Identification of yeast and human mitochondrial S-adenosylmethionine transporters

Introduction: In S. cerevisiae, S-adenosylmethionine (SAM) is synthesized from ATP and methionine by two synthetases, Sam1p and Sam2p, that are both localized exclusively in the cytosol (Kumar A et al. (2002) Genes Dev., 16, 707– 719). Therefore SAM must be transported into the mitochondria where it is required, for methylation reactions of DNA, RNA and proteins. Moreover in yeast SAM is required as essential cofactor in the last step of biotin and lipoic acid biosyntheses both localized in the mitochondria. Here we report the identification and functional characterization of the yeast mitochondrial SAM carrier (Sam5p), encoded by YNL003c, and of its human ortholog named SAMC, encoded by SLC25A26. They have the characteristic features of the mitochondrial carrier family (Palmieri F., Pflugers Arch-Eur J Physiol 2004, 447, 689– 709). The corresponding proteins were overexpressed in bacteria, reconstituted into phospholipid vesicles and identified from their transport properties. The subcellular localization of the GFP fused to Sam5p or SAMC, the phenotype of SAM5 knock-out yeast strain, and the tissue distribution of the human SAMC were also investigated. Results: Recombinant Sam5p and SAMC catalyzed an active influx of [ 3 H]SAM into proteoliposomes in exchange for internal SAM. Sam5p catalyzed also an influx of [ 3 H]SAM into empty proteoliposomes whereas SAMC catalysed virtually only a countertransport. High activities were also found with internal adenosylornithine (sinefungin), S-adenosylcysteine (SAC) and S-adenosylhomocysteine (SAHC). The Km and Vmax values of Sam5p were 75.6 F 8.8 AM and 105.5 F 30.2 Amol/min per g protein, respectively. The Km and Vmax of the recombinant purified SAMC were 23.0 F 2.5 AM and 463 F 95 Amol/min per g protein, respectively. SAHC, SAC and adenosylornithine inhibited [ 3 H]SAM/SAM exchange competitively. Sam5p-GFP and SAMC-GFP expressing cells showed a green fluorescence of the mitochondrial network. Yeast cells lacking SAM5 were unable to grow on synthetic minimal medium supplemented with glucose in the absence of biotin or on rich medium with nonfermentable carbon sources. Both phenotypes of the knock-out mutant were reversed by expressing the cytosolic SAM synthetase (Sam1p).