Introducing the human Leigh syndrome mutation T9176G into Saccharomyces cerevisiae mitochondrial DNA leads to severe defects in the incorporation of Atp6p into the ATP synthase and in the mitochondrial morphology

Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.

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Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

Genetics ◽

10.1093/genetics/154.1.147 ◽

2000 ◽

Vol 154 (1) ◽

pp. 147-154 ◽

Cited By ~ 1

Author(s):

Douglas J Kominsky ◽

Peter E Thorsness

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Schizosaccharomyces Pombe ◽

Atp Synthase ◽

Kluyveromyces Lactis ◽

Blot Hybridization ◽

Inner Mitochondrial Membrane ◽

Yeast Saccharomyces Cerevisiae ◽

Mitochondrial Atp Synthase ◽

Petite Negative Yeast

Abstract Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the α- and γ-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

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Functional Analysis of Subunit e of the F1Fo-ATP Synthase of the Yeast Saccharomyces cerevisiae: Importance of the N-Terminal Membrane Anchor Region

Eukaryotic Cell ◽

10.1128/ec.4.2.346-355.2005 ◽

2005 ◽

Vol 4 (2) ◽

pp. 346-355 ◽

Cited By ~ 46

Author(s):

Valerie Everard-Gigot ◽

Cory D. Dunn ◽

Brigid M. Dolan ◽

Susanne Brunner ◽

Robert E. Jensen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Atp Synthase ◽

Mitochondrial Membrane ◽

Critical Role ◽

Coiled Coil ◽

Mitochondrial Morphology ◽

Hydrophobic Region ◽

Inner Membrane ◽

E Protein ◽

Subunit E

ABSTRACT Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.

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A model of mitochondrial ATP synthase deficiencies. The role of mitochondrial carriers.

10.1101/2021.07.13.452228 ◽

2021 ◽

Author(s):

Jean-Pierre Mazat ◽

Anne Devin ◽

Edgar Yoboue ◽

Stephane Ransac

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Atp Synthase ◽

Yeast Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Complex Iv ◽

Mitochondrial Atp Synthase ◽

Atp6 Gene ◽

Substrate Level

The m.8993T>G mutation of the mitochondrial MT-ATP6 gene is associated with NARP syndrome (Neuropathy, Ataxia and Retinitis Pigmentosa). The equivalent point mutation introduced in yeast Saccharomyces cerevisiae mitochondrial DNA considerably reduced the activity of ATP synthase and of cytochrome-C-oxidase preventing yeast growth on oxidative substrates. The overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) is able to rescue the growth on oxidative substrate in stimulating the substrate-level phosphorylation of ADP cou-pled to conversion of α-ketoglutarate (AKG) into succinate with an increase in Complex IV activity. In order to better understand the mechanism of ATP synthase mutation bypass, we developed a core model of mitochondrial metabolism based on AKG as respiratory substrate. We describe the different possible metabolite output and the ATP/O ratio values as a function of ATP synthase inhibition.

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Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism

Human Molecular Genetics ◽

10.1093/hmg/ddab299 ◽

2021 ◽

Author(s):

Sebastian Kenvin ◽

Ruben Torregrosa-Muñumer ◽

Marco Reidelbach ◽

Jana Pennonen ◽

Jeremi J Turkia ◽

...

Keyword(s):

Cell Fate ◽

Atp Synthase ◽

Motor Neurons ◽

Mitochondrial Diseases ◽

Leigh Syndrome ◽

Cellular Reprogramming ◽

Patient Specific ◽

Mitochondrial Morphology ◽

Cell Fate Decisions ◽

Truncating Mutation

Abstract Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

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Mitochondrial DNA duplication, recombination, and introgression during interspecific hybridization

Scientific Reports ◽

10.1038/s41598-021-92125-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Silvia Bágeľová Poláková ◽

Žaneta Lichtner ◽

Tomáš Szemes ◽

Martina Smolejová ◽

Pavol Sulo

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Interspecific Hybridization ◽

Mobile Elements ◽

Variable Length ◽

Mitochondrial Genomes ◽

Free Standing ◽

Saccharomyces Paradoxus ◽

Mtdna Recombination

AbstractmtDNA recombination events in yeasts are known, but altered mitochondrial genomes were not completed. Therefore, we analyzed recombined mtDNAs in six Saccharomyces cerevisiae × Saccharomyces paradoxus hybrids in detail. Assembled molecules contain mostly segments with variable length introgressed to other mtDNA. All recombination sites are in the vicinity of the mobile elements, introns in cox1, cob genes and free standing ORF1, ORF4. The transplaced regions involve co-converted proximal exon regions. Thus, these selfish elements are beneficial to the host if the mother molecule is challenged with another molecule for transmission to the progeny. They trigger mtDNA recombination ensuring the transfer of adjacent regions, into the progeny of recombinant molecules. The recombination of the large segments may result in mitotically stable duplication of several genes.

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Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa119 ◽

2020 ◽

Vol 85 (4) ◽

pp. 895-901

Author(s):

Takamitsu Amai ◽

Tomoka Tsuji ◽

Mitsuyoshi Ueda ◽

Kouichi Kuroda

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Ethidium Bromide ◽

Nuclear Genome ◽

Target Sequence ◽

Guide Rna ◽

Crispr System ◽

Mitochondrial Target ◽

Gene Treatment ◽

Mtdna Deletion

ABSTRACT Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a “mito-CRISPR system” that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

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