期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 345, 期 4, 页码 1315-1325出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2006.05.026
关键词
fatty acid oxidation; carnitine/organic cation transporters; OCTN1; 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-[H-3]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K-m of 422 mu M 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 in tact L-[H-3]carnitine uptake (K-m 1324 mu M), 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. (c) 2006 Elsevier Inc. All rights reserved.
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