A vital function for mitochondrial DNA in the petite-negative yeastKluyveromyces lactis

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeastKluyveromyces lactis, specific mutations in the nuclear genesMGI2 andMGI5 encoding the- and-subunits of the mitochondrial F₁-ATPase, allow mtDNA to be lost. In this study we show that wild-typeK. lactis does not survive in the absence of its mitochondrial genome and that the function ofmgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing amgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned theK. lactis homologue of theSaccharomyces cerevisiae mitochondrial genome maintenance geneMGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption ofMGM101 is not lethal in a haploidmgi strain with a specific mutation in a subunit of the mitochondrial F₁-ATPase. These observations suggest that mtDNA inK. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F₀.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle. Received: 7 April 1998 / Accepted: 10 June 1998     

Cloning and characterization of the lipoyl-protein ligase gene LIPB from the yeast Kluyveromyces lactis : synergistic respiratory deficiency due to mutations in LIPB and mitochondrial F1-ATPase subunits

The mgi1-4 and mgi2-1 mutants of the petite-negative yeast Kluyveromyces lactis have mutations in the β- and α-subunits of the mitochondrial F₁-ATPase, respectively. The mutants are respiratory competent but can form petites with deletions in mitochondrial DNA. In this study a cryptic nuclear mutation (lipB-1) was identified which, in combination with the mgi alleles, displays a synergistic respiratory-deficient phenotype on glycerol medium. The gene defined by the mutation was cloned and shown to encode a polypeptide of 332 amino acids with an N-terminal sequence characteristic of a mitochondrial targeting signal. The deduced protein shares 27% sequence identity with the product of the Escherichia coli lipB gene, which encodes a lipoyl-protein ligase involved in the attachment of lipoyl groups to lipoate-dependent apoproteins. A K. lactis strain carrying a disrupted lipB allele is severely compromised for growth on glycerol medium. The growth defect cannot be rescued by addition of lipoic acid, but cell growth can be restored on medium containing ethanol plus succinate. In addition, it was observed that lipB mutants of K. lactis, unlike the wild-type, are unable to utilize glycine as sole nitrogen source, indicating that activity of the glycine decarboxylase complex (GDC) is also affected. Taken together, these findings suggest that LIPB is the main determinant of the lipoyl-protein ligase activity required for lipoylation of enzymes such as α-ketoacid dehydrogenases and GDC.     

Cloning and characterization of the lipoyl-protein ligase gene LIPB from the yeast Kluyveromyces lactis : synergistic respiratory deficiency due to mutations in LIPB and mitochondrial F1-ATPase subunits

The mgi1-4 and mgi2-1 mutants of the petite-negative yeast Kluyveromyces lactis have mutations in the β- and α-subunits of the mitochondrial F₁-ATPase, respectively. The mutants are respiratory competent but can form petites with deletions in mitochondrial DNA. In this study a cryptic nuclear mutation (lipB-1) was identified which, in combination with the mgi alleles, displays a synergistic respiratory-deficient phenotype on glycerol medium. The gene defined by the mutation was cloned and shown to encode a polypeptide of 332 amino acids with an N-terminal sequence characteristic of a mitochondrial targeting signal. The deduced protein shares 27% sequence identity with the product of the Escherichia coli lipB gene, which encodes a lipoyl-protein ligase involved in the attachment of lipoyl groups to lipoate-dependent apoproteins. A K. lactis strain carrying a disrupted lipB allele is severely compromised for growth on glycerol medium. The growth defect cannot be rescued by addition of lipoic acid, but cell growth can be restored on medium containing ethanol plus succinate. In addition, it was observed that lipB mutants of K. lactis, unlike the wild-type, are unable to utilize glycine as sole nitrogen source, indicating that activity of the glycine decarboxylase complex (GDC) is also affected. Taken together, these findings suggest that LIPB is the main determinant of the lipoyl-protein ligase activity required for lipoylation of enzymes such as α-ketoacid dehydrogenases and GDC. Received: 16 December 1996 / Accepted: 24 February 1997     

The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA

The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.     

Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria

The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F₀F₁-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.     

Interspecies sex-specific growth responses inKluyveromyces

The sex-specific growth factors produced by thea and mating types ofKluyveromyces lactis were examined for interspecies cross-reactivity on growing cells ofKluyveromyces aestuarii andKluyveromyces delphensis. Taxonomically,K. aestuarii is closely related toK. lactis;K. delphensis is more distantly related. Reciprocal growth responses occurred betweenK. aestuarii and the a mating type ofK. lactis; no response was evident betweenK. delphensis and either sex ofK. lactis.     

Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component,Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ_m) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F_oF₁-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ_m that manifests as a decreased growth phenotype, indicating that the F_oF₁-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F_o-moiety. These Dk cells generate the Δψ_m by combining the hydrolytic activity of the matrix-facing F₁-ATPase and the electrogenic exchange of ATP⁴- for ADP³- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F_oF₁-ATPase complexes were identified. In fact, the immunoprecipitation of a F₁-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ_m of Dk cells. Thus, even in the absence of subunit a, a portion of the F_oF₁-ATPase is assembled in Dk cells.     

α and β subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ^?) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ^? and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ? subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ^? or ρ^o mutations in S. cerevisiae.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F₁ ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β_DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.     

Identification of the F1-ATPase at the Cell Surface of Colonic Epithelial Cells: ROLE IN MEDIATING CELL PROLIFERATION*

F1 domain of F₁F_o-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F₁-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F₁-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F₁-ATPase and the amino acids in the F₁-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F₁-ATPase and the loss of its biological activity. In addition we demonstrated that F₁-ATPase mediates the growth effects of the peptide. Indeed, blocking F₁-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F₁-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F₁-ATPase in the pro-proliferative action of this gastrointestinal peptide.     

Genetic structure of natural populations ofDryas iulia (Lepidoptera: Nymphalidae) revealed by enzyme polymorphism and mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP)

Dryas iulia appears to have undergone a mode of evolution different from that of other members of its subfamily (Heliconiinae). While other species constitute highly subdivided and inbred populations, those ofD. iulia are thought to be large and uniform. Analyzing six samples from Southern Brazil (state of Rio Grande do Sul) in relation to three enzyme systems (EST, LAP, and PGM) and their mtDNA RFLP patterns, we found that they are very similar at the molecular level. TheF statistics for enzyme polymorphism data revealed that inbreeding makes a great contribution to the population homozygosity, sinceF _IS equals 0.1322 andF _ST equals 0.0023. Since the chi-square test showed thatF _ST is not significant, we conclude that all localities belong to the same population. The mtDNA differentiation was about 12 times greater than for nuclear genes;F _ST was equivalent to 0.0265. We suggest that this difference is due to a higher dispersal of males, in relation to females.     

Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

The mitochondrial genome of Chlamydomonas. II. Genetic analysis of non-mendelian obligate photautotrophic mutants

Summary Among a collection of obligate photoautotrophic (dark-dier,dk) mutants isolated inChlamydomonas reinhardtii, two have been found which are inherited in crosses to wild type in a non-Mendelian, biparental and apparently random fashion. F₁ progeny include not only cells which show thedk and wildtype parental phenotypes but also many which possess intermediate phenotypes between wild type anddk. When F₁ progeny withdk, intermediate or wild-type phenotype were backcrossed to wild type, thedk phenotype continued to be inherited in a biparental and random fashion. Upon selection, neither mutant formed stable clones producing onlydk progeny, suggesting that the two mutants segregatedk and wild-type progeny somatically and that the homozygousdk condition may be lethal. The biparental transmission of these two non-Mendeliandk mutations resembles the transmission of acriflavin-inducedminute mutations ofChlamydomonas and is distinct from the uniparentally inherited chloroplast mutations of this alga. Both thedk andminute mutations may alter mitochondrial DNA and thereby alter mitochondrial functions.     

Specific mutations in alpha- and gamma-subunits of F1-ATPase affect mitochondrial genome integrity in the petite-negative yeast Kluyveromyces lactis.

We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits.     

The serum- and glucocorticoid-induced protein kinase-1 (Sgk-1) mitochondria connection: Identification of the IF-1 inhibitor of the F1F0-ATPase as a mitochondria-specific binding target and the stress-induced mitochondrial localization of endogenous Sgk-1

The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F₁F₀-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F₁F₀-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F₁F₀-ATPase, as part of the cellular stressed induced program.