Molecular cloning and characterization of MT-ACT48, a novel mitochondrial acyl-CoA thioesterase.
While characterizing Eps15 partners, we identified a 48-kDa polypeptide (p48) which was precipitated by Eps15-derived glutathione S-transferase fusion proteins. A search in a murine expressed sequence tag data base with N-terminal microsequences of p48 led to the identification of two complete cDNA clones encoding two isoforms of a 439-amino acid protein sharing 95% nucleic and amino acid identity. Northern blot and immunoblotting studies showed that p48 was ubiquitously expressed. A significant homology (19% identity and 40% similarity) between p48 and rat brain cytosolic acyl-CoA thioesterase was observed in an 80-amino acid C-terminal domain, retrieved from proteins from human, nematode, and plants. The thioesterase function of p48 was further demonstrated against long chain acyl-CoAs in a spectrophotometric assay. Furthermore, data obtained from sequence analysis showed that p48 contained a mitochondrial targeting signal, cleaved in mature protein as assessed by microsequencing. The mitochondrial localization of both endogenous and transfected p48 was confirmed by confocal microscopy. These results indicate that p48, called MT-ACT48 (mitochondrial acyl-CoA thioesterase of 48 kDa), defines a novel family of mitochondrial long chain acyl-CoA thioesterases.
Poupon
V
V
CJF 97-10 INSERM, Faculté Necker-Enfants Malades, 156 rue de Vaugirard, 75756 Paris Cedex 15, France.
Bègue
B
B
Gagnon
J
J
Dautry-Varsat
A
A
Cerf-Bensussan
N
N
Benmerah
A
A
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013379:Substrate Specificity; D014162:Transfection
D010170:Palmitoyl-CoA Hydrolase