Identification of the Mitochondrial NAD+ Transporter in Saccharomyces cerevisiae

Abstract

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD ؉ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD ؉ carrier protein (Ndt1p) is described. Mitochondria contain in their matrix the universal hydrogen transfer coenzyme NAD ϩ , which serves to transfer hydrogen from substrates to the respiratory chain by oxidative phosphorylation. In addition to its well known role as a coenzyme in redox reactions, NAD ϩ exerts other important functions in mitochondria. In Saccharomyces cerevisiae, mitochondrial NADH has been shown to participate in Fe/S protein biogenesis (1) and to be the source for NADPH (2), which is required in mitochondria for oxidative stress protection and for specific biogenesis reactions. NAD ϩ has been shown also to serve critical regulatory functions in gene transcription, enzyme activity, and other important processes through ADP-ribosylation and deacetylation reactions (3-5). In addition, the increase in the NAD ϩ /NADH ratio in mitochondria seems to be important for the extension of the life-span of yeast by calorie restriction (6). The enzymes of NAD ϩ biosynthesis are generally believed to be localized outside the mitochondria (Refs. 7-9, but see Refs. 10 and 11), therefore, NAD ϩ must be imported into these organelles. For a long time nicotinamide adenine dinucleotides were known to be unable to cross the inner membranes of mitochondria (12). However, NAD ϩ has been shown to be taken up by intact plant mitochondria, the uptake being concentration-and temperature-dependent and specifically inhibited by an azido derivative of NAD ϩ (13, 14). Moreover, using human cultured cells harvested under quiescent conditions (6 -8 days after medium change), Rustin et al. (15) observed a depletion of mitochondrial NAD ϩ and an influx of NAD ϩ into the mitochondrial matrix of these cells after adding external NAD ϩ to digitonin-permeabilized cells.These studies contradicted the notion of mitochondrial inner membrane impermeability to pyridine coenzymes and led to the hypothesis that NAD ϩ is transported into mitochondria by a carrier-mediated system. However, the one or more proteins responsible for the observed transport activities have not been hitherto isolated or identified. In this study we provide evidence that the gene products of YIL006W and YEL006W, named Ndt1p and Ndt2p, respectively, are two isoforms of the mitochondrial NAD ϩ transporter in S. cerevisiae. These proteins are 373 and 335 amino acids long, respectively, possess the characteristics of the MCF, 2 and display a high degree (70%) of homology. Ndt1p was overexpressed in Escherichia coli, purified, reconstituted in phospholipid vesicles,...

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

“…The previously proposed pyruvate carrier, YIL006w, has now been identified and functionally characterized as the mitochondrial NAD carrier protein (Ndt1p) by using transport assays with purified protein reconstituted into liposomes (40). Compared with the pyruvate substrate, the adenine-containing NAD substrate is consistent with the "G-L" motif found at contact point II and aromatic stacking at contact point I.…”

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

“…8B), suggesting that in wildtype cells reducing equivalents of NADPH can be transferred from the mitochondria to the cytosol through the activity of Yhm2p. In untreated wild-type and knock-out cell mitochondria, the NADH level was too low, as observed previously in wild-type cells (15), to assess the NADH/NAD ϩ ratio. Upon addition of H 2 O 2 , both the low NADH/ NAD ϩ ratio and total amount of NADH ϩ NAD ϩ were not significantly different among the strains tested (supplemental Fig.…”

“…Transport at 25°C was started by adding [ 14 C]citrate or [ 14 C]oxoglutarate to proteoliposomes and terminated by the addition of 30 mM pyridoxal 5Ј-phosphate and 10 mM bathophenanthroline, which in combination and at high concentrations inhibit the activity of several mitochondrial carriers completely and rapidly (see, for example, Refs. [13][14][15][16]. In controls, the inhibitors were added with the labeled substrate.…”

The malate–aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast

“…5E). Perhaps the mitochondrial NAD carriers (Todisco et al 2006) are more active under CR, increasing the inward flux of NAD. It is noteworthy that these values represented relative differences in nucleotides among matched samples rather than precise quantification.…”