Abstract
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.
Author Contributions
Conceptualization, L.P. and F.P.; methodology, D.V.M., M.M. and M.A.D.N., formal analysis, D.V.M., M.M., M.A.D.N., L.P. and F.P.; investigation, D.V.M., M.M., M.A.D.N., L.P. and F.P.; writing—original draft preparation, D.V.M. and M.M.; writing—review and editing, M.M., L.P. and F.P. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
No new data were created.
Conflicts of Interest
The authors declare no conflict of interest.
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