The solute carrier 44A1 is a mitochondrial protein and mediates choline transport.
Choline oxidation to betaine takes place in the mitochondria; however, a protein regulating mitochondrial choline transport was never identified. The purpose of this study was to analyze subcellular localization of the solute carrier 44A1 (SLC44A1), a plasma membrane choline transporter sensitive to inhibition by hemicholinium-3. We generated N- and C-terminal-SLC44A1-specific antibodies and analyzed localization of endogenous and overexpressed SLC44A1 in C2C12 mouse muscle cells, MCF7 human breast cancer cells, and mouse tissues using confocal microscopy, differential centrifugation, and Western blotting. We further performed choline uptake competition studies on isolated mitochondria using the specific inhibitor hemicholinium-3 and SLC44A1 antibodies, and analyzed mitochondria of FL83B hepatocytes after the targeted knock-down of SLC44A1 using siRNA technology. In addition, we analyzed SLC44A1 expression during choline deficiency. Localization studies revealed plasma membrane, cytosolic, microsomal, and mitochondrial localization of endogenous and His-tagged SLC44A1. Uptake studies in isolated mitochondria show an accumulation of (3)H-choline, which is strongly inhibited by hemicholinium-3 (60%), by an excess of unlabeled choline (97%), and by both SLC44A1 antibodies. SLC44A1 mRNA and protein expression were down-regulated during choline deficiency. These data clearly establish SLC44A1 as an important mediator of choline transport across both the plasma membrane and the mitochondrial membrane.
Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada. vmichel@uoguelph.ca
Vera
V
Michel
Marica
M
Bakovic
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D002794:Choline / Q000378:metabolism*; D002796:Choline Deficiency / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006426:Hemicholinium 3 / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; C494981:CTL1 protein, mouse; D017931:DNA Primers; D024101:Mitochondrial Proteins; D027701:Organic Cation Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C510732:SLC44A1 protein, human; D006426:Hemicholinium 3; D002794:Choline