Biochimica et Biophysica Acta, 1018 (1990) 147-150 147 Elsevier BBAEBC 00034 Structural and functional properties of mitochondrial anion carriers F. Palmieri, F. Bisaccia, L. Capobianco, V. Iacobazzi, C. Indiveri and V. Zara Department of Pharmaco-Biology, Laboratory of Biochemistry, University of Bari and CNR Unit for the Study of Mitochondria and Bioenergetics, Bari (Italy) (Received 1 May 1990) Key words: Mitochondrial anion carrier; Ion transport; Carnitine carrier; Metabolite transport The inner mitochondrial membrane contains several transport systems for metabolites, which are necessary for oxidative phosphorylation, the tricarboxylic acid cycle and the amino acid metabolism, as well as for the transfer of reducing equivalents and for important metabolic pathways, whose enzymes are partitioned be- tween the cytosol and the mitochondtia. The carriers which have been most extensively studied are: the AAC, the PIC, the UNC, the OGC, the DIC, the AGC, the PYC, the CIC and the CAC. Although these carriers have been identified in intact rnitochondria, the state of their characterization is very different. With the excep- tion of the CAC, all the other carriers indicated above have been purified and functionally reconstituted into liposomes [1,2]. The primary structure of the first three carriers has been determinated by amino-acid analysis and/or by DNA sequencing. These proteins are formed by three segments of about 100 amino acids, which are homologous one to another. The clear homology led to the development of a concept of a carrier family, which is supposed to have originated from a common ancestor gene [3,4]. It is proposed that the other mitochondrial carriers also fall into the same family, an idea which is supported by the very similar M r of around 30 kDa for most of the carriers so far identified. With respect to elucidation of transport mechanism and regulation, the AAC, the PIC and the AGC are the best characterized. In this paper several structural and functional proper- ties of previously purified carriers are reported, together with the first pure preparation of the CAC from rat liver mitochondria. Abbreviations: AAC, ADP/ATP carrier; AGC, aspartate/glutamate carrier; CAC, camitine carrier; CIC, citrate (tricarboxylate) carrier; DIC, dicarboxylate carrier; OGC, oxoglutarate carrier; PIC, phos- phate carder, PYC, pyruvate cartier; UNC, uncoupling protein; HTP, hydroxyapatite; SMP, submitochondrial particles; SDS, sodium dodecylsulfate. Correspondence: F. Palmieri, Dipartimento Farmaco-Biologico, Uni- versith di Bari, Traversa 200 Re David 4, 70125 Bari, Italy. Purification of the carnitine carrier. One of our aims in the last year was to provide the molecular identifica- tion of the last remaining important mitochondrial car- tier, namely the CAC. When comparing the procedures for purification of various mitochondrial carrier pro- teins, it becomes obvious that all have been isolated by the use of HTP and often celite columns as well, al- though different experimental conditions and various special tricks for every single carrier protein were neces- sary [1,2]. Fig. 1, lane A shows the polypeptide pattern of a characteristic HTP eluate from solubilized rat liver mitochondria. All the major bands have been clearly correlated to definite carrier proteins mainly by our group. Partial purification of the CAC has been re- M A B 30 kOa mm Fig. 1. Purification of the camitine carrier from rat liver mito- chondria. SDS gel electrophoresis of (A) HTP eluate (10 #1) obtained from Triton X-100 solubilized rat liver mitochondria; (B) fraction (300 #1) containing pure CAC eluted from celite with Triton X-100 buffer supplemented with 1.6 mg/ml cardiolipin; (M) protein markers (bovine serum albumin, carbonic anhydrase and cytochrome c). 0005-2728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)