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.

Competing interests: None declared

Appendices

Sentence Take-Home Message

Heterologous yeast expression of the human ornithine transporters reveals that ORNT2 cannot vicariate for the deletion of the yeast homolog suggesting that ORNT1 and ORNT2 have different functions determined by few key residues; moreover this system can be used to distinguish pathogenic ORTN1 mutations from neutral polymorphisms.

Compliance with Ethics and Guidelines

This article does not contain any studies with human or animal subjects performed by any of the authors.

Conflict of Interest Statement

Mara Doimo, Raffaele Lopreiato, Valentina Basso, Raissa Bortolotto, Alessandra Tessa, Filippo M. Santorelli, Eva Trevisson, and Leonardo Salviati declare that they have no conflict of interest.

Author Contribution

MD concepted, designed, and performed experiments, analyzed data, and drafted the article; RL concepted the experiments, analyzed data, and drafted the article; VB performed experiments; RB performed experiments; AT critically revised the article; FMS critically revised the article; ET drafted and critically revised the article; and LS concepted the experiment and drafted the article.