Biochimica et Biophysica Acta, 1189 (1994) 65-73 65 © 1994 Elsevier Science B.V. All rights reserved 0005-2736/94/$07.00 BBAMEM 76178 The reconstituted carnitine carrier from rat liver mitochondria: evidence for a transport mechanism different from that of the other mitochondrial translocators Cesare Indiveri a,b, Annamaria Tonazzi a and Ferdinando Palmieri a,, a Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Traversa 200 Re David, 4, 70125 Bari (Italy) and b CNR Unit for the Study of Mitochondria and Bioenergetics, Bari (Italy) (Received 8 June 1993) Key words: Mitochondrion; Reconstitution; Carnitine carrier; Transport mechanism;(Rat liver) The transport mechanism of the reconstituted carnitine carrier purified from rat liver mitochondria was investigated kinetically. The half-saturation constant (K m) for carnitine on the internal side of the liposomal membrane (8.7 mM) was found to be much higher than that determined for the external surface (0.45 mM). The exclusive presence of a single transport affinity for carnitine on each side of the membrane indicated a unidirectional insertion of the carnitine carrier into the proteoliposomes, most probably right-side-out with respect to mitochondria. Under these defined conditions bisubstrate initial velocity studies of homologous (carnitine/carnitine) and heterologous (carnitine/acylcarnitine) antiport were performed by varying both the internal and external substrate concentrations. The kinetic patterns obtained showed that the ratio Km/Vma x is not influenced by the second (non-varied) substrate, which indicates a ping-pong mechanism. The carnitine carrier thus differs from all other mitochondrial carriers analyzed so far in the reconstituted state, for which a common sequential type of reaction mechanism has been found. Introduction The inner mitochondrial membrane contains a spe- cific carrier system for the transport of carnitine and acylcarnitines, known as the carnitine carrier (for re- view, see Ref. 1). This system plays a central role in the translocation of fatty acids via acylcarnitines into the mitochondrial matrix, where the acyl groups are re- leased to be used for fatty acid oxidation. The proper- ties of the carnitine carrier have been extensively inves- tigated in intact mitochondria [2-10]. This carrier cat- alyzes a 1 : 1 exchange between acylcarnitines and car- nitine, which is inhibited by SH reagents like mersalyl and N-ethylmaleimide and by substrate analogues like sulphobetaines. Furthermore, besides the exchange re- action a unidirectional transport of carnitine is cat- alyzed with lower activity. Recently, we have identified and purified the carnitine carrier protein from rat liver mitochondria [11]. When functionally reconstituted, the purified protein with an apparent molecular mass of 32.5 kDa mediated the exchange reaction between * Corresponding author. Fax + 39 80 242770. Abbreviations: DTE, dithioerythritol; Pipes, 1,4-piperazinediethane- sulphonic acid. carnitine and acylcarnitines, as well as the uniport mode of transport [12,13]. The substrate specificity and inhibitor sensitivity were similar to those found in intact mitochondria [11-13]. In addition, in the recon- stituted system we were able to discriminate two classes of SH-groups that are functionally important for the transport activity of the carnitine carrier protein [14]. Mainly based on studies in reconstituted systems it has been proposed that the mitochondrial carriers, besides forming a structural family [15-17], also consti- tute a homogenous functional group of proteins, char- acterized by a common sequential type of reaction mechanism [1]. For a detailed functional characteriza- tion of the reconstituted carnitine carrier, and for comparison with other members of the mitochondrial carrier family, it is important to establish the orienta- tion of the inserted protein. Moreover, investigation of the transport mechanism is of particular interest in the case of the carnitine carrier since it is the only known mitochondrial transport system which is able to cat- alyze both electroneutral exchange and uniport in a non H+-compensated manner. In this paper, by apply- ing the two-substrate kinetic analysis, it was possible to determine whether one (ping-pong mechanism) or two binding sites (sequential mechanism) have to be occu- SSDI 0005-2736(93)E0288-L