12 research outputs found
Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins".
The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane α-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established. © 2006 Elsevier B.V. All rights reserved
Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection
The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms
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Microfluidic Formulation of Diazoxide-Loaded Solid Lipid Nanoparticles as a Novel Approach for Friedreich's Ataxia Treatment
Data availability:
Data will be made available on request.Friedreich ataxia (FRDA) is a hereditary autosomal recessive disorder characterized by frataxin deficiency, impacting mitochondrial function and causing oxidative damage. Diazoxide (DZX), a vasodilating drug used in the management of systemic hypertension, has shown promise in preclinical models but faces challenges in crossing the blood-brain barrier and potential toxicity at higher doses. This study aimed to create solid lipid nanoparticles (SLNs) loaded with DZX by microfluidic technique to improve blood-brain barrier (BBB) penetration and reduce side effects. Employing an in vitro BBB model, SLN-DZX demonstrated enhanced permeability compared to plain DZX. Cell viability assays carried out on FRDA fibroblast cells indicated enhanced viability with 1 μM SLN-DZX. Cellular uptake studies confirmed SLN internalization in FRDA fibroblasts, and subsequent treatment with SLN-DZX significantly reduced both total and mitochondrial ROS levels compared to control and empty SLN-treated cells. These findings suggest SLN-DZX as a potential therapeutic approach for FRDA, mitigating oxidative stress with improved BBB penetration and reduced toxicity.M.I.U.R.—Programma Operativo Nazionale (PON) “Ricerca e Innovazione” 2014–2020 Tematica IV.4 “Dottorati e Contratti di ricerca su tematiche dell’innovazione”
Heterologous Expression in Yeast of Human Ornithine Carriers ORNT1 and ORNT2 and of ORNT1 Alleles Implicated in HHH Syndrome in Humans
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