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

2-Methylactyl-CoA racemase is an auxiliary enzyme required for the peroxiso mal beta -oxidative breakdown of (2R)-pristanic acid and the (25R)-isomer o f C-27 bile acid intermediates. The enzyme activity is found not only in pe roxisomes but also is present in mitochondria of human liver and fibroblast s. 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 t argeting signal (PTS1). From the in vitro interaction between recombinant r acemase and recombinant human PTS1 receptor (Pex5p), and the peroxisomal lo calization of green fluorescent protein (GFP) fused to the N terminus of fu ll-length racemase or its last six amino acids in tranfected Chinese hamste r ovary (CHO) cells, we concluded that ASL is a new PTSI. variant. To be re cognized by Pex5p, however, the preceding lysine residue is critical. As sh own in another series of transfection experiments with GFP fused to the C t erminus of the full-length racemase or racemase with deletions of the N ter minus, mitochondrial targeting information is localized between amino acids 22 and 85. Hence, our data show that a single transcript gives rise to a r acemase protein containing two topogenic signals, explaining the dual cellu lar localization of the activity.