TY - JOUR
T1 - Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae
AU - Todisco, Simona
AU - Agrimi, Gennaro
AU - Castegna, Alessandra
AU - Palmieri, Ferdinando
PY - 2006/1/20
Y1 - 2006/1/20
N2 - 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. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not α-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD +-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The Δndt1Δndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.
AB - 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. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not α-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD +-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The Δndt1Δndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.
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U2 - 10.1074/jbc.M510425200
DO - 10.1074/jbc.M510425200
M3 - Article
C2 - 16291748
AN - SCOPUS:33644513561
VL - 281
SP - 1524
EP - 1531
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 3
ER -