Lamhonwah Anne-Marie, Tein Ingrid
Division of Neurology, Department of Pediatrics, University of Toronto, Ont., Canada.
Biochem Biophys Res Commun. 2006 Jul 14;345(4):1315-25. doi: 10.1016/j.bbrc.2006.05.026. Epub 2006 May 12.
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.
肉碱是脂肪酸β氧化所必需的两性离子。我们报告了有机阳离子/肉碱转运体OCTN1在线粒体中的新定位。我们构建了绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）标记的人OCTN1 cDNA构建体，并在几种转染的哺乳动物细胞系中展示了hOCTN1的表达。用不同的细胞内标记物对GFP-hOCTN1转染细胞进行免疫染色并结合共聚焦荧光显微镜观察，证实了OCTN1在线粒体中的表达。在转染的MEF-3T3细胞中，RFP-hOCTN1融合蛋白与线粒体-GFP标记构建体有明显的共定位，而在转染的人皮肤成纤维细胞中，GFP-hOCTN1与其他细胞内标记物没有共定位。在GFP-hOCTN1转染的HepG2新鲜分离线粒体中，L-[(3)H]肉碱摄取的米氏常数（K(m)）为422微摩尔，用抗GFP抗体进行的蛋白质印迹法鉴定出预期的GFP-hOCTN1融合蛋白（90 kDa）。我们用抗GST-hOCTN1抗体在HepG2线粒体中显示了天然OCTN1的内源性表达。此外，我们明确证实，在肉碱/脂酰肉碱转位酶活性（另一种线粒体肉碱转运体）低于1%的突变人皮肤成纤维细胞的分离线粒体中，完整的L-[(3)H]肉碱摄取（K(m) 1324微摩尔）完全归因于OCTN1。用高特异性兔抗GST-hOCTN1抗体和免疫金标记的山羊抗兔抗体孵育小鼠心脏，通过透射电子显微镜（TEM）证实了这种线粒体定位。这表明OCTN1在细胞内肉碱稳态中具有与其他OCTN家族成员不同的重要作用。
