title: Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2.
abstract: Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Carola
authors.initials: C
authors.lastname: Muñoz-Montesino
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Roa
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Peña
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Mauricio
authors.initials: M
authors.lastname: González
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Kirsty
authors.initials: K
authors.lastname: Sotomayor
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Eveling
authors.initials: E
authors.lastname: Inostroza
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Carolina A
authors.initials: CA
authors.lastname: Muñoz
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Iván
authors.initials: I
authors.lastname: González
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Mafalda
authors.initials: M
authors.lastname: Maldonado
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Carlos
authors.initials: C
authors.lastname: Soliz
authors.affiliation: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
authors.forename: Alejandro M
authors.initials: AM
authors.lastname: Reyes
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Vera
authors.affiliation: Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile. Electronic address: corivas@udec.cl.
authors.forename: Coralia I
authors.initials: CI
authors.lastname: Rivas
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics*; D005609:Free Radicals / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D010084:Oxidation-Reduction; D034741:RNA, Small Interfering; D060347:Sodium-Coupled Vitamin C Transporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D034741:RNA, Small Interfering; C555162:SLC23A1 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D001205:Ascorbic Acid
keywords: Ascorbic acid transport; Free radicals; Intracellular ascorbate transporter; Mitochondria; Mitochondrial ascorbate transporter; SVCT2; Transport asymmetry