Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2 |
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| Institution: | 1. Departamento de Fisiopatología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile;2. Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile;1. University of Alabama at Birmingham, Department of Pathology, Division of Molecular and Cellular Pathology, Birmingham, AL 35294, USA;2. University of Alabama at Birmingham, Department of Biology, Birmingham, AL 35294, USA;3. Veterans Administration Medical Center, Birmingham, AL 35294, USA;1. Monash Centre for Health Research and Implementation, School of Public Health and Preventive Medicine, Monash University, MHRP, 43-51 Kanooka Grove, Clayton, VIC 3168, Australia;2. Diabetes and Vascular Medicine Unit, Monash Health, Locked Bag 29, Clayton, VIC 3168, Australia;3. Centre for Chronic Disease, Victoria University, Melbourne, Australia;4. School of Population Health, The University of Auckland, New Zealand;1. Department of Urology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA;2. Department of Genetics University of Alabama at Birmingham, Birmingham, AL, 35294, USA;1. Department of Urology, Medical University Innsbruck, Innsbruck, Austria;4. Department of Pathology, Medical University Innsbruck, Innsbruck, Austria;6. Department of Internal Medicine V, Medical University Innsbruck, Innsbruck, Austria;2. Department of Pathology, Hôspital Tenon, HUEP, Sorbonne University, Paris, France;3. Pathology Laboratory Obrist and Brunhuber, Zams, Austria;5. Department of Urology, Eberhard Karls University, Tübingen, Germany;7. Tyrolean Cancer Research Institute, Innsbruck, Austria |
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| 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 0.6 mM. 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. |
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| Keywords: | SVCT2 Mitochondria Ascorbic acid transport Intracellular ascorbate transporter Mitochondrial ascorbate transporter Transport asymmetry Free radicals |
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