title: Pathogenic potential of SLC25A15 mutations assessed by transport assays and complementation of Saccharomyces cerevisiae ORT1 null mutant.
abstract: HHH syndrome is an autosomal recessive urea cycle disorder caused by alterations in the SLC25A15 gene encoding the mitochondrial ornithine carrier 1, which catalyzes the transport of cytosolic ornithine into the mitochondria in exchange for intramitochondrial citrulline. In this study the functional effects of several SLC25A15 missense mutations p.G27R, p.M37R, p.N74A, p.F188L, p.F188Y, p.S200K, p.R275Q and p.R275K have been tested by transport assays in reconstituted liposomes and complementation of Saccharomyces cerevisiae ORT1 null mutant in arginine-less synthetic complete medium. The HHH syndrome-causing mutations p.G27R, p.M37R, p.F188L and p.R275Q had impaired transport and did not complement ORT1∆ cells (except p.M37R slightly after 5 days in solid medium). The experimentally produced mutations p.N74A, p.S200K and p.R275K exhibited normal or considerable transport activity and complemented ORT1∆ cells after 3 days (p.N74A, p.S200K) or 5 days (p.R275K) incubation. Furthermore, the experimentally produced p.F188Y mutation displayed a substantial transport activity but did not complement the ORT1∆ cells in both liquid and solid media. In view of the disagreement in the results obtained between the two methods, it is recommended that the method of complementing the S. cerevisiae ORT1 knockout strain is used complimentary with the measurement of the catalytic activity, in order to distinguish HHH syndrome-causing mutations from isomorphisms.
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Carlo M T
authors.initials: CM
authors.lastname: Marobbio
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Punzi
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Ciro L
authors.initials: CL
authors.lastname: Pierri
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Palmieri
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Rosa
authors.initials: R
authors.lastname: Calvello
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy.
authors.forename: Maria A
authors.initials: MA
authors.lastname: Panaro
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy. Electronic address: ferdpalmieri@gmail.com.
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics* / Q000378:metabolism*; D001120:Arginine; D001692:Biological Transport; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D022124:Hyperammonemia / Q000235:genetics; D008081:Liposomes / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics*; D020125:Mutation, Missense; D009952:Ornithine / Q000172:deficiency / Q000235:genetics; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D056806:Urea Cycle Disorders, Inborn / Q000235:genetics
chemical_list: D026923:Amino Acid Transport Systems, Basic; D003470:Culture Media; D008081:Liposomes; D033681:Mitochondrial Membrane Transport Proteins; C101833:ORT1 protein, S cerevisiae; D011994:Recombinant Proteins; C442901:SLC25A15 protein, human; D029701:Saccharomyces cerevisiae Proteins; D001120:Arginine; D009952:Ornithine
keywords: HHH syndrome; Membrane transport; Mitochondrial carrier; Mitochondrial ornithine carrier; Mitochondrial transporter; Mutations, SLC25A15