Mitochondrial and peroxisomal targeting of 2-methylacyl-CoA racemase in humans

• Published in: Journal of lipid research Vol. 41; no. 11; pp. 1752 - 1759
• Main Authors: Amery, L, Fransen, M, De Nys, K, Mannaerts, G P, Van Veldhoven, P P
• Format: Journal Article
• Language: English
• Published: United States Elsevier 01.11.2000
• Subjects: Amino Acid Sequence; Animals; Base Sequence; bile acids; CHO Cells; cholestanoic acid; Cricetinae; epimerase; Green Fluorescent Proteins; Humans; Liver - enzymology; Liver - ultrastructure; Luminescent Proteins - metabolism; Microscopy, Fluorescence; Mitochondria, Liver - enzymology; Molecular Sequence Data; peroxins; Peroxisome-Targeting Signal 1 Receptor; Peroxisomes - enzymology; pristanic acid; Racemases and Epimerases - chemistry; Racemases and Epimerases - genetics; Racemases and Epimerases - metabolism; Rats; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; Sequence Homology; Transfection; β-oxidation
• ISSN: 0022-2275
• DOI: 10.1016/s0022-2275(20)31968-4

2-Methylacyl-CoA racemase is an auxiliary enzyme required for the peroxisomal beta-oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer of C(27) bile acid intermediates. The enzyme activity is found not only in peroxisomes but also is present in mitochondria of human liver and fibroblasts. The C terminus of the human racemase, a protein of 382 amino acids with a molecular mass of 43,304 daltons as deduced from its cloned cDNA, consists of KASL. Hitherto this sequence has not been recognized as a peroxisomal targeting signal (PTS1). From the in vitro interaction between recombinant racemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal localization of green fluorescent protein (GFP) fused to the N terminus of full-length racemase or its last six amino acids in tranfected Chinese hamster ovary (CHO) cells, we concluded that ASL is a new PTS1 variant. To be recognized by Pex5p, however, the preceding lysine residue is critical. As shown in another series of transfection experiments with GFP fused to the C terminus of the full-length racemase or racemase with deletions of the N terminus, mitochondrial targeting information is localized between amino acids 22 and 85.Hence, our data show that a single transcript gives rise to a racemase protein containing two topogenic signals, explaining the dual cellular localization of the activity.