Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans

Abstract

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.

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

“…The crucial role of arginine 179 was elucidated which likely affects the substrate-binding pocket altering the affinity for ornithine. All the disease-causing alleles had a detrimental effect on the ability of the hORNT1 to complement the deletion of Arg11 [ 81 ]. Then, mutated TMEM165 (SLC64A1) has been expressed in S. cerevisiae strain deleted of the yeast Golgi Ca 2+ and Mn 2+ transporters, Gdt1p and Pmr1 [ 105 ].…”

Motor axonal neuropathy associated with GNE mutations

“…Pathogenicity prediction was performed using VarSome (https://varsome.com/). 6 Molecular modeling was performed as described 7 using the Protein Data Bank 4ZHT structure.…”

Learning from Yeast about Mitochondrial Carriers

“…All mutations with exception of Gln246Lys reduced or abrogated the ability of Orc1p to support growth of the cells. Only the Met37Arg and Leu71Gln Orc1 mutants partially restored growth ability [199].…”

“…S. cerevisiae was used to study the role of SLC25A2 (Orc2, Ornt2) and the function of Orc1 missense mutations [199]. Orc2p, which has the 88% identity with Orc1p, was suggested to act as a second mitochondrial ornithine carrier, in part responsible for the milder phenotype in HHH patients [200].…”