absence of anionic phospholipids in kluyveromyces lactis cells is fatal...

Absence of anionic phospholipids inKluyveromyces lactis cells is fatal without F1-catalysed ATP hydrolysis

Viktoria Palovicova, Annamaria Bardelcikova, and Margita Obernauerova

Abstract: We have shown in previous research that the loss of phosphatidylglycerol and cardiolipin caused by disruption ofthe PGS1 gene is lethal for the petite-negative yeast Kluyveromyces lactis. This present study demonstrates the role andmechanism of atp2.1 in the suppression of pgs1 lethality in K. lactis cells. Phenotypic characterization has shown that astrain lacking the phosphatidylglycerolphosphate synthase (atp2.1pgs1D) possessed a markedly impaired respiratory chain,very low endogenous respiration, and uncoupled mitochondria. As a result the mutant strain was unable to generate a suffi-cient mitochondrial membrane potential via respiration. The atp2.1 suppressor mutation enabled an increase in the affinityof F1-ATPase for ATP in the hydrolytic reaction, resulting in the maintenance of sufficient membrane potential for the bio-genesis of mitochondria and survival of cells lacking anionic phospholipid biosynthesis.

Key words: Kluyveromyces lactis, anionic phospholipids, PGS1 gene, phosphatidylglycerolphosphate synthase, mitochondria.

Résumé : Nous avons précédemment montré que la perte de phosphatidylglycérol et de cardiolipine causée par l’interrup-tion du gène PGS1 est létale pour la levure Kluyveromyces lactis négative à petite. Cette étude démontre le rôle et le méca-nisme d’action d’atp2.1 dans la suppression de la létalité de pgs1 chez K. lactis. La caractérisation phénotypique a montréqu’une souche dépourvue de phosphatydylglycérolphosphate synthase (atp2.1pgs1D) était sévèrement affectée sur le plan dela chaine respiratoire, avec une respiration endogène très faible et un découplage des mitochondries. En conséquence, lasouche mutante était incapable de générer un potentiel membranaire mitochondrial suffisant par l’intermédiaire de la respira-tion. La mutation suppressive atp2.1 permettait d’augmenter l’affinité de la F1-ATPase pour l’ATP lors de la réaction d’hy-drolyse, résultant au maintien d’un potentiel membranaire suffisant à la biogenèse des mitochondries et à la survie descellules dépourvues de biosynthèse de phospholipides anioniques.

Mots‐clés : Kluyveromyces lactis, phospholipides anioniques, gène PSG1, phosphatidylglycérolphosphate synthase,mitochondrie.

[Traduit par la Rédaction]

Introduction

Cardiolipin (CL) and its precursor, phosphatidylglycerol(PG), despite their low levels (0.1%–30%) in the biologicalmembranes, play an essential role in fundamental physiologi-cal processes in eukaryotic cells. CL is the major anionicphospholipid in the mitochondrial inner membrane. It is spe-cifically associated with a number of mitochondrial proteins,many of which require CL to function optimally. It wasfound that CL is essential for the biogenesis and proper func-tioning of the respiratory chain (complexes I, III, IV) (Fryand Green 1981; Gomez and Robinson 1999; Yu and Yu1980; Hayer-Hartl et al. 1992; Robinson et al. 1990; Sedlákand Robinson 1999), ATP synthase (complex V) (Eble et al.1990), and transport proteins, such as the ATP–ADP translo-cator and phosphate translocator (Koshkin and Greenberg2002; Schlame 2008). CL is involved in maintaining the op-

timal mitochondrial internal structure (Koshkin andGreenberg 2000; Pfeiffer et al. 2003), in protein import(Jiang et al. 2000), in mitochondrial outer-membrane proteinbiogenesis (Gebert et al. 2009), and in maintaining mitochon-drial DNA (mtDNA) (Zhong et al. 2004; Chen et al. 2010). Itis also important for some nonbioenergetic functions such ascell wall synthesis (Zhong et al. 2005), the translational regu-lation of nuclear gene COX4 expression (Su and Dowhan2006), and the mitochondrion–vacuole signalling pathway(Chen et al. 2008). Several clinical studies carried out in re-cent years indicated that a reduction in CL level correlateswith negative aspects of several physiological states, such asBarth’s syndrome (Bione et al. 1996; Claypool et al. 2008),ischemia and (or) reperfusion, and the stimulation of pathwaysinitiating ageing and apoptosis (McMillin and Dowhan 2002).The mutation or disruption of the PGS1 gene (encoding

phosphatidylglycerolphosphate synthase (EC 2.7.8.5), an es-

Received 19 December 2011. Revision received 1 March 2012. Accepted 1 March 2012. Published at www.nrcresearchpress.com/cjm on14 May 2012.

V. Palovicova, A. Bardelcikova, and M. Obernauerova. Department of Microbiology and Virology, Faculty of Natural Sciences,Comenius University, Mlynská dolina B-2, Bratislava 842 15, Slovak Republic.

Corresponding author: Margita Obernauerova (e-mail: [email protected]).

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Can. J. Microbiol. 58: 694–702 (2012) doi:10.1139/W2012-040 Published by NRC Research Press

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sential enzyme of the CL biosynthetic pathway) results in theloss of PG and CL (Chang et al. 1998; Džugasová et al.1998). The absence of anionic phospholipids has no dramaticeffect in the petite-positive yeast Saccharomyces cerevisiae.Its cells display reduced growth characteristics, a diminishedrespiratory activity, and a reduced membrane potential (Šubík1974; Janitor and Šubík 1993; Koshkin and Greenberg2002). The reduction in growth at higher temperature pro-vides support to the theory that the CL biosynthesis pathwayplays an important role in the survival of S. cerevisiae cellsunder stressful conditions (Koshkin and Greenberg 2002).The simultaneous loss of both phospholipids and mtDNAhas a significant impact on S. cerevisiae physiology. This le-thal event converts the petite-positive strain of S. cerevisiaeinto a petite-negative one (Janitor et al. 1996).The yeast Kluyveromyces lactis is a petite-negative spe-

cies, in which the simultaneous loss of the mtDNA-controlled genes responsible for the respiratory chain and ox-idative phosphorylation components is lethal (Chen andClark-Walker 1996). However, several years ago, a numberof studies indicated that specific mutations in the ATP1,ATP2, and ATP3 nuclear genes encoding the a-, b-, and g-subunits, respectively, of F1-ATPase allow the petite-negativeyeast K. lactis to steadily survive deletions, or even the lossof mtDNA (Chen and Clark-Walker 1995, 1996; Clark-Walker et al. 2000). These atp mutations (also described asmgi, mitochondrial genome integrity, mutations) cause struc-tural changes in the F1 part of the F1F0-ATP-synthase com-plex, resulting in an increased activity of ATPase. Theincreased hydrolysis of ATP then prevents the imminent col-lapse of the mitochondrial inner membrane potential, whichnormally occurs upon the loss of the mtDNA-encoded F0subunit (Clark-Walker and Chen 2001). It was also shownthat not all atp mutant alleles support a suppressor pheno-type (Clark-Walker et al. 2000). Moreover, our laboratoryhas shown that the loss of both anionic phospholipids PGand CL, caused by deletion of the PGS1 gene, also has a le-thal effect on the viability and multiplication of this yeaststrain (Tyciakova et al. 2004). Based on the postulated anddemonstrated activities of CL in many mitochondrial pro-cesses and the activity of the atp suppressor, we previouslycreated an atp2.1pgs1D K. lactis mutant disrupted in itsPGS1 gene (Obernauerova and Polakovicova 2009). Theatp2.1pgs1D mutant exhibits impaired growth on rich andminimal media containing nonfermentable carbon and energysources. These mutant cells are thermosensitive, but they areable to form colonies even after the induction of mtDNA de-letions, indicating a central role of these lipids in oxidativephosphorylation (Obernauerova and Polakovicova 2009). Asimilar phenotype was demonstrated by Patràšová et al.(2010) for the K. lactis atp mutant, incapable of synthesizinganionic phospholipids. The aim of this study was to analyzethe mechanism of atp2.1 suppression in K. lactis mutantcells lacking anionic phospholipids.

Materials and methods

Strains and vectorsThe following strains of K. lactis were used in this study:

laboratory strain JBD 100 (MATa, trp1, ura3-10, lac4-1),University of Leiden, the Netherlands; CW75 (MATa/MATa,

ade1/ADE1, LYS1/lys1, ura3.1/ura3.1, atp2.1/atp2.1) (Clark-Walker and Chen 2001); CW75-1D (MATa, ade1, lys1,ura3.1, atp2.1) (this study); CW75-1D-63 (MATa, ade1,lys1, ura3.1, atp2.1, pgs1::KanMX) (this study); CW75-1D-pATP2 (MATa, ade1, lys1, atp2.1, pRS306K-ATP2); Escher-ichia coli XL-1 Blue was used as a host for construction, am-plification, and preparation of plasmid DNA.

Media and growth conditionsYeast strains were grown aerobically at 28 °C in rich YPD

medium (10 g·L–1 yeast extract, 20 g·L–1 bactopeptone,20 g·L–1 glucose) or in minimal YNB medium (6.7 g·L–1

yeast nitrogen base without amino acids, 20 g·L–1 glucose,and supplemented with the auxotrophic requirements(40 µg·mL–1)). Sporulation medium contained 10 g·L–1 potas-sium acetate, 1.4 g·L–1 yeast extract, 0.5 g·L–1 glucose, and20 g·L–1 Difco agar. G418 medium is the medium supple-mented with the drug at 200 µg·mL–1. Solid media were pre-pared with 20 g·L–1 Difco agar. The bacterial strain wasgrown in Luria–Bertani medium (10 g·L–1 tryptone, 5 g·L–1

yeast extract, 5 g·L–1 NaCl, pH 7.4) at 37 °C. For the plas-mid maintenance, ampicillin was added to a final concentra-tion of 100 µg·mL–1.

Ascus dissection and phenotype determinationAsci were dissected with a micromanipulator following a

brief treatment with Zymolyase (Seikagaku, Japan). Pheno-types were determined by dropping spore suspensions ontominimal medium containing glucose supplemented with ap-propriate nutritional requirements. Genotype was inferredfrom the phenotype of the wild-type strain K. lactis CW75alleles.

PCR amplifications, recombinant DNA techniques, DNAsequencingStandard protocols for generating recombinant DNA, re-

striction enzyme analysis, and gel electrophoresis, as is de-scribed by Sambrook et al. (1989), were used.

Construction of the atp2.1pgs1D mutantThe Klpgs1::KanMX disruption cassette (Tyciakova et al.

2004), flanked by about 1760 bp upstream and 785 bp down-stream of KlPGS1 chromosomal sequences, was excised byEcoRI and SalI and used for disruption of the KlPGS1 genein the haploid strain CW75-1D. The K. lactis strain was trans-formed by electroporation using a BioRad gene pulser at1.0 kV, 25 µF, and 400 U in 0.2 cm cuvettes (Sánchez et al.1993). Genomic DNA from G418 resistant transformants wasused as a template for the verification of correct PGS1 genedisruption by PCR. For PCR amplifications, Hot Start TaqDNA polymerase (Qiagen) and the following primers wereused: A (KlPGS1up) 5′-GCGGTTTCAAACTGAAGCTC-3′,B (KanMXup) 5′-GGACGAGGCAAGCTAAACAG-3′, C(KanMXdown) 5′-TGGTCGCTATACTG-CTGTCG-3′, D(KlPGS1down) 5′-GGCTTTCTGTTACGCTCGTC-3′.The atp2.1 mutation in strain CW75-1D was verified by se-

quence determination (ABI Prism 3100; Applied Biosystems,Foster City, California) using the primers KlATP2S 5′-ATTGC-CAAGGCTCATGGTGG-3′ and KlATP2R 5′-AAAGAGCA-GAGGGAGTGAGAGGTT-3′.

Palovicova et al. 695

Published by NRC Research Press

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Integration of the ATP2 geneReintroduction of the KlATP2 gene into the atp2.1 mutant

(CW75-1D) was achieved as follows: genomic DNA fromK. lactis JBD100 was extracted (Holm et al. 1986) and usedas a template for amplification of the KlATP2 gene. PCR wascarried out using the Hot Start Taq DNA polymerase (Qiagen)and the following primers: F (KlATP2) 5′-ATCTCAC-CTTCAATTTCAGCACACC-3′ (forward) and R (KlATP2)5′-CGATATGTGATCCTCTAAACGGGTAC-3′ (reverse).The 3.304 kb PCR product, containing the KlATP2 gene,flanked by 607 bp upstream and 1183 bp downstream chro-mosomal sequences, was introduced into the pRS306K vec-tor opened using SpeI and XhoI. The resulting construct(9.292 kb) was used to transform the CW75-1D (atp2.1)strain by electroporation (Sánchez et al. 1993).

Determination of cytochrome spectra and oxygen uptakeDifferential cytochrome spectra (reduced and oxidized) of

yeast cells (of about 30 mg dry mass of cells·mL–1) grownfor 24 h in YPD medium were recorded using a PerkinElmer557 spectrophotometer. Oxygen uptake rates were determinedin a 3 mL vessel containing 50 mmol·L–1 potassium gluta-rate, 10 mmol·L–1 potassium phosphate, 100 mmol·L–1 KCl,50 mmol·L–1 glucose, pH 4.3, at 30 °C, as described byŠubík et al. (1977).

Isolation of mitochondriaMitochondria were isolated from yeast cells, grown to

early stationary phase in minimal medium with glucose at28 °C. Spheroplasts were received using 10 000 U ofZymolyase-20T (Seykagaku, Japan), resuspended in ice-coldbuffer containing 0.6 mol·L–1 sorbitol, 0.5 mmol·L–1 EDTA,1 g·L–1 BSA, 10 mmol·L–1 Tris–HCl, 1 mmol·L–1 PMSF,pH 7.4, and broken in a French press at 1000 psi (1 psi =6.894 747 kPa). Mitochondria were recovered by centrifuga-tion at 10 500g for 20 min in a Sorvall SS34 rotor after priorremoval of cellular debris by 2 rounds of centrifugation at1500g and 3000g for 5 min. Mitochondrial pellets were re-suspended in the buffer containing 0.6 mol·L–1 mannitol and10 mmol·L–1 Tris–HCl, pH 7.4.

ATPase activity and kinetic parametersATPase activity was estimated in freshly prepared mito-

chondria (Law et al. 1995). Mitochondrial suspension (1 mgwet mass) was added to 1 mL of 10 mmol·L–1 Tris–HCl(pH 8.0), 2 mmol·L–1 MgCl2, 200 mmol·L–1 KCl, and ATPat final concentration of 2.0 mmol·L–1. After incubation(10 min at 30 °C), 0.5 mL aliquots were added to 50 µL of3 mol·L–1 trichloroacetic acid. After 30 min of incubation onice the suspension was centrifugated (10 min, 13 500g). A500 µL aliquot from the supernatant was added to 1 mL ofSumner buffer and used for inorganic phosphate concentra-tion determination. Mitochondrial proteins were determinedaccording to the Bradford method, using bovine serum albu-min as standard. ATPase activity was expressed as nano-moles of inorganic phosphate hydrolysed per minute permilligram of protein. Km and Vmax were obtained from aLineweaver–Burk plot. Oligomycin-sensitive ATPase activitywas determined after incubation (10 min) in the presence ofoligomycin (5 µg·mL–1).

Membrane potential measurementMitochondrial membrane potential was determined using

the potential-dependent uptake of the fluorescent dye Rhod-amine 123 (125 µg·mL–1) in buffer (0.4 mol·L–1 mannitol,0.36 mmol·L–1 EDTA, 10 mmol·L–1 Tris–maleate, pH 6.8) at30 °C. Each reaction was performed using 0.5 mg of mito-chondria in a final volume of 1.5 mL. Fluorescence wasmonitored using a Shimadzu RF 5301 spectrophotometer (ex-citation wavelength 507 nm and emission wavelength529 nm). Changes in membrane potential were monitored inresponse to added NADH (2 mmol·L–1) as the substrate caus-ing the polarization of mitochondrial membrane and to10 µmol·L–1 CCCP (carbonyl cyanide m-chlorophenylhydra-zone), inducing depolarization of the membrane. The mito-chondrial membrane potential was determined in response toadded ATP (0.7 mmol·L–1), and CCCP (10 µmol·L–1) aswell.

Results

Construction of a yeast system for investigation ofatp2.1pgs1D lethality suppression by atp2.1 mutationTo study the effect of the atp2.1 mutation on the suppres-

sion of pgs1 lethality, the K. lactis diploid strain, CW75,homozygous for the atp2.1 mutation, was subjected to sporu-lation, ascus dissection, and tetrad analysis. One of the iso-lated haploid spores having an Ade– Lys– Ura– phenotypeand containing the atp2.1 mutation (nucleotide mutationAGA→GGA leading to amino acid substitution Arg435→Glywas verified by sequence determination, data not shown),named CW75-1D, was transformed by a 1.7 kb disruptioncassette containing KanMX flanked by an extensive chromo-somal DNA sequence of KlPGS1, as described by Tyciakovaet al. (2004) (Fig. 1). The fragment was integrated into thegenome of K. lactis by homologous recombination, and thedeletion mutants were selected for resistance against G418(geneticin). The correct disruption of the PGS1 gene in trans-formants was confirmed by PCR analysis using the primersA, B, C, and D (Fig. 2).

Loss of anionic phospholipids results in markedly reducedrespiratory activity of atp2.1pgs1D mutantPrevious studies indicated that the K. lactis atp2.1pgs1D

mutant exhibits a reduced growth on rich and minimal glu-cose medium and does not grow on media containing nonfer-mentable carbon and energy sources (Obernauerova andPolakovicova 2009; Patràšová et al. 2010). Cytochrome spec-tra of both atp2.1pgs1D and atp2.1 mutants and of a wild-type strain grown in glucose medium showed that theatp2.1pgs1D mutant does not produce cytochrome b, and itslevels of cytochrome c and cytochrome a are also reducedcompared with the atp2.1 mutant and the wild-type strain(Fig. 3). This fact, together with the inability of theatp2.1pgs1D mutant to utilize glycerol, correlates with thestrain’s respiratory activity. The oxygen uptake of theatp2.1pgs1D mutant, atp2.1 mutant, and wild-type strain isdocumented in Table 1. Our results, as well as those ofPatràšová et al. (2010), indicate that the oxygen uptake ofthe atp2.1pgs1D mutant is markedly lower than that of thewild-type strain (about 69% reduction). Moreover, its oxygenuptake is not stimulated by membrane uncoupler CCCP.

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These data demonstrate that the respiration of theatp2.1pgs1D mutant is defective. In addition, the drop inRCR (respiratory control ratio) from 3.2 for the wild type to1.36 for the atp2.1 mutant and 0.98 for the atp2.1pgs1D mu-tant shows that the activity of the respiratory chain in mito-chondria with depleted anionic phospholipids is notcontrolled by phosphorylation reactions (Table 1).

ATPase activity of atp2.1 mutant mitochondriaOligomycin-sensitive ATPase activity can also be an useful

indicator of the ATP synthase coupling. Oligomycin binds to

Fig. 1. Disruption of KlPGS1 gene. (a) Klpgs1::KanMX cassette. (b) Intact chromosomal PGS1 gene. (c) Chromosomal PGS1 gene disruptedwith Klpgs1::KanMX cassette. Primers used in PCR analysis derived from chromosomal DNA sequences (A and D) or KanMX cassette (Band C) are indicated with arrows.

Fig. 2. PCR analysis of DNA fragments amplified from chromoso-mal DNA of the atp2.1 Kluyveromyces lactis strain. Lanes: 1, intactPGS1 gene (4.5 kb); 2, KlPGS1 gene disrupted with Klpgs1::KanMX cassette (1.9 and 1.1 kb); 3, l DNA–HindIII molecularmass marker.

Fig. 3. Differential cytochrome spectra of yeast cells. Cells weregrown for 24 h in rich medium with glucose, 30 mg dry mass ofcells·mL–1 were used for analysis.



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the F0 subunit and inhibits ATP hydrolysis by the ATP syn-thase, but it does not inhibit ATP hydrolysis by the F1-ATPase, by virtue of the fact that it is not associated withF0. The ATPase activities of both atp2.1pgs1D and atp2.1mitochondrial mutants were not markedly different, but theywere more than double the activity of the wild-type strain.Moreover, the total mitochondrial ATPase activity of thewild-type mitochondria exhibited an approximately 2-fold re-duction in ATP hydrolysis upon the addition of oligomycin, aproton-translocation inhibitor, whereas the activity of ATPhydrolysis in both mutant strains was unresponsive to oligo-mycin (95% resistance for the ATPase from atp2.1pgs1D and89% resistance for the ATPase from atp2.1) (Fig. 4). Thesefindings indicate that the increase in ATP hydrolysis in bothmutant strains is the result of the atp2.1 mutation, and astriking reduction in F1-ATPase sensitivity to oligomycin inthe atp2.1pgs1D and atp2.1 mutants enables us to concludethat F1 complexes are not bound to F0, and most likely donot assume the form of a coupled enzyme.

Kinetic parameters of F1-ATPase of atp2.1 mutantmitochondriaThe kinetic parameters of F1-ATPase in the analyzed

strains showed that the Km and Vmax values of theatp2.1pgs1D and atp2.1 strains were not significantly differ-ent (Table 2) from each other but were markedly differentfrom the wild-type strain. The Km values of mutant F1-ATPases were about 72% lower than those in the wild-typestrain, whereas the Vmax for both mutant enzymes were morethan 2-fold higher than the wild-type strain. On the basis ofthese results, it can be assumed that an increased affinity ofthe F1 mutant for ATP in the hydrolytic reaction is causedby the involvement of the atp2.1 suppressor.

Inner mitochondrial potential is diminished inatp2.1pgs1D mitochondriaThe analyzed strains were also tested in terms of quantify-

ing their mitochondrial membrane potential. The membranepotential in isolated mitochondria was monitored in responseto the addition of NADH or ATP by measuring the uptake ofthe fluorescent dye rhodamine 123. As expected, in theatp2.1pgs1D mitochondria, the addition of NADH inducedminimal fluorescence quenching of rhodamine 123, the mem-brane potential was very low, in contrast to the atp2.1 andwild-type mitochondria, and not sensitive to CCCP (Fig. 5a).In the second instance (Fig. 5b), the membrane potential cre-ated by the F1-catalysed hydrolysis of ATP in the

atp2.1pgs1D mutant was comparable to that of the atp2.1mutant. The membrane potential of both mutants was higherthan that of the wild type; a membrane potential response toCCCP was not observed (Fig. 5b). Finally, the results ob-tained indicate that the lethality of K. lactis cells caused bythe absence of anionic phospholipids is suppressed by an im-proved hydrolysis of ATP by F1-ATPase containing a b sub-

Table 1. Respiratory rates of wild type, atp2.1, and atp2.1pgs1D Kluy-veromyces lactis strains grown in YNB glucose medium.

Oxygen uptake (µmol O·min–1·(mg dry mass)–1)

StrainEndogenousrespiration CCCP RCR

atp2.1PGS1pATP2 102.15±1.3 326.88±2.5 3.2±0.25atp2.1PGS1 71.0±0.9 96.7±1.5 1.36±0.15atp2.1pgs1D 32.0±1.1 31.5±1.8 0.98±0.18

Note: Respiratory activities in cells were measured as endogenous respiration, aswell as in the presence of CCCP (carbonyl cyanide m-chlorophenylhydrazone,10 µmol·L–1). RCR, respiratory control ratio, indicates the coupling of respirationwith ATP synthesis. The experiment was performed in triplicate for each strain.

Fig. 4. ATPase activity in Kluyveromyces lactis cells. Mitochondriaisolated from the atp2.1pgs1D mutant, atp2.1PGS1 strain, and thewild-type atp2.1PGS1pATP2 were incubated without (– oli) or with(+ oli) oligomycin (5 µg·mL–1) to determine the fraction of inhibitedATPase activity. Values are averages from 3 separate mitochondrialpreparations in each case.

Table 2. Kinetic parameters of Kluyveromyces lactis strains mito-chondrial F1-ATPase.

StrainKm

(mmol·L–1 ATP)Vmax

(µmol Pi·min–1·(mg protein)–1)atp2.1PGS1pATP2 1.7±0.1 14.36±1.3atp2.1PGS1 0.48±0.05 33.97±2.1atp2.1pgs1D 0.47±0.09 32.23±2.5



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unit with the atp2.1 mutation, which in cells under nonrespir-ing conditions can help create and maintain a proper mem-brane potential, indispensable for the mitochondrialbiogenesis and the viability of K. lactis cells.

Discussion

This study presents our observations on the role and mech-anisms of pgs1-lethality suppression in K. lactis cells byatp2.1 mutation. We have shown that the F1-ATPase activityof the atp2.1 suppressor mutation is essential foratp2.1pgs1D nonrespiring cells, which lack anionic phospho-lipid synthesis, because it is involved in the maintenance ofmitochondrial membrane potential, essential for the biogene-sis of mitochondria and viability of K. lactis cells.CL and its precursor PG have been demonstrated to partic-

ipate in many critical mitochondrial processes (Fry and Green1981; Gomez and Robinson 1999; Yu and Yu 1980; Hayer-Hartl et al. 1992; Robinson et al. 1990; Sedlák and Robinson1999; Eble et al. 1990; Koshkin and Greenberg 2002;Schlame 2008; Pfeiffer et al. 2003; Jiang et al. 2000). Thestudies performed using Saccharomyces cerevisiae or Can-dida glabrata pgs1 mutants have shown that the absence ofthe anionic phospholipids PG and CL seriously, but not le-thally, compromises mitochondrial biogenesis and the func-tioning of cells. These pgs1 mutant cells exhibited decreasedgrowth on rich glucose or glycerol media and did not growon minimal media containing the respiratory substrate.

Anionic phospholipid deficiency also resulted in reducedamounts of cytochrome b and cytochrome a, as well as de-creased respiratory activity (Chang et al. 1998; Šubík 1974;Janitor and Šubík 1993; Chen and Clark-Walker 1993). De-spite reduced respiratory activity, the oxygen uptake of pgs1cells was stimulated by the uncoupler CCCP. This indicatesthat the activity of their mitochondrial respiratory chain wascontrolled by phosphorylation reactions and that the mito-chondrial inner membrane potential created by the mecha-nism of oxidative phosphorylation was still sufficient for themitotic growth of these petite-positive mutant strains(Koshkin and Greenberg 2002; Chen and Clark-Walker1993; Batova et al. 2008).The results presented in this study show that compared

with the afore-mentioned petite-positive yeast, theatp2.1pgs1D mutant still has significantly damaged compo-nents of the respiratory chain, caused by cytochrome b defi-ciency and decreased amounts of cytochrome a andcytochrome c (Fig. 3). This fact and the inability of the pgs1mutant cells to grow on rich and minimal glycerol media in-dicates a central role for the anionic lipids (CL, PG) in theoxidative phosphorylation of aerobic K. lactis yeast. Thelevel of endogenous respiration showed that deletion of thePGS1 gene caused a marked reduction in oxygen consump-tion in mutant cells. Based on the minimal oxygen uptake inthe atp2.1pgs1D mutant strain not responsive to CCCP addi-tion (Table 1), we propose that in contrast to the petite-positive yeast, energy coupling does not operate in K. lactis

Fig. 5. Generation of inner mitochondrial membrane potential monitored by fluorescent quenching of rhodamine 123. Mitochondrial protein(0.5 mg) isolated from the atp2.1pgs1D mutant, atp2.1 strain, and the wild-type atp2.1PGS1pATP2 was added to 1.5 mL of buffer, pH 6.8,containing 125 µg·mL–1 rhodamine 123. Excitation was carried out at 507 nm, and fluorescence emission was monitored at 529 nm. In addi-tion, the solution contained 2 mmol·L–1 NADH (a) or 0.7 mmol·L–1 ATP (b), and 10 µmol·L–1 CCCP. The experiment was performed intriplicate for each strain.



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mutant mitochondria. In addition, since the respiratory chainwas not functional in the atp2.1pgs1D mutant, the mitochon-drial membrane potential generated using NADH becomesnegligible (Fig. 5a), and thus most likely insufficient tomaintain the growth of cells lacking anionic phospholipids.An alternative process that could maintain the mitochon-

drial membrane potential involves the reversal of the activityof the F1F0-complex from ATP synthesis to hydrolysis. Thismechanism of mitochondrial membrane potential generationis typical for S. cerevisiae grown under strictly anaerobicconditions (Criddle and Schatz 1969; Groot et al. 1971), forrho0/rho– and aac-lethality suppressor mutants of K. lactis(Chen et al. 1998; Clark-Walker and Chen 2001), and formammalian cells lacking mtDNA (Buchet and Godinot1998).Based on these data and our results, which showed that the

atp2.1pgs1D mutant is not able to generate a membrane po-tential through respiration, it has been hypothesized that thesurvival and growth of the K. lactis mutant depends on thegeneration of a voltage gradient across the mitochondrial in-ner membrane by a reverse F1-ATPase activity (Castrejón etal. 1997; Koshkin and Greenberg 2000). F1-ATPase assaysshowed that the increased activity of ATP hydrolysis in theatp2.1pgs1D mutant was nearly the same as in the isogenicatp2.1 mutant but was markedly higher than in the wild type(Fig. 4). The F1-ATPase of both mutants exhibited a de-creased Km for ATP compared with the wild type. As a con-sequence it is likely that F1 mutants are able to hydrolyzesufficient ATP to allow the ADP–ATP translocase to electro-genically exchange ADP in the matrix for the ATP in the cy-tosol, establishing a membrane potential across themitochondrial inner membrane. Subsequent analysis showedthat the mitochondrial membrane potential in both mutantswas significantly higher than in the wild-type strain (Fig. 5b).Moreover, the membrane potential of both mutants was sen-sitive to CCCP, which along with reduced ATPase sensitivityto oligomycin (Fig. 3), might indicate that mutant mitochon-dria contain an ineffective F0 sector and that the transmem-branous potential is generated by the active F1 part of theabove-mentioned organelle enzyme.This finding is in agreement with previous studies that

have shown a significant role of anionic phospholipids in theorganization and optimal activity of oxidative phosphoryla-tion complexes as well as in the ability of atp mutations toalter the coupling of the ATP synthase (Koshkin and Green-berg 2000, 2002; Wang et al. 2007). The particular contribu-tion of the pgs1 and atp2.1 mutations for the functionality ofF1 or F0 in the atp2.1pgs1D mutant remain to be clarifiedand will require further experiments.In summary, (i) aerobic K. lactis yeasts are unable to toler-

ate the absence of both electron–proton transport pumpingand the ATP synthesis components of oxidative phosphoryla-tion (Clark-Walker and Chen 2001), (ii) CL provides stabilityand (or) activity to respiratory chain supercomplexes (Fryand Green 1981; Gomez and Robinson 1999; Sedlák andRobinson 1999), and (iii) the loss of mtDNA (Clark-Walkerand Chen 1996), or the absence of PG and CL caused by dis-ruption of the KlPGS1 gene (Tyciakova et al. 2004) in thepetite-negative yeast K. lactis, is lethal. We have presentedhere the biochemical basis of the mechanisms by which theatp2.1 mutation in the atp2.1pgs1D mutant, lacking the oxi-

dative phosphorylation reactions, increases the viability ofaerobic K. lactis yeasts lacking anionic phospholipids by in-creasing the hydrolysis of ATP to provide the potential re-quired for the import of mitochondrial proteins indispensablefor mitochondrial biogenesis. The pgs1 mutation in petite-negative K. lactis cells is another example from the series ofmutations (rho–/rho0 or aac) reported in petite-negative yeast,whose lethality to cells is suppressed by a mutation of atp2.1.

AcknowledgementsWe are grateful to G.D. Clark-Walker for the K. lactis

strain used in this study and Pavol Sulo for tetrad analysis.This publication is the result of the project implementation:“Centre of excellence for exploitation of informational bio-macromolecules in the disease prevention and improvementof quality of life”, ITMS 26240120027, supported by the Re-search & Development Operation Programme funded by theERDF, grant from the Slovak Grant Agency of Science(VEGA 1/0287/10) and Slovak Grant Agency of ScienceAPVV-0282-10.

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absence of anionic phospholipids in kluyveromyces lactis cells is fatal...

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