A Novel Member of Solute Carrier Family 25 (SLC25A42) Is a Transporter of Coenzyme A and Adenosine 3′,5′-Diphosphate in Human Mitochondria

“…We were therefore technically unable to assess the effect of these ionophores on the kinetic constants of SLC25A42 or to answer the question as to whether SLC25A42 is electrogenic or electroneutral as was the case for the NAD ϩ carrier (26) but not for other reconstituted transporters (see, for example, Refs. 4,12,27,28).…”