Functional Analysis of Mutant Human Carnitine Acylcarnitine Translocases in Yeast L. IJlst,* ,1 C. W. T. van Roermund,* ,1 V. Iacobazzi,‡ W. Oostheim,* J. P. N. Ruiter,* J. C. Williams,§ F. Palmieri,‡ and R. J. A. Wanders* , † ,2 *Department of Clinical Chemistry and †Department of Pediatrics, Academic Medical Centre, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands; ‡Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, 70125 Bari, Italy; and §Division of Medical Genetics, Children’s Hospital, Los Angeles, P.O. Box 90, Los Angeles, California 90027 Received December 19, 2000 Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity (< 1%) in the milder patient. Palmitate oxidation activity in cells from the severe patient was less than 5% but in the milder patient 27% residual activity was found. Sequencing of the CACT cDNAs revealed a c.241G> A (G81R) in the severe and a c.955insC mutation (C- terminal extension of 21 amino acids (CACT(21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression sys- tem based on the ability of human CACT to function- ally complement a CACT-deletion strain of yeast. Ex- pression in this strain revealed significant residual activity for CACT(21aa), while the CACT(G81R) was inactive. © 2001 Academic Press Key Words: fatty acid; carnitine acylcarnitine translo- case; Mendelian disorder; hereditary disease; gene ex- pression; yeast expression; functional complementation. Mitochondrial -oxidation of long-chain fatty acids (LCFA) is the major source of energy production in man. Since the mitochondrial inner membrane is im- permeable for LCFAs or their CoA esters, the LCFAs are transported as acylcarnitines. Three different gene products are involved in this carnitine-dependent transport shuttle: carnitine palmitoyltransferase I (CPT I), carnitine acylcarnitine translocase (CACT) and carnitine palmitoyltransferase II (CPT II). After activation of LCFAs to their CoA-esters, CPT I con- verts the fatty acyl-CoA esters to their corresponding carnitine esters, which are subsequently translocated across the mitochondrial inner membrane in exchange for free carnitine by CACT. Once inside the mitochon- drial matrix, CPT II reconverts the carnitine esters back to the CoA esters which then can serve as a substrate for the -oxidation spiral. In the past, many patients have been identified with a defect in het carnitine cycle either at the level of CPT I, CACT or CPT II. Of these diseases, CPT II deficiency is the most frequent and several mutations have been reported. Recently, the molecular basis of hepatic CPT I deficiency was resolved in a single patient (1). The identified mutations in both CPT I (1) and CPT II deficiency (2) are disease-causing as shown by heterol- ogous expression of the mutant proteins. Since the first report of CACT deficiency (MIM entry 212138) by Stan- ley et al. (3), 12 additional cases have been reported (4 –13). As with patients affected in CPT I and CPT II, patients with CACT deficiency suffer from hypoketotic hypoglycaemia and hyperammonemia due to the defi- cient fatty acid oxidation. Among the various fatty acid -oxidation disorders, LCFA -oxidation is usually most profoundly impaired in case of CACT deficiency (residual activity  5%). Most patients show a severe phenotype characterised by generalised muscle weakness, cardiomyopathy, hep- atomegaly and impaired liver function usually with fatal outcome, although three patients have been re- ported with a milder presentation. The cDNA encoding rat CACT was cloned in 1997 by Indiveri et al. (14) and the human homolog was cloned shortly thereafter (10). In the same paper the first mutation in the cDNA of a CACT-deficient patient with a mild presentation was reported (10). Abbreviations used: CACT, carnitine acylcarnitine translocase; LCFA, long chain fatty acid; CPT I, carnitine palmitoyltransferase I; CPT II, carnitine palmitoyltransferase II; cit 2, citrate synthase 2. 1 Equal first authors. 2 To whom correspondence should be addressed. Fax: +31 20 6962596. E-mail: Wanders@AMC.UVA.NL. Biochemical and Biophysical Research Communications 280, 700 –706 (2001) doi:10.1006/bbrc.2000.4178, available online at http://www.idealibrary.com on 700 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.