The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p
The mitochondrial carrier family (MCF) consists of nuclear-encoded proteins which catalyze the transport of a wide variety of compounds across the mitochondrial inner membrane. These proteins present common structural features, which consist of three repeats of two transmembrane helices enclosing a...
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2024-04-01
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| author | Daniela Valeria Miniero Magnus Monné Maria Antonietta Di Noia Luigi Palmieri Ferdinando Palmieri |
| author_facet | Daniela Valeria Miniero Magnus Monné Maria Antonietta Di Noia Luigi Palmieri Ferdinando Palmieri |
| author_sort | Daniela Valeria Miniero |
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| description | The mitochondrial carrier family (MCF) consists of nuclear-encoded proteins which catalyze the transport of a wide variety of compounds across the mitochondrial inner membrane. These proteins present common structural features, which consist of three repeats of two transmembrane helices enclosing a translocation pore with a single substrate binding site. Access to the pore from the matrix side is controlled by a network of salt bridges formed by conserved charged residues of the signature motifs PX[D/E]XX[R/K] (M-gate) on the transmembrane helices H1, H3, and H5. On the cytosolic side, a less-conserved network is formed by the residues of the motifs [F/Y][D/E]XX[R/K] (C-gate) on H2, H4, and H6. In this work, to test the role of the charged residues of the C-gate in transport, we analyzed the charged residues of the cytoplasmic motifs of the yeast mitochondrial NAD+ transporter (Ndt1p). Single cysteine mutations of the negatively and positively charged residues were introduced by site-directed mutagenesis and only three of them (H4:E258, H4:K261, and H6:E359) completely inactivated the carrier. The double cysteine salt-bridge pair mutant H4-H6:K261C/E359C exhibited a higher transport rate than the corresponding single mutants as well as when the charged residues were swapped in these positions (H4-H6:K261E/E359K). The double mutant H2-H4:K164C/E258C and the swapped H2-H4:K164E/E258K exhibited transport rates at similar levels to the single K164C. The sextuple mutant with all the charged residues inverted was inactive. These preliminary results suggest that not all the charged C-gate residues are essential for transport and that some of them may have additional roles in transport besides forming salt-bridges. |
| format | Article |
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| issn | 2504-3900 |
| language | English |
| publishDate | 2024-04-01 |
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| spelling | doaj-art-8900a5764e644ebaa991a36b2daf66df2025-08-20T02:43:02ZengMDPI AGProceedings2504-39002024-04-0110315110.3390/proceedings2024103051The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1pDaniela Valeria Miniero0Magnus Monné1Maria Antonietta Di Noia2Luigi Palmieri3Ferdinando Palmieri4Department of Biosciences, Biotechnologies and Environment, University of Bari, 70125 Bari, ItalyDepartment of Science, University of Basilicata, 85100 Potenza, ItalyDepartment of Biosciences, Biotechnologies and Environment, University of Bari, 70125 Bari, ItalyDepartment of Biosciences, Biotechnologies and Environment, University of Bari, 70125 Bari, ItalyDepartment of Biosciences, Biotechnologies and Environment, University of Bari, 70125 Bari, ItalyThe mitochondrial carrier family (MCF) consists of nuclear-encoded proteins which catalyze the transport of a wide variety of compounds across the mitochondrial inner membrane. These proteins present common structural features, which consist of three repeats of two transmembrane helices enclosing a translocation pore with a single substrate binding site. Access to the pore from the matrix side is controlled by a network of salt bridges formed by conserved charged residues of the signature motifs PX[D/E]XX[R/K] (M-gate) on the transmembrane helices H1, H3, and H5. On the cytosolic side, a less-conserved network is formed by the residues of the motifs [F/Y][D/E]XX[R/K] (C-gate) on H2, H4, and H6. In this work, to test the role of the charged residues of the C-gate in transport, we analyzed the charged residues of the cytoplasmic motifs of the yeast mitochondrial NAD+ transporter (Ndt1p). Single cysteine mutations of the negatively and positively charged residues were introduced by site-directed mutagenesis and only three of them (H4:E258, H4:K261, and H6:E359) completely inactivated the carrier. The double cysteine salt-bridge pair mutant H4-H6:K261C/E359C exhibited a higher transport rate than the corresponding single mutants as well as when the charged residues were swapped in these positions (H4-H6:K261E/E359K). The double mutant H2-H4:K164C/E258C and the swapped H2-H4:K164E/E258K exhibited transport rates at similar levels to the single K164C. The sextuple mutant with all the charged residues inverted was inactive. These preliminary results suggest that not all the charged C-gate residues are essential for transport and that some of them may have additional roles in transport besides forming salt-bridges.https://www.mdpi.com/2504-3900/103/1/51mitochondrial carriersliposomemutagenesistransport |
| spellingShingle | Daniela Valeria Miniero Magnus Monné Maria Antonietta Di Noia Luigi Palmieri Ferdinando Palmieri The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p Proceedings mitochondrial carriers liposome mutagenesis transport |
| title | The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p |
| title_full | The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p |
| title_fullStr | The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p |
| title_full_unstemmed | The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p |
| title_short | The Role of the Charged Residues in the C-Gate of the Yeast Mitochondrial NAD<sup>+</sup> Transporter Ndt1p |
| title_sort | role of the charged residues in the c gate of the yeast mitochondrial nad sup sup transporter ndt1p |
| topic | mitochondrial carriers liposome mutagenesis transport |
| url | https://www.mdpi.com/2504-3900/103/1/51 |
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