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.     

Coupling factor activity of the purified pea mitochondrial F1-ATPase

The pea cotyledon mitochondrial F₁-ATPase was released from the submitochondrial particles by a washing procedure using 300 mM sucrose /2 mM Tricine (pH 7.4). The enzyme was purified by DEAE-cellulose chromatography and subsequent sucrose density gradient centrifugation. Using polyacrylamide gel electrophoresis under non-denaturing conditions, the purified protein exhibited a single sharp band with slightly lower mobility than the purified pea chloroplast CF₁-ATPase. The molecular weights of pea mitochondrial F₁-ATPase and pea chloroplast CF₁-ATPase were found to be 409 000 and 378 000, respectively. The purified pea mitochondrial F₁-ATPase dissociated into six types of subunits on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Most of these subunits had mobilities different from the subunits of the pea chloroplast CF₁-ATPase. The purified mitochondrial F₁-ATPase exhibited coupling factor activity. In spite of the observed differences between CF₁ and F₁, the mitochondrial enzyme stimulated ATP formation in CF₁-depleted pea chloroplast membranes. Thus, the mitochondrial F₁ was able to substitute functionally for the chloroplast CF₁ in reconstituting photophosphorylation.     

The coupled chemomechanics of the F1-ATPase molecular motor

The enzyme F₁-ATPase is a rotary nanomotor in which the central γ subunit rotates inside the cavity made of α₃β₃ subunits. The experiments showed that the rotation proceeds in steps of 120° and each 120° step consists of 80° and 40° substeps. Here the Author proposes a stochastic wave mechanics of the F₁-ATPase motor and combines it with the structure-based kinetics of the F₁-ATPase to form a chemomechanic coupled model. The model can reproduce quantitatively and explain the experimental observations about the F₁ motor. Using the model, several rate-limited situations about γ subunit rotation are proposed, the effects of the friction and the load on the substeps are investigated and the chemomechanic coupled time during ATP hydrolysis cycle is determined.     

F1-ATPase of Micrococcus lysodeikticus is not a glycoprotein

It has been claimed (Andreu, J.M., Warth, R. and Muñoz, E. (1978) FEBS Lett. 86, 1–5) that the F₁-ATPase of Micrococcus lysodeikticus is a glycoprotein containing mannose and glucose as the principal sugars. Even after extensive purification of M. lysodeikticus F₁-ATPase by DEAE-Sephadex A25 chromatography, carbohydrate contents varying from 2.7 to 10.8% have been found. Concanavalin A-reactive components corresponding to the succinylated lipomannan have been detected and separated from the ATPase in purified F₁ preparations by immunoelectrophoresis (rocket and two-dimensional) through agarose gels containing concanavalin A. Passage of the purified F₁-ATPase through concanavalin A-Sepharose 4B columns removed the carbohydrate component(s) without loss of the specific activity of the ATPase. Mannose was the only sugar detectable by gas-liquid chromatography of the F₁-ATPase before Con A-Sepharose 4B chromatography and it was completely eliminated after chromatography. No qualitative or quantitative changes in the subunit (, β, γ, δ and ε) profiles were detectable when the sodium dodecyl sulfate polyacrylamide gels were scanned by densitometry of F₁-ATPase before and after Con A-Sepharose 4B chromatography. We conclude that there is no evidence of carbohydrate covalently linked to this F₁-ATPase and that this membrane protein is not a glycoprotein. The presence of carbohydrate is attributable to contamination with lipomannan.     

Respirofermentative metabolism in Kluyveromyces lactis: Ethanol production and the Crabtree effect

Kluyveromyces lactis is a yeast widely used in processes related to milk whey use and lactose fermentation. However, contradictory information about some aspects related to the respirofermentative metabolism of this yeast is found in the literature. We have studied ethanol production and oxygen use in discontinuous and continuous cultures of K. lactis under hypoxic and aerobic conditions. Growth in nonfermentable carbon sources reflects a more efficient respiratory capacity of K. lactis in relation to Saccharomyces cerevisiae; however, in both species, similar glucose fermentation levels under aerobic oxygen-limited conditions are found. Continuous K. lactis cultures in fully oxidative conditions show the oxygen and substrate uptake rates typical of a respiration-unlimited Crabtree-negative yeast; however, a small residual fermentation is present even when respiration is not limited. Some aspects of the Crabtree effect in K. lactis are discussed. The impossibility of including K. lactis in any group of the metabolism-based classification from Alexander and Jeffries (1990) has led us to the formulation of a new group which incorporates the peculiarities of this and other related yeasts.     

Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F₁ from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F₁-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with ¹²⁵I-labelled purified monoclonal antibodies specific to various peptides. The ¹²⁵I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H₂O₂ and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F₁.     

Evolution of the alcohol dehydrogenase (ADH) genes in yeast: characterization of a fourth ADH in Kluyveromyces lactis

Summary Three alcohol dehydrogenase (ADH) genes have recently been characterized in the yeast Kluyveromyces lactis. We report on a fourth ADH in K. lactis (KADH II: KADH2 gene) which is highly similar to other ADHs in K. lactis and Saccharomyces cerevisiae. KADH II appears to be a cytoplasmic enzyme, and after expression of KADH2 in S. cerevisiae enzyme activity comigrated with a K. lactis ADH present in cells grown in glucose or in ethanol. KADH I was also expressed in S. cerevisiae and it comigrated with a major ADH species expressed under glucose growth conditions in K. lactis. The substrate specificities for KADH I and KADH II were shown to be more similar to that of SADH II than to SADH I. SADH I cannot efficiently utilize long chain alcohols, in contrast to other cytoplasmic yeast ADHs, presumably because of the presence of a methionine (residue 271) in its substrate binding cleft. A comparison of the DNA sequences of ADHs among K. lactis, S. cerevisiae and Schizosaccharomyces pombe suggests that the ancestral yeast species contained one cytoplasmic ADH. After divergence from S. pombe, the ADH in the ancestor to K. lactis and S. cerevisiae was duplicated, and one ADH became localized to the mitochondrion, presumably for the oxidative use of ethanol. Following the speciation of S. cerevisiae and K. lactis, the gene encoding the cytoplasmic ADH in S. cerevisiae duplicated, which resulted in the development of the SADH II protein as the primary oxidative enzyme in place of SADH III. In contrast, the K. lactis mitochondrial ADH duplicated to give rise to the highly expressed KADH3 and KADH4 genes, both of which may still play primary roles in oxidative metabolism. These data suggest that K. lactis and S. cerevisiae use different compartments for their metabolism of ethanol. Our results also indicate that the complex regulatory circuits controlling the glucose-repressible SADH2 in S. cerevisiae are a recent acquisition from regulatory networks used for the control of genes other than SADH2.

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Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

Structure of the phosphopeptidomannans from flocculent and non-flocculent yeast Kluyveromyces lactis

After extraction from whole cells, and purification by gel filtration, the chemical composition and molecular mass estimation of the cell-wall phosphopeptidomannan (PPM) showed no significant difference respectively between flocculent, weakly, very weakly and non-flocculent Kluyveromyces lactis yeast strains. However, when PPMs were tested as ligands of a lectin, extracted from the flocculent strain, the PPM isolated from the flocculent and weakly flocculent strain were recognized to a higher degree than those isolated from the non and very weakly flocculent strains. Acetolysis of PPM extracted from the four strains produced five oligosaccharide fractions corresponding to mono-, di-, tri-, penta-and hexa-saccharides. The flocculent strain was characterised by a high content of di-and penta-saccharides. The ¹H NMR analysis of the oligosaccharides demonstrated that the flocculent strain contained equivalent levels of the two mannobioses: Man( 1 → 2)Man and Man( 1 → 3)Man and of the two mannotrioses Man( 1 → 2)Man( 1 → 2)Man and Man( 1 → 3)Man( 1 → 2)Man. In contrast, the non-flocculent and the very weakly flocculent strains contained a single type of mannobiose Man( 1 → 2)Man and one type of mannotriose Man( 1 → 2)Man( 1 → 2)Man.     

Acceleration of the ATP-binding rate of F1-ATPase by forcible forward rotation

F₁-ATPase (F₁) is a reversible ATP-driven rotary motor protein. When its rotary shaft is reversely rotated, F₁ produces ATP against the chemical potential of ATP hydrolysis, suggesting that F₁ modulates the rate constants and equilibriums of catalytic reaction steps depending on the rotary angle of the shaft. Although the chemomechanical coupling scheme of F₁ has been determined, it is unclear how individual catalytic reaction steps depend on its rotary angle. Here, we report direct evidence that the ATP-binding rate of F₁ increases upon the forward rotation of the rotor, and its binding affinity to ATP is enhanced by rotation.     

The complex of mitochondrial F1-ATPase with the natural inhibitor protein is unable to catalyze single-site ATP hydrolysis

Interaction of mitochondrial F₁-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [¹⁴C]nucleotides in F₁-ATPase during steady-state [¹⁴C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.     

Thiol oxidation is crucial in the desensitization of the mitochondrial F1FO-ATPase to oligomycin and other macrolide antibiotics

Background

The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.

Methods

The inhibition mechanism of macrolides was explored on swine heart mitochondrial F₁F_O-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction.

Results

When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I₁ and I₂ pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI₁I₂), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE.

Conclusions

The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of F_O. The oxidation of highly conserved thiols in the ATP synthase c-ring of F_O weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.

General significance

The findings, by elucidating the macrolide inhibitory mechanism on F_O, indirectly cast light on the F₁F_O torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy.     

The synthesis of enzyme-bound ATP by the F1-ATPase from the thermophilic bacterium PS3 in the presence of organic solvents

Synthesis of enzyme-bound ATP was demonstrated with purified TF₁ (F₁-ATPase from thermophilic bacterium PS3) from medium inorganic phosphate (P_i) and enzyme-bound ADP in the presence of organic solvents such as dioxane, ethanol, dimethylformamide, methanol, acetone, acetonitrile or ethyleneglycol. The optimal concentrations of dimethylformamide, ethanol or methanol were 50%, 30% and 40% and the half-maximal concentrations of P_i were 13 mM, 20 mM and 18 mM, respectively. Thus it is evident that the effect of dimethylsulfoxide on TF₁ to form enzyme-bound ATP [8] is not due to a specific interaction between dimethylsulfoxide and the enzyme, but to a decrease in polarity of the medium. In the presence of methanol, the dependence of ATP synthesis on various divalent metal ions was compared to that for the ATP-hydrolyzing activity and the ATP-driven proton-translocating activity of TF₁. While Mn²⁺, Co²⁺, Zn²⁺ and Cd²⁺ are as effective as Mg²⁺ for the ATP-hydrolyzing activity of TF₁, Zn²⁺ and Cd²⁺ are either less or not effective for proton translocation and for ATP synthesis. This result appears to be consistent with the idea that the TF₁-ATP complex formed in organic solvents represents one of the intermediates in the reaction sequences of ATP synthesis by H⁺-ATPase using the proton gradient.     

The proton pore in the Escherichia coli F0F1-ATPase: A requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F₀ portion of the F₀F₁-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F₀F₁-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F₁-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F₀ (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62–69).     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997