The use of yeast mitochondria to study the properties of wild-type and mutant human mitochondrial ornithine transporter.

Abstract

Yeast deletion models have general utility for the study of a variety of inherited metabolic disorders. Mutations in the mitochondrial ornithine transporter result in hyperammonemia, hyperornithinemia, homocitrullinuria syndrome, a disorder of the urea cycle. To study the effects of mutations in a model system that more closely resembles the in vivo environment, we have developed an expression system based on a yeast strain lacking its endogenous ornithine transporter homologue. Wild-type human ornithine transporter and a recurrent mutation, DeltaF 188, were expressed and characterized with this system. The wild-type transporter appeared to insert into yeast mitochondria in the same orientation as in mammalian mitochondria. It showed stereospecificity, strong antiport activity and ornithine transport was competed by citrulline and arginine. The DeltaF 188 mutant was not incorporated into the membrane to the same extent as wild type, but retained significant residual activity and lost stereospecificity. In these isolated mitochondria, samarium chloride was found to be a potent blocker of transport compared to previously reported sulfhydryl-based inhibitors. A low-affinity background transport activity that promoted the exchange of ornithine for either acidic or basic amino acids was observed. This yeast model can readily be extended to the study of protein:protein interactions. In this manner, the use of yeast deletion strains can serve as a general framework to perform metabolic pathway analysis.

• Authors: Hiroki Morizono, Joelle E Woolston, Marco Colombini, Mendel Tuchman
• Journal: Molecular genetics and metabolism (Mol Genet Metab) Vol. 86 Issue 4 Pg. 431-40 (Dec 2005) ISSN: 1096-7192 [Print] United States
• PMID: 16256388 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Amino Acid Transport Systems, Basic
• Chlorides
• DNA, Complementary
• Mitochondrial Membrane Transport Proteins
• Proteins
• Recombinant Proteins
• SLC25A15 protein, human
• ornithine transporter
• Samarium
• Ornithine

Topics

• Amino Acid Transport Systems, Basic (antagonists & inhibitors, chemistry, genetics, metabolism)
• Base Sequence
• Biological Transport, Active (drug effects)
• Chlorides (pharmacology)
• Cloning, Molecular
• DNA, Complementary (genetics)
• Humans
• In Vitro Techniques
• Mitochondria (metabolism)
• Mitochondrial Membrane Transport Proteins
• Models, Molecular
• Mutation
• Ornithine (metabolism)
• Protein Conformation
• Proteins (antagonists & inhibitors, chemistry, genetics, metabolism)
• Recombinant Proteins (antagonists & inhibitors, chemistry, genetics, metabolism)
• Saccharomyces cerevisiae (genetics, metabolism)
• Samarium (pharmacology)