Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome is an autosomal recessive metabolic disorder usually presenting in the neonatal period with intermittent episodes of hyperammonemia, psychomotor delay, and progressive encephalopathy. Adult cases usually evolve into frank spastic paraparesis. The syndrome is caused by mutations in SLC25A15/ORNT1 encoding the mitochondrial ornithine transporter; a second ornithine transporter, ORNT2 of unknown function, is also present in most placental mammals. ORNT2 is believed to originate from an ancient retro-transposition event. In yeast Saccharomyces cerevisiae the major function of the transporter (encoded by Arg11) is to shuttle ornithine from the mitochondrial matrix to the cytosol. Its inactivation abolishes growth in the absence of arginine.
In this work, we used functional complementation in S. cerevisiae to characterize the function of human ORNT2 and to test the pathogenicity of ORNT1 mutations found in HHH patients. Notably, we found that human ORNT1 but not ORNT2 complements the deletion of the yeast gene, despite their high level of homology. However, we identified some key residues in ORNT2, which may recover its functional competence when replaced with the corresponding residues of ORNT1, suggesting that roles of the two transporters are different. Moreover, we used this system to test a series of missense mutations of ORNT1 identified in patients with HHH syndrome. All mutations had a detrimental effect on the functionality of the human gene, without however clear genotype–phenotype correlations. Our data support yeast as a simple and effective model to validate missense mutations occurring in patients with HHH.
HHH Ornithine ORNT1 ORNT2 Urea cycle disorder Yeast model
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This work was supported in part by grants from Telethon Italy (GGP13222), the University of Padova (CPDA123573/12), and Fondazione CARIPARO (to L.S.); we are grateful to Martina Frizzarin and Paolo Lorenzon for their experimental support.
Camacho JA, Rioseco-Camacho N (2009) The human and mouse SLC25A29 mitochondrial transporters rescue the deficient ornithine metabolism in fibroblasts of patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome. Pediatr Res 66:35–41CrossRefPubMedGoogle Scholar
Camacho J, Rioseco-Camacho N (2012) Hyperornithinemia-hyperammonemia-homocitrullinuria. In: Gene reviews at gene tests medical genetics information resource (database online). Copyright, University of Washington, Seattle. 1997–2013. http://www.genetests.org. Accessed 6 Nov 2015
Camacho JA, Obie C, Biery B et al (1999) Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter. Nat Genet 22:151–158CrossRefPubMedGoogle Scholar
Camacho JA, Rioseco-Camacho N, Andrade D, Porter J, Kong J (2003) Cloning and characterization of human ORNT2: a second mitochondrial ornithine transporter that can rescue a defective ORNT1 in patients with the hyperornithinemia-hyperammonemia-homocitrullinuria syndrome, a urea cycle disorder. Mol Genet Metab 79:257–271CrossRefPubMedGoogle Scholar
Cheung CW, Cohen NS, Raijman L (1989) Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J Biol Chem 264:4038–4044PubMedGoogle Scholar
Crabeel M, Soetens O, De Rijcke M, Pratiwi R, Pankiewicz R (1996) The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis. J Biol Chem 271:25011–25018CrossRefPubMedGoogle Scholar
Ersoy Tunali N, Marobbio CM, Tiryakioglu NO et al (2014) A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: functional analysis of the mutant protein. Mol Genet Metab 112:25–29CrossRefPubMedPubMedCentralGoogle Scholar
Fecarotta S, Parenti G, Vajro P et al (2006) HHH syndrome (hyperornithinaemia, hyperammonaemia, homocitrullinuria), with fulminant hepatitis-like presentation. J Inherit Metab Dis 29:186–189CrossRefPubMedGoogle Scholar
Fiermonte G, Dolce V, David L et al (2003) The mitochondrial ornithine transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms. J Biol Chem 278:32778–32783CrossRefPubMedGoogle Scholar
Marobbio CM, Punzi G, Pierri CL, Palmieri L, Calvello R, Panaro MA, Palmieri F (2015) Pathogenic potential of SLC25A15 mutations assessed by transport assays and complementation of Saccharomyces cerevisiae ORT1 null mutant. Mol Genet Metab 115:27–32CrossRefPubMedGoogle Scholar
Monne M, Miniero DV, Daddabbo L, Robinson AJ, Kunji ER, Palmieri F (2012) Substrate specificity of the two mitochondrial ornithine carriers can be swapped by single mutation in substrate binding site. J Biol Chem 287:7925–7934CrossRefPubMedPubMedCentralGoogle Scholar
Porcelli V, Fiermonte G, Longo A, Palmieri F (2014) The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids. J Biol Chem 289:13374–13384CrossRefPubMedPubMedCentralGoogle Scholar
Soetens O, Crabeel M, El Moualij B, Duyckaerts C, Sluse F (1998) Transport of arginine and ornithine into isolated mitochondria of Saccharomyces cerevisiae. Eur J Biochem 258:702–709CrossRefPubMedGoogle Scholar
Tessa A, Fiermonte G, Dionisi-Vici C et al (2009) Identification of novel mutations in the SLC25A15 gene in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome: a clinical, molecular, and functional study. Hum Mutat 30:741–748CrossRefPubMedGoogle Scholar
Trevisson E, Burlina A, Doimo M, Pertegato V, Casarin A, Cesaro L, Navas P, Basso G, Sartori G, Salviati L (2009) Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations. J Biol Chem 284:28926–28934CrossRefPubMedPubMedCentralGoogle Scholar
Valle D, Simell O (eds) (2001) The hyperornithinemias. McGraw Hill, New YorkGoogle Scholar