title: The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.
abstract: The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: Sreevidya
authors.initials: S
authors.lastname: Aluvila
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: Rusudan
authors.initials: R
authors.lastname: Kotaria
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: Jiakang
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: June A
authors.initials: JA
authors.lastname: Mayor
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: D Eric
authors.initials: DE
authors.lastname: Walters
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: David H T
authors.initials: DHT
authors.lastname: Harrison
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064. Electronic address: ronald.kaplan@rosalindfranklin.edu.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Kaplan
mesh_terms: D019943:Amino Acid Substitution; D000838:Anions / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019343:Citric Acid / Q000737:chemistry / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000838:Anions; D002352:Carrier Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C020723:citrate-binding transport protein; D019343:Citric Acid