OCTN3 IS A MAMMALIAN PEROXISOMAL MEMBRANE CARNITINE TRANSPORTER: IMPLICATIONS FOR PEROXISOMAL DISORDERS

Objectives: To determine the cellular localization of the organic cation/carnitine transporter, OCTN3. Carnitine is a zwitterion essential for the ßoxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes and microsomes.

Methods: We applied 10 nm immunogold labelled anti-mOctn3 antibodies and 3 nm immunogold labelled anti-catalase antibodies to murine liver and visualized them with TEM. We applied anti-mOcnt3 antibody to control human skin fibroblasts and Zellweger patient fibroblasts (Pex1 and Pex19 deficient cells) and studied OCTN3 cellular expression by immunostaining with a secondary antibody (Cy3-goat anti-rabbit IgG) and by Western blot analysis.

Results: We demonstrated expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20µM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrated expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast in two peroxisomal biogenesis disorders, we showed reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane.

Conclusion: Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for OCTN3 on peroxisomal membranes. This localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism. The predicted clinical phenotype for a defect in OCTN3 may parallel those seen in peroxisomal disorders.