Molecular cloning and characterization of MT-ACT48, a novel mitochondrial Acyl-CoA thioesterase

While characterizing Eps15 partners, we identified a 48-kDa polypeptide (p4 8) which was precipitated by Eps15-derived glutathione S-transferase fusion proteins. A search in a murine expressed sequence tag data base with N-ter minal microsequences of p48 led to the identification of two complete cDNA clones encoding two isoforms of a 439-amino acid protein sharing 95% nuclei c and amino acid identity. Northern blot and immunoblotting studies showed that p48 was ubiquitously expressed. A significant homology (19% identity a nd 40% similarity) between p48 and rat brain cytosolic acyl-CoA thioesteras e was observed in an 80-amino acid C-terminal domain, retrieved from protei ns from human, nematode, and plants. The thioesterase function of p48 was f urther demonstrated against long chain acyl-CoAs in a spectrophotometric as say. Furthermore, data obtained from sequence analysis showed that p48 cont ained a mitochondrial targeting signal, cleaved in mature protein as assess ed by microsequencing. The mitochondrial localization of both endogenous an d transfected p48 was confirmed by confocal microscopy. These results indic ate that p48, called MT-ACT48 (mitochondrial acyl-CoA thioesterase of 48 kD a), defines a novel family of mitochondrial long chain acyl CoA thioesteras es.