THE MITOCHONDRIAL TRICARBOXYLATE CARRIER

The tricarboxylate carrier has recently been purified from rat liver m itochondria by three distinct scientific groups using different method s. A 37-38-kDa protein has been prepared by silca gel 60 chromatograph y by our group (Claeys and Azzi, 1989; Glerum et al., 1990). The speci fic citrate transport activity of this preparation is not significantl y different from that measured in mitochondria and it is inhibitable b y 1,2,3- benzenetricarboxylic acid. Bisaccia et al. (1990) have report ed the isolation of a 30-kDa protein by Celite 535 chromatography, and Kaplan's group (Kaplan et al., 1990) have isolated a 32.5-kDa protein by Matrex Orange, Matrex Blue, and Affi-Gel chromatography. Peptide m apping has failed to support any structural homologies between the 37- 38-kDa and the 30-32.5-kD proteins. The 38-kD protein is N-terminally blocked. The peptides obtained by several cleavage procedures have bee n partially sequenced. Their sequence information has been used to obt ain different cDNA clones by a dual approach, the polymerase chain rea ction and screening of a lambda ZAP cDNA library. The largest cDNA whi ch could be isolated is 2,986 bp in length and contains a 1071-bp-long open reading frame and an unusually long 3' untranslated region, both of which have been completely sequenced. The protein sequence of the carrier from the first in-frame methionine is 322 amino acids in lengt h and exhibits a molecular mass of 35,546. Comparison of the protein s equence to the sequences of the four members of the mitochondrial carr ier protein family (ADP/ATP carrier, phosphate carrier, 2-oxoglutarate /malate carrier, and uncoupling protein) does not reveal significant s imilarity (cf. Walker et al., 1987). A tripartite internal homology, w hich is a characteristic of these proteins, is not present in the sequ ence of the tricarboxylate carrier protein. The mRNA for the tricarbox ylate carrier is expressed in rat liver and brain, but not in rat hear t.