Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria.
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.
Lamhonwah
Anne-Marie
AM
Division of Neurology, Department of Pediatrics, University of Toronto, Ont., Canada.
Tein
Ingrid
I
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002331:Carnitine / Q000493:pharmacokinetics; D002333:Carnitine Acyltransferases / Q000172:deficiency / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D008929:Mitochondria, Heart / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation; D027701:Organic Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027981:Symporters; D014162:Transfection
D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C109807:SLC22A4 protein, human; D027981:Symporters; D049452:Green Fluorescent Proteins; D002333:Carnitine Acyltransferases; D002331:Carnitine