PMID:16729965

Citation	Lamhonwah, AM and Tein, I (2006) Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria. Biochem. Biophys. Res. Commun. 345:1315-25
Abstract	Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.
Links	PubMed Online version:10.1016/j.bbrc.2006.05.026
Keywords	3T3 Cells; Adult; Animals; Blotting, Western; Carnitine/pharmacokinetics; Carnitine Acyltransferases/deficiency; Carnitine Acyltransferases/genetics; Cell Line; Cell Line, Tumor; Fibroblasts/cytology; Fibroblasts/enzymology; Fibroblasts/metabolism; Gene Expression Profiling; Green Fluorescent Proteins/genetics; Green Fluorescent Proteins/metabolism; Humans; Male; Mice; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria/metabolism; Mitochondria/ultrastructure; Mitochondria, Heart/metabolism; Mitochondria, Heart/ultrastructure; Mutation; Organic Cation Transport Proteins/genetics; Organic Cation Transport Proteins/metabolism; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transfection
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