High Level Expression and Characterization of the Mitochondrial Citrate Transport Protein from the Yeast Saccharomyces cerevisiae

Abstract

The gene encoding the mitochondrial citrate transport protein (CTP) in the yeast Saccharomyces cerevisiae has been identified, and its protein product has been overexpressed in Escherichia coli. The expressed CTP accumulates in inclusion bodies and can be solubilized with sarkosyl. Approximately 25 mg of solubilized CTP at a purity of 75% is obtained per liter of E. coli culture. The function of the solubilized CTP has been reconstituted in a liposomal system where both its kinetic parameters (i.e. Km = 0.36 mM and Vmax = 2.5 mumol/min/mg protein) and its substrate specificity have been determined. Notably, the yeast CTP displays a stricter specificity for tricarboxylates than do CTPs from higher eukaryotic organisms. Dot matrix analysis of the yeast CTP sequence indicates the presence of three homologous sequence domains (each approximately 100 residues in length), which are also related to domains in other CTPs. Thus, the yeast CTP displays the tripartite structure characteristic of other mitochondrial transporters. Alignment of the yeast CTP sequence with CTPs from other sources defines a consensus sequence that displays 89 positions of amino acid identity, as well as the more generalized mitochondrial transporter-associated sequence motif. Based on hydropathy analysis, the yeast CTP contains six putative membrane-spanning alpha-helices. Finally, Southern blot analysis indicates that the yeast genome contains a single gene encoding the mitochondrial CTP. Our data indicate that, based on both its structural and functional properties, the expressed yeast CTP can be assigned membership in the mitochondrial carrier family. The identification of the yeast CTP gene, and the expression and purification of large quantities of its protein product, pave the way for investigations into the roles of specific amino acids in the CTP translocation mechanism, as well as for the initiation of crystallization trials.

The Yeast Mitochondrial Citrate Transport Protein

“…Thus, it has been purified (6, 7), kinetically characterized (8), cloned (9), and overexpressed (10). More recently we have extended our studies of the CTP to the yeast homologue of the higher eukaryotic protein (11). An advantage afforded by the yeast mitochondrial CTP is that following overexpression in Escherichia coli and subsequent isolation, CTP function can be reconstituted with high specific activity.…”

“…2 Moreover, we have demonstrated that upon isolation, both the isolated wild-type and Cys-less CTPs exist as functional dimers. 3 Based on hydropathy analysis, each monomer of the homodimer is predicted to contain six membrane-spanning domains (11).…”

The mitochondrial citrate carrier: Metabolic role and regulation of its activity and expression

“…Besides the rat, CiC cDNA has been isolated and sequenced from a variety of species (15,38,39). Palmieri and coworkers (40) determined the complete sequence of the human Cic gene.…”

“…CiC was first discovered in liver by Chappel and Haarhoff (9), so far different groups have purified this carrier to homogeneity from hepatic mitochondria of rat (10)(11)(12), bovine (13), eel (14), yeast (15), and maize (16). The first purification of CiC from rat-liver mitochondria was carried out in the presence of the nonionic detergent Triton X-100 followed by chromatography on hydroxyapatite/celite (10).…”

Manganese activation of superoxide dismutase 2 in Saccharomyces cerevisiae requires MTM1 , a member of the mitochondrial carrier family

“…Eukaryotes typically express Ϸ30-40 distinct MCF proteins, and although substrates for many have been identified [e.g., components of the tricarboxylic acid cycle, ADP͞ATP, flavins, etc. (24)(25)(26)], the substrates for the bulk of MCF transporters are unknown. In addition to the MCF proteins, 10 ATP-binding cassette transporters, 6 P-type ATPases, and 3 other transporters were tested for their effects on SOD2 activity (Table 1).…”