In mouse alpha-methylacyl-CoA racemase, the same gene product is simultaneously located in mitochondria and peroxisomes

alpha-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subc ellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatogra phy, The isolated proteins mere of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with alpha- methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15 B1. The putative initial ATG in the racemase gene was preceded by a functio nal promotor as shown with the luciferase reporter gene assay. The correspo nding cDNAs were isolated from rat and mouse liver. The recombinant rat pro tein was overexpressed in active form in Pichia pastoris. The presented dat a suggest that the polypeptide encoded by the racemase gene can alternative ly be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxis omal targeting signal.