The mitochondrial citrate transport protein: Evidence for a steric interaction between glutamine 182 and leucine 120 and its relationship to the substrate translocation pathway and identification of other mechanistically essential residues

Abstract

Previous examination of the accessibility of a panel of single-Cys mutants in transmembrane domain III (TMDIII) of the yeast mitochondrial citrate transport protein to the hydrophilic, cysteine-specific methanethiosulfonate reagent MTSES enabled identification of the water-accessible surface of this TMD. Further studies on the effect of citrate on MTS reagent accessibility, indicated eight sites within TMD III at which citrate conferred temperature-independent protection, thus providing strong evidence for participation of these residues in the formation of a portion of the substrate translocation pathway. Unexpectedly, citrate did not protect against inhibition of the Leu120Cys variant, despite its location on a water- and citrate-accessible surface of the TMDIII helix. This led to the hypothesis that in the 3-dimensional CTP structure, TMDIV packs against TMDIII in a manner such that the Leu120 side-chain folds behind the side-chain of Gln182. The present investigations addressed this hypothesis by examining the properties of the Gln182Cys single mutant and the Leu120Cys/Gln182Ala double mutant. We observed that in contrast to our findings with the Leu120Cys mutant, citrate did protect the Gln182Cys variant against MTSES-mediated inhibition. Importantly, truncation of the Gln182 side-chain to Ala enabled citrate to protect the Leu120Cys double mutant against inhibition. In combination these data support the idea that the Gln182 side-chain lines the transport path and sterically blocks access of citrate to the Leu120 side-chain. In a parallel series of investigations, we constructed 24 single-Cys substitution mutants that were chosen based on their hypothesized importance in substrate binding and/or translocation. We observed that substitution of Cys for residues E34, K37, K83, R87, Y148, D236, K239, T240, R276, and R279 resulted in > or =98% inactivation of CTP function, suggesting an essential structural and/or mechanistic role for these native residues. Superposition of this functional data onto a detailed 3-dimensional homology model of the CTP structure indicates that the side-chains of each of these residues project into the putative transport pathway. We hypothesize that a subset of these residues, in combination with four previously identified essential residues, define the citrate binding site(s) within the CTP.

Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein

“…Thus, Leu-116, Gly-119, Leu-120, Ser-123, Gln-182, Asn-185, and Gln-186 were predicted to be near binding site one and residues Glu-34, Phe-76, Glu-131, Lys-134, Thr-228, Val-229, Asp-236, Thr-240, and Gln-243 were predicted to be near binding site two. The effect of Cys substitution at several of these locations had been characterized previously as part of our studies into the role of TMDs III and IV in CTP function (15,18). The third group consisted of selected residues in matrix loops A (i.e.…”

“…For each cluster, the most energetically favorable orientation was selected, producing two final proposed binding sites as described below. (11,14,18) utilizing the Cys-less CTP as the template. Mutations of four groups of residues were characterized.…”

“…With this background in mind, the current studies focused on characterizing the kinetic properties of a panel of singleCys mutants that we had previously constructed based on their predicted importance in the CTP mechanism (11,14,18). We identified ten mutations that display both substantially elevated K m values and reduced V max values, nine of which localize to two topographically distinct clusters within the CTP structure.…”

“…Thus we have cloned (6), overexpressed (7,8), and purified (9, 10) the functional form of this transporter. Recently, employing a Cys-less yeast mitochondrial CTP construct that displays native functional properties (11) as the template, we have: (i) demonstrated that the transporter exists as a homodimer in detergent micelles (12); (ii) utilized cysteine-scanning mutagenesis combined with probing the accessibility of single-Cys mutants to MTS reagents, in both the absence and presence of citrate, to identify those residues in transmembrane domains III and IV that line the substrate translocation pathway (13-15); (iii) developed a detailed homology model of the three-dimensional structure of the CTP (16) based on the crystal structure of the mitochondrial ADP/ATP carrier (17); and (iv) superimposed our functional data onto this homology model to delineate substantial portions of the translocation pathway within the structure (15,16).With this background in mind, the current studies focused on characterizing the kinetic properties of a panel of singleCys mutants that we had previously constructed based on their predicted importance in the CTP mechanism (11,14,18). We identified ten mutations that display both substantially elevated K m values and reduced V max values, nine of which localize to two topographically distinct clusters within the CTP structure.…”

The Yeast Mitochondrial Citrate Transport Protein

“…Gly-119 is the sole exception as its role in substrate binding is indirect; its lack of a side chain permits the proper packing of TMDs II and III in the formation of site 1, thereby allowing space for the Arg-87 side chain to interact with citrate. Furthermore, we believe that the assertion by Robinson and Kunji (32, 37) that Gln-182 is a key residue in substrate binding is likely to be incorrect based on our earlier findings that the Q182C mutant displays reduced K m and V max values, with the reduction in the latter parameter being considerably more pronounced (22,23). Our findings suggest that Gln-182 is primarily involved in the conformational changetriggering mechanism that allows citrate to be transported, rather than in substrate binding.…”