Dual Mutations Reveal Interactions Between Components of Oxidative Phosphorylation in Kluyveromyces Lactis

Dual Mutations Reveal Interactions Between Components of Oxidative Phosphorylation in Kluyveromyces lactis

G. D. Clark-Walker and X. J. Chen Molecular Genetics and Evolution Group, Research School of Biological Sciences, The Australian National University, Canberra, ACT, 2601, Australia Manuscript received April 10, 2001 Accepted for publication August 3, 2001

ABSTRACT Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for ␳0-lethality has been identiﬁed by disruption of nuclear genes

encoding electron transport and F0-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, ⌬⌿, across the mitochondrial inner

membrane due to lack of proton pumping by the electron transport chain or reversal of F1F0-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or ␳0-lethality can be suppressed by the atp2.1 ␤ mutation in the -subunit of F1-ATPase. Suppression is viewed as occurring by an increased hydrolysis of

ATP by mutant F1, allowing sufﬁcient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain ⌬⌿. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered

to occur by mutant F1 acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of ␳0-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.

OLLOWING the discovery that mutations in Kluy- ated with a mitochondrial inner membrane complex, F veromyces lactis can convert this petite-negative yeast F0. Under conditions permitting oxidative phosphoryla- into a petite-positive form resembling Saccharomyces cere- tion, the F1F0-ATP synthase catalyzes the formation of visiae (Chen and Clark-Walker 1993), progress has ATP from ADP in response to proton translocation from been made in understanding differences between the the intermembrane space to the mitochondrial matrix wild types of these yeasts (Chen and Clark-Walker (Boyer 1997; Weber and Senior 1997; Saraste 1999).

1999). S. cerevisiae, as a representative of petite-positive The site for ATP synthesis is F1, which is composed of yeasts, readily loses its mitochondrial DNA (mtDNA) to 3␣-, 3␤-, 1␥-, 1␦-, and 1ε-subunits that in S. cerevisiae and form respiratory-deﬁcient petite mutants when treated K. lactis are all encoded by nuclear genes (Attardi and with DNA targeting drugs (Ferguson and von Borstel Schatz 1988; Poyton and McEwen 1996; Chen et al. 1992; Contamine and Picard 2000), whereas loss of 1998). mtDNA and mitochondrial protein synthesis is lethal The F0 complex, on the other hand, contains three for K. lactis (␳0-lethality; Clark-Walker and Chen 1996; subunits encoded by mtDNA (ATP6,-8, and -9; Attardi Murray et al. 2000). Mutations in K. lactis that suppress and Schatz 1988; Cox et al. 1992; Poyton and McEwen ␳0-lethality, initially termed mgi, for mitochondrial ge- 1996; Foury et al. 1998) as well as three major nuclear- nome integrity, occur in the ATP1,-2, and -3 genes encoded components (ATP4,-5, and -7; Velours et al. ␣ ␤ ␥ encoding the -, -, and -subunits of F1-ATPase (Chen 1988; Uh et al. 1990; Norais et al. 1991). Thus loss of and Clark-Walker 1995, 1996; Clark-Walker et al. mtDNA inactivates the F0 complex. Furthermore, by

2000). Hence K. lactis strains containing a suppressor disrupting the three nuclear genes for F0 in K. lactis,it mutation resemble S. cerevisiae by readily losing mtDNA has been demonstrated that suppression of ␳0-lethality and forming petite mutants when treated with ethidium by mutant F1 can occur in the absence of all six F0 bromide. subunits (Chen et al. 1998). In addition to encoding

F1-ATPase, the site for suppressor mutations, is associ- F0 subunits, mtDNA contains genes for the electron transport complexes III, ubiquinol-cytochrome c reduc- tase (CYB), and IV, cytochrome c oxidase (COX1,-2, Corresponding author: G. D. Clark-Walker, Molecular Genetics and and -3). Both complexes pump protons out of mito- Evolution Group, Research School of Biological Sciences, The Austra- lian National University, PO Box 475, Canberra, ACT, 2601, Australia. chondria during passage of electrons to oxygen. There- E-mail: [email protected] fore loss of mtDNA removes both the electron trans-

Genetics 159: 929–938 (November 2001) 930 G. D. Clark-Walker and X. J. Chen port/proton pumping and ATP synthesis components genic exchange of ATP for ADP to maintain ⌬⌿ at a of oxidative phosphorylation. Such a profound change level sufﬁcient for protein import into mitochondria. can be tolerated by S. cerevisiae but not by wild-type K. In view of the postulated role for ADP/ATP translocase lactis. Accordingly, we want to know what determines for survival of S. cerevisiae petite mutants, we investigated ␳0-lethality of K. lactis and how wild-type S. cerevisiae and whether this enzyme participates in suppression of ␳0-

F1 mutants of K. lactis survive loss of oxidative phosphory- lethality in K. lactis. lation. In relation to the death of K. lactis on loss of mtDNA, it appears a priori that the mitochondrial genome contains MATERIALS AND METHODS one or more vital genes. Identification of the genes Strains and media: Yeast strains used in this study are listed required for survival involved solving a conundrum. For in Table 1. Diploid strain CW75, homozygous for atp2.1, was instance, it is known that K. lactis can sustain deletions obtained as a zygotic colony from CK429-5D a atp2.1 lysA1 in mtDNA removing ATP9 (Clark-Walker et al. 1997) ura3.1 crossed with CK429-7C ␣ atp2.1 ade1 ura3.1. Complete and, in a separate strain, COX1 and -2 (Hardy et al. medium (GYP) contains 0.5% Bacto yeast extract, 1% Bacto Peptone, and 2% glucose. Glycerol medium (GlyYP) contains 1989). Likewise, K. lactis can survive disruptions in nu- 2% glycerol in place of glucose. Minimal medium (GMM) ␦ clear genes that inactivate F1 (ATP1,-2, and -3, ATP , contains 0.67% Difco (Detroit) yeast nitrogen base without ε and ATP ) and F0 (ATP4,-5, and -7), as well as genes amino acids and 2% glucose. Nutrients essential for auxotro- of the electron transport chain [CYC1, QCR7, QCR8, phic strains were added at 25 ␮g/ml for bases and 50 ␮g/ml PETIII, and COX18 (for review see Chen and Clark- for amino acids. Ethidium bromide (EB) medium is GYP plus EB at 16 ␮g/ml. For sporulation of K. lactis, ME medium Walker 1999)]. From these results it can be concluded contains 5% malt extract and 2% Bacto agar. S. cerevisiae was that neither electron transport nor ATP synthesis/hy- sporulated on 1.5% K-acetate agar. Antimycin (Sigma, Castle drolysis is essential for K. lactis. Consequently, it would Hill, New South Wales) was added to GYP medium after auto- seem that either K. lactis mtDNA contains a novel but claving to give a concentration of 1 ␮m. G418 (Geneticin; essential gene not found in petite-positive yeasts or le- GIBCO BRL, Grand Island, NY) was added to GYP medium after autoclaving to give a concentration of 200 ␮g/ml. thality could be due to simultaneous loss of genes for Gene disruption: Isolation of K. lactis genes and their disrup- both electron transport and ATP synthesis/hydrolysis tion have been described in detail in previous publications components of oxidative phosphorylation. The second (Table 1). Genes coding for the OSCP (ATP5) and d (ATP7) possibility, termed the two-component model, has been subunits of F0 were disrupted by insertion of kan and URA3 investigated by a genetic test where disruptions in nu- (Chen et al. 1998). The gene for cytochrome c (CYC1) was disrupted by insertion of URA3 (Chen and Clark-Walker clear genes for electron transport and the F0 complex 1993) and PET111, encoding a translational activation factor are brought together in haploid spores by meiosis. for COX2, was disrupted by insertion of kan (Costanzo et al. The second question addressed in this study concerns 2000). The unique gene for ADP/ATP translocase (AAC)in the mechanism of ␳0-lethality suppression in K. lactis. plasmid pUC19-KlAAC (Viola et al. 1995) and a copy of AAC Suppression may occur by a process invoked to explain disrupted by URA3 in plasmid pUC-KlAAC-URA were gifts from Cesira Galeotti. The disruption, in addition to removing survival of S. cerevisiae petite mutants. In wild-type cells, the majority of the AAC gene, deletes 453 bp of downstream the electrochemical potential across the mitochondrial sequence containing many stop codons in all three reading inner membrane, ⌬⌿, is generated by proton export frames. during electron transport or, in some circumstances, by Disruption of genes in K. lactis was performed by the one- back pumping of protons through F following hydroly- step replacement method (Rothstein 1983). Verification that 0 diploids were heterozygous for single-copy disruption was un- sis of ATP by F1-ATPase (Mitchell 1979; Nicholls dertaken by digestion of genomic DNA followed by Southern and Ferguson 1992; Boyer 1997). Neither of these blotting and hybridization according to the protocols de- mechanisms for generation of ⌬⌿ is available to petite scribed in previous publications. Construction of strains het- mutants. It has been proposed that the combined activi- erozygous for two disruptions was by transformation of a strain with a single disruption. Strain CK11/5 atp7::URA3/ϩ pet111 ties of F1-ATPase and ADP/ATP translocase are respon- ⌬ ϩ ⌬⌿ ::kan/ was formed by disruption of ATP7 in CK11/4, sible for maintenance of an adequate (Dupont et and strain CK11/6 atp5⌬::kan/ϩ cyc1::URA3/ϩ was obtained al. 1985). ADP/ATP translocase, located in the mito- by disruption of ATP5 in CK11/3. Likewise, CW75/3 atp7:: chondrial inner membrane, is the third component of URA3/ϩ pet111⌬::kan/ϩ was obtained by disruption of PET111 oxidative phosphorylation that, under aerobic condi- in CW75/1. Strains CW75/5 aac⌬::URA3/ϩ atp5⌬::/ϩ and ⌬ ϩ ⌬ ϩ tions, exchanges ATP in the mitochondrial matrix for CW75/6 aac ::URA3/ pet111 ::kan/ were isolated from CW75/4 aac⌬::URA3/ϩ following transformation with the re- ADP in the cytosol (Klingenberg 1984). In petite mu- spective gene disruption cassettes. tants, it is thought that ADP/ATP translocase reverses Complementation of aac-disruptant lethality by wild-type to import ATP with four negative charges and export AAC: To confirm disruption of AAC, the wild-type gene was ADP with three negative charges (Klingenberg and reintroduced into CK11/7. The 1661-bp MscI-HpaI fragment Rottenberg 1977; Laris 1977; Dupont et al. 1985). from pUC19-KlAAC containing KlAAC (Figure 3) was ligated with pCXJ30 cut with SmaI. pCXJ30 is a multicopy vector of The action of F1-ATPase is to hydrolyze ATP to ADP in 5579 bp containing the S11 K. lactis origin of DNA replication the absence of F0. It is presumed that the combined and a kan gene conferring resistance to G418 (X. J. Chen, action of the two enzymes provides sufficient electro- unpublished observations). The resulting plasmid, pCXJ30- Oxidative Phosphorylation in K. lactis 931

TABLE 1 Genotype and source of yeast strains

Strain Relevant genotype Source or reference K. lactis Haploids CK350 a atp5⌬::kan adeT600 ura3.1 Chen et al. (1998) CW36-ATP2 ␣ ade1 adeX his7 ura3.2, LEU2::pCXJ4-ATP2 Clark-Walker et al. (2000) CW36-H ␣ ade1 adeX his7 ura3.2 LEU2::pCXJ4-atp2.1 Clark-Walker et al. (2000) GlyϪ3.9 ␣ ade1 adeX his7 ura3.2 [⌬atp9] Clark-Walker et al. (1997) PH1 a atp7::URA3, adeT600 ura3.1 Chen et al. (1998) PM6-7A a adeT600 ura3.1 Chen and Clark-Walker (1993) Diploids CK11 ␣/a ϩ/adeT600 lysA1/ϩura3.1/ura3.1 This study CK11/1 CK11 with atp5⌬::kan/ϩ This study CK11/2 CK11 with atp7::kan/ϩ This study CK11/3 CK11 with cyc1::URA3/ϩ This study CK11/4 CK11 with pet111⌬::kan/ϩ This study CK11/5 CK11 with atp7::URA3/ϩ pet111⌬::kan/ϩ This study CK11/6 CK11 with atp5⌬::kan/ϩ cyc1::URA3/ϩ This study CK11/7 CK11 with aac⌬::URA3/ϩ This study CW75 ␣/a, ade1/ϩϩ/lysA1 ura3.1/ura3.1 atp2.1/atp2.1 This study CW75/1 CW75 with atp7::URA3/ϩ This study CW75/2 CW75 with pet111⌬::kan/ϩ This study CW75/3 CW75 with atp7::URA3/ϩ pet111⌬::kan/ϩ This study CW75/4 CW75 with aac⌬::URA3/ϩ This study CW75/5 CW75 with aac⌬::URA3/ϩ atp5⌬::kan/ϩ This study CW75/6 CW75 with aac⌬::URA3/ϩpet111⌬::kan/ϩ This study S. cerevisiae Haploids CS133-2C MAT␣ cyt1⌬::kan his4 leu2-3 ura3-52 This study CS171 MATa atp4⌬::kan ade2-1 leu2-3 ura3-52 This study Diploid CWS7 MATa/MAT␣ atp4⌬::kan/ϩϩ/cyt1⌬::kan ade2/ϩϩ/his4 leu2/leu2 ura3/ura3 This study

AAC containing a single copy of AAC, was introduced into medium at 28Њ. Spheroplasts were produced by digestion with CK11/7 by selecting for resistance to G418 following transfor- 10,000 units Zymolyase-20T (Seikagaku) for 60 min at 30Њ, mation. A selected transformant, CK11/7-pCXJ30-AAC, was resuspended in ice-cold 0.6 m sorbitol/10 mm TES [N-Tris sporulated on ME agar containing 200 ␮g/ml G418 to main- (hydroxymethyl) methyl-2-aminoethanesulfonic acid] pH 7.4/ tain the plasmid. 0.1 mm 4-(2-aminoethyl)-benzene-sulfonyfluoride (AEBSF), an Ascus dissection and phenotype determination: Asci were HCl protease inhibitor (Calbiochem, La Jolla, CA), and bro- dissected with a Singer MSM Series 200 System (Singer Instru- ken in a French press at 1000 p.s.i. Mitochondria were recov- ments, Somerset, UK) following brief treatment with Zymoly- ered by centrifugation at 9000 rpm for 20 min in a Sorvall ase (Seikaguku, Tokyo). Tetrad colonies were photographed SS34 rotor after prior removal of cellular debris by two rounds after 4 days at 28Њ. Phenotypes were determined by replica of centrifugation at 3500 and 5000 rpm for 5 min. Mitochon- plating to G418 medium for kan, GMM Ade Lys lacking uracil drial pellets were resuspended in 500–700 ␮lof5mm TES (pH for URA3, and to GlyYP for glycerol growth. Genotypes have 7.5), 15% w/v glycerol, 0.4 mm EDTA, 0.4 mm dithiothreitol been inferred from phenotypes according to alleles listed in containing 0.2 mm AEBSF protease inhibitor. ATPase activity Table 1. was estimated according to Law et al. 1995. Freshly prepared Preparation of AAC probe: A 514-bp segment of the K. lactis mitochondria (50 ␮l) were added to 1.2 ml of 10 mm Tris- AAC gene was amplified from pUC19-KlAAC by the polymer- HCl (pH 8.2), 2 mm MgCl2, 200 mm KCl, and to final ATP ase chain reaction using primers 5Ј CGATAACTAACAACG concentrations of 150, 200, 300, 500, 1000, and 3000 ␮m. TACC 3Ј in the forward direction and 5Ј GTAGACATCGAC Incubation was at 30Њ and 0.5-ml aliquots were removed at 1 TAGACC 3Ј in the reverse direction. Location of the amplified and 2 min and added to 50 ␮l3m trichloroacetic acid. Inor- segment is shown in Figure 3. ganic phosphate concentration was measured with Sumner DNA manipulation: Standard conditions, described in detail buffer against a standard curve and protein was estimated in previous publications (Chen and Clark-Walker 1993, by Dc-Protein Assay (Bio-Rad Laboratories, Richmond, CA). 1995, 1996), were used for DNA isolation, restriction enzyme ATPase activity is expressed as micromoles of ATP hydrolyzed digestion, electrophoresis, transfer of DNA to Nylon, hybrid- per minute per milligram protein. Vmax and Km estimations, ization, and autoradiography. according to the Michaelis-Menton equation, were obtained Kinetic parameters of F1-ATPase: Mitochondria were pre- from a Linweaver-Burk plot. Values are averages from three pared from 750-ml cultures grown to stationary phase in GYP separate mitochondrial preparations in each case. 932 G. D. Clark-Walker and X. J. Chen

Figure 1.—Response of K. lactis wild type (PM6-7A) and strains lacking components of F0, encoded by mtDNA, Gly- 3.9 [⌬ATP9] or nuclear DNA, CK350 (atp5⌬::kan), and PH1 (atp7::kan) to antimycin (1 ␮m) in the presence of 2% glucose. Aliquots (10 ␮l) of cultures, serially diluted to 10Ϫ2–10Ϫ5, were added to plates that were incubated for 3 days at 28Њ before photography.

RESULTS

Sensitivity of F0 mutants to antimycin: K. lactis strains lacking F0 subunits, coded by either the mitochondrial or nuclear genomes, were examined for their response to antimycin on glucose medium. Antimycin speciﬁcally inhibits electron transport but does not affect glycolysis

(Slater 1973). As shown in Figure 1, strains lacking F0 subunit 9, (Gly-3.9), the OSCP protein coded by ATP5 (CK350), or subunit d, coded by ATP7 (PH1), are sensitive to 1 ␮m antimycin, whereas the wild type, PM6- 7A, is less affected. Hence inhibition of respiration by antimycin is more severe for strains lacking a functional

F0 complex, which suggests that K. lactis may not tolerate simultaneous inhibition of both electron transport and Figure 2.—Tetrad dissection of asci and phenotype deter- F F -ATP synthase. mination of surviving colonies obtained from CK11/5 (atp7⌬:: 1 0 ϩ ⌬ ϩ Њ Colethality of disruptions for two components of oxi- URA3/ pet111 ::kan/ ). After 4 days at 28 , colonies were replica plated to GYP medium containing G418 (200 ␮g/ dative phosphorylation: In the absence of mutations in ml), GlyYP, and GMM minimal medium supplemented with mtDNA that inactivate genes for both electron transport adenine and lysine. and the F0 complex, an indirect genetic test involving disrupted nuclear genes was applied. Diploid strains of K. lactis were constructed by serial inactivation of genes GlyYP containing glycerol (Figure 2; Table 2). For the required for the two components of oxidative phosphor- 95 tetrads dissected from CK11/5 the results are ylation. As a preliminary step in the construction of straightforward. There is no surviving colony containing double-disruptant strains, the survival of spores from both G418 resistance, as a marker of PET111 disruption, strains containing single disruptions was examined. Strains and growth in the absence of uracil, as a marker for with disruptions in genes specifying electron transport ATP7 inactivation. Microscopic examination shows that components cytochrome c, cyc1::URA3 (CK11/3, 32/35 spores inferred to contain two disruptions have germi- give 4:0 viable colonies) or the translational activation nated to form microcolonies containing between 100 factor for Cox2p, pet111⌬::kan (CK11/4, 36/39) show and 1000 cells that do not proliferate. From the illustra- a slight decrease in spore survival compared to ones tion it can be seen that large colonies growing on glyc- ⌬ inactivated in F0 subunits OSCP, atp5 ::kan (CK11/1, erol do not contain inactivated genes (as they require 38/39) or subunit d, atp7::URA3 (CK11/2, 39/40). The uracil and are sensitive to G418). Consequently the non- small decrease in spore survival revealed by these num- viable colonies in these tetrads can be inferred to con- bers provides a background for studies using double tain two inactivated genes. Likewise, in tetrads showing disruptants. three surviving colonies, the nonviable colony can be Following meiosis and ascus dissection of strains con- inferred to contain two inactivated genes on the basis taining two disruptions, CK11/5 (atp7::URA3 pet111 of the phenotypes of the viable colonies. In tetrads lack- ⌬::kan) and CK11/6 (atp5⌬::kan cyc1::URA3), pheno- ing a Glyϩ colony, G418ϩ and Uraϩ phenotypes segre- types were examined by replica plating to medium con- gate from one another in the four viable spores. The taining G418, minimal medium lacking uracil, and number of asci showing 4:0, 3:1, or 2:2 survival of Oxidative Phosphorylation in K. lactis 933 74 dissected Total asci b 1 a a a 00 00 a 0 a 01 200 a a a a 121 a 1 101 a Ratio of viable:nonviable spores (No. of asci) TABLE 2 00000 211 00000 00 0 21002 211 21201 012121 2 0 0 01020 01021 0000000 211 0100210 2121011 01 200 21 0210 01 20111 21 01 Spore viability and allelic segregation from double disruptant strains ::kan ⌬ ::URA3 ⌬ ::URA3 ⌬ ::kan ::URA3 ::kan ⌬ ⌬ ⌬ ::URA3 ⌬ ::kan aac ::kan AAC ::kan ⌬ ⌬ ::kan aac ::kan AAC ⌬ ⌬ ⌬ ::kan cyc1::URA3 ::kan CYC1 ⌬ ⌬ ATP5 cyc1::URA3 atp2.1 ATP5 aac atp2.1 ATP7 PET111 atp5 atp2.1 atp5 ATP5 CYC1 ATP7 PET111 atp7::URA3 PET111 ATP7 pet111 atp7::URA3 pet111 atp5 atp2.1 ATP5 AAC atp2.1 atp5 atp2.1 ATP7 pet111 atp2.1 atp7::URA3 pet111 atp2.1 atp7::URA3 PET111 atp2.1 pet111 atp2.1 PET111 AAC atp2.1 pet111 atp2.1 PET111 aac Tetrads with nonviable colonies inferred to contain a single gene disruption (see text). Segregation of disrupted alleles cannot be inferred. a b CW75/6 (8) (46) (2) (1) (8) (8) (1) CW75/3 (7) (36) (11) (2) (1) (1) (1) (1) 60 CK11/6 (12) (47) (2) (11) (3) 75 StrainCK11/5 Inferred genotype 4:0CW75/5 (16) 3:1 (59) (7) 2:2 (20) (22) (2) 95 (5) (2) 38 934 G. D. Clark-Walker and X. J. Chen

16:59:20 is close to the ratio of 1:4:1 for parental ditype, unlinked. Another aspect of results from sporulation of tetratype, and nonparental ditype, respectively. As a 1:4:1 CK11/5 is that all spores containing single disruptions ratio indicates independent assortment, it can be con- survive. cluded that the ATP7 and PET111 genes of K. lactis are Some nonviable colonies inferred to contain a single gene disruption have been observed on dissection of CK11/6. Examination of phenotypes from CK11/6, containing disruptions in ATP5 and CYC1, shows that ﬁve spores inferred to contain cyc1::URA3 do not survive (Uraϩ/G418s; Table 2). The relevant asci show 3:1 and 2:2 ratios of viable to nonviable spores. However, the results from CK11/6 support the previous observation from CK11/5 that spores inferred to contain double disruptions do not survive. Thus not a single surviving colony, lacking both components of oxidative phosphorylation, has been found in 170 asci. Suppression of double-disruptant lethality: To ascer- tain whether a ␳0-lethality suppressor mutation can com- pensate for loss of both oxidative phosphorylation components due to disruption of nuclear genes, a diploid strain, CW75, was constructed that is homozygous for the suppressor allele atp2.1. Subsequently, a strain, CW75/3, that is heterozygous for disruptions in both ATP7 and PET111 was obtained. Sporulation and ascus dissection of CW75/3 revealed that 54 out of 60 tetrads produced four viable colonies, while 6 tetrads contained one nonviable spore (Table 2). Phenotypic analysis showed that two nonviable spores were inferred to contained atp7::URA3, one had pet111⌬::kan, two contained both disruptions, and one spore lacked any disruption. From these results it is apparent that the atp2.1 allele can suppress colethality of atp7::URA3 and pet111⌬::kan. Although S. cerevisiae can survive lack of both components of oxidative phosphorylation due to loss of mtDNA, it has not been determined if it can tolerate loss of nuclear genes encoding these complexes. Conse- quently, to determine whether S. cerevisiae is directly analogous to ␳0-lethality suppressor mutants of K. lactis, segregants from a diploid strain, CWS7, heterozygous ⌬ for disruptions in genes for cytochrome c1, cyt1 ::kan, ⌬ and F0 subunit b, atp4 ::kan, were examined for survival. As anticipated, no lethality of colonies containing double disruptions was observed in 20 tetrads (not illustrated), thereby formally demonstrating that baker’s yeast behaves as though it contains a suppressor analogous to atp2.1 of K. lactis. Disruption of AAC is lethal in K. lactis: Examination

Figure 3.—Disruption of AAC in K. lactis. (A) Restriction site map of AAC and replacement of a BclI fragment containing the majority of the gene by URA3 from S. cerevisiae. The PCR- ampliﬁed DNA of 514 bp used as a probe contains a small portion of the AAC gene from the 5Ј region. (B) Autoradio- graph of DNA from CK11 (wild type) and CK11/7, heterozygous for disruption of AAC. Genomic DNA was digested with MscI-HpaIorMscI before separation of fragments in 0.8% agarose. Hybridization was performed with the AAC-PCR prod- uct labeled with [32P]dATP. (C) Ascus dissection of CK11/7 (aac⌬::URA3/ϩ). The photo was taken after 4 days at 28Њ. Oxidative Phosphorylation in K. lactis 935

one of the colonies in these tetrads had a Uraϩ phenotype representing aac⌬::URA3. Colonies having a Uraϩ phenotype also grew on G418, marking the retention of the plasmid containing the wild-type AAC gene. In addition, some UraϪ colonies also retained the plasmid as they grew on G418 (Figure 4). From these data it appears that lethality of haploids following meiosis of CK11/7 is a consequence of AAC disruption and is not due to inactivation of a gene at an extraneous site. This conclusion is supported by observations that all three AAC genes of S. cerevisiae can restore viability of K. lactis disrupted in AAC (G. D. Clark-Walker, unpublished observations). Suppression of aac-disruptant lethality by atp2.1: To examine participation of the ADP/ATP translocase in suppression of ␳0-lethality, strain CW75, homozygous for atp2.1, was disrupted in one copy of AAC to produce CW75/4. Sporulation and ascus dissection of CW75/4 showed that 67 of the 78 tetrads gave four viable colonies while the other 11 had one nonviable colony. In each case the nonviable spore corresponded to the inferred presence of aac⌬::URA3. The survival of haploid strains containing a disrupted AAC gene in the presence of the suppressor allele atp2.1 Figure 4.—Tetrad dissection of asci and phenotype deter- permitted an assessment of whether such strains were mination of colonies obtained from CK11/7 (aac⌬::URA3/ dependent on mtDNA. Examination of whether four ϩ) transformed with the multicopy plasmid pCXJ30-AAC con- haploid strains from a single ascus could survive loss of Њ taining the wild-type gene from K. lactis. After 4 days at 28 , mtDNA was determined by growth on a GYP plate con- colonies were replica plated to GYP medium containing G418 ␮ (200 ␮g/ml) and GMM minimal medium supplemented with taining 16 g/ml ethidium bromide (not illustrated). adenine and lysine. Two strains, with wild-type AAC as determined by South- ern blotting (not illustrated), and the suppressor allele atp2.1, grew on EB where they lose mtDNA, whereas of whether ADP/ATP translocase is required for sup- the two segregants, with a disrupted AAC gene, did not pression of ␳0-lethality was performed in the first in- grow. Furthermore, prolonged incubation of AAC-dis- stance by disrupting one copy of the gene, AAC,inthe ruptants on EB did not lead to the appearance of resis- wild-type diploid CK11. Correct disruption of AAC in tant papillae. In other words, strains containing atp2.1 strain CK11/7 was ascertained by hybridization with a are not able to lose mtDNA when they lack ADP/ATP PCR probe specific to the 5Ј region of the sequence translocase. that is retained in the disrupted gene (Figure 3). The Suppression of aac-disruptant lethality by atp2.1 re- wild type, CK11, has a single hybridizing fragment of quires both F0 and electron transport: The demonstra- 1661 bp, produced by MscI and HpaI digestion, whereas tion that mtDNA cannot be lost from haploid strains the heterozygous disruptant, CK11/7, contains an addi- containing aac⌬::URA3 and atp2.1 raised a question as tional band of 2950 bp from MscI cleavage, correspond- to whether electron transport/proton pumping or F1F0- ing to the expected size for disruption of AAC that has ATP synthase activities are required. To examine this lost the HpaI site due to replacement by URA3 (Figure question, two strains, CW75/5 and CW75/6, were made 3). Sporulation and ascus dissection of CK11/7 gave by disruption of one copy of ATP5 and one copy of only two viable spores per tetrad (Figure 3). In some PET111, respectively, in CW75/4 containing aac⌬::URA3. tetrads, minicolonies could be seen that did not grow Spore viability and phenotypes of surviving colonies fol- on subculture. Replica plating showed that all viable lowing dissection of asci are shown in Table 2. In both spores in 51 tetrads examined were UraϪ and conse- cases there are no viable colonies containing two disrup- quently lacked a disrupted AAC gene (not illustrated). tions in 38 asci from CW75/5 and 74 asci from CW75/6. To confirm that lethality is a consequence of AAC With CW75/5, 2 asci have a 3:1 ratio and 2 have a 2:2 disruption, the wild-type gene on a multicopy plasmid, ratio of viable to nonviable colonies with one nonviable pCXJ30, was transformed into CK11/7. Sporulation on spore inferred to contain only aac⌬::URA3 in each case. ME agar containing G418 to maintain the plasmid, fol- Likewise, in tetrads derived from CW75/6 there are lowed by ascus dissection, showed that some tetrads nonviable spores inferred to contain single disruptions produced four or three viable colonies (Figure 4). Rep- in AAC: 2 with a 3:1 ratio and 8 with a 2:2 ratio. The 2 lica plating to minimal medium indicated that two or remaining tetrads have a nonviable spore inferred to 936 G. D. Clark-Walker and X. J. Chen

TABLE 3 or ATP7 genes encoding the OSCP and d subunits, respectively (Chen et al. 1998). By contrast, in S. cerevis- Kinetic parameters of F1-ATPase iae, combined loss of electron transport and F0 com- ␮ Vmax mol Pi plexes, by either elimination of mtDNA or inactivation ␮ Strain Km m ATP min/mg protein of nuclear genes, is not lethal. Hence the combined evidence supports the conclusion that loss of mtDNA CW36-ATP2 1052 Ϯ 263 5.08 Ϯ 1.12 (wild type) from wild-type K. lactis is lethal because both electron CW36-H (atp2.1) 204 Ϯ 10 0.90 Ϯ 0.02 transport and F0 components of oxidative phosphorylation are missing. Lethality is thought to be due to lack of a mechanism for generating sufﬁcient ⌬␺ for import of proteins through the inner membrane. contain a single disruption in PET111. Despite the de- In accord with expectations from previous studies, creased viability of spores containing single disruptions, introduction of the atp2.1 mutation results in survival these results are in accord with the number of nonviable of spores containing double disruptions. A possible ex- spores derived from the parental strain, CW74/4, con- planation for suppression of ␳0-lethality and double dis- taining just an AAC disruption, where 11 nonviable spores ruptant-lethality by this allele comes from the observa-

were found in 78 tetrads (Table 2). However, the conclu- tion that mutant F1-ATPase has a decreased Km for ATP sion obtained from the above analysis is that both elec- compared to wild type. In other words, the atp2.1 allele

tron transport and F1F0-ATP synthase activities are re- has greatly increased the afﬁnity of F1 for ATP. As a quired to support viability of strains lacking ADP/ATP consequence it is likely that mutant F1 can hydrolyze translocase, even though the suppressor allele, atp2.1, sufﬁcient ATP to allow the ADP/ATP translocase to is present. electrogenically exchange ADP in the matrix for ATP

Kinetic parameters of F1-ATPase: Previous studies in the cytosol. Conversely, because the wild-type enzyme

have shown that F1-ATPase activity does not correlate has a lower afﬁnity for ATP, it may not be able to supply with ability of atp alleles to suppress ␳0-lethality (Clark- an adequate level of ADP to support exchange. Walker et al. 2000). However, ␳0-suppression was shown In the second part of this study we used coincidence to require some ATPase activity, as a double mutant, of mutations to investigate the role of ADP/ATP translo- lacking ability to hydrolyze ATP, yet containing atp3.1, case in suppression of ␳0-lethality. It has been proposed could not lose mtDNA. In view of these results it was that if ADP/ATP translocase participates in suppression ␳0 decided to compare kinetic parameters of F1-ATPase of -lethality then disruption of the AAC gene in K. from wild-type and atp 2.1 mutant strains. For this com- lactis would not allow mtDNA to be lost from cells con- parison we used the previously constructed haploid iso- taining a F1 suppressor allele because there would be genic strains, CW36-ATP2 containing wild-type ATP2 no way to maintain ⌬␺. In other words, cells lacking and CW36-H (atp2.1), which differ only by the intro- ADP/ATP translocase would need to maintain ⌬␺ by duced mutation (Clark-Walker et al. 2000). As shown proton pumping from electron transport and/or rever-

in Table 3, F1-ATPase of mitochondria from the atp2.1 sal of F1F0-ATP synthase.

strain has a ﬁvefold lower Km for ATP than the wild However, in an initial experiment we found that dis-

type, although the latter strain has a greater Vmax. The ruption of AAC is lethal and that introduction of the

increased afﬁnity of mutant F1 for ATP in the hydrolysis wild-type gene restores viability. This result was unantici- reaction suggests an explanation for suppressor activity pated as it has been reported that inactivation of AAC as discussed below. is nonlethal in haploid K. lactis (Viola et al. 1999), although attempts by another group to disrupt the gene in haploid K. lactis were not successful (Trezeguet et DISCUSSION al. 1999). A possible explanation for the conﬂicting results could rest on genetic differences between strains. The assembly of two mutated genes into a haploid Because the atp2.1 allele can suppress AAC-disruption nucleus by meiosis has allowed us to examine their com- lethality in our strain, it is possible that some genes bined effect on dissected spores. By this means, it has been required for energy transduction in mitochondria could found that loss of the electron transport and F0 compo- be the most likely candidates for allelic differences. nents of oxidative phosphorylation are synergistically Indeed, differences may exist between genes required

lethal in K. lactis. Inactivation of electron transport has for the efﬁciency of the F1F0-ATP synthase that could been achieved either by disruption of the PET111 gene, be connected to the mechanism of atp2.1 suppression encoding a translational activation factor for Cox2p syn- of AAC-disruption lethality. For instance, according to thesis (Costanzo et al. 2000), or by disruption of the observations from other organisms, it is believed that unique gene for cytochrome c, CYC1 (Chen and Clark- disruption of ADP/ATP translocase leads to hyperpolar-

Walker 1993). Elimination of a functional F0 complex ization of the mitochondrial inner membrane (Espo- has been accomplished by disruption of either the ATP5 sito et al. 1999; Vander Heiden et al. 1999). Relief Oxidative Phosphorylation in K. lactis 937 of hyperpolarization by atp2.1 is proposed to occur by Contamine, V., and M. Picard, 2000 Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the partial uncoupling in the forward direction of ATP syn- budding yeast. Microbiol. Mol. Biol. Rev. 64: 281–315. thesis. Support for this view comes from the knowledge Costanzo, M. C., N. Bonnefoy, E. H. Williams, G. D. Clark-Walker that atp suppressor mutants are all less efficient at mak- and T. D. Fox, 2000 Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators ing ATP than wild type as evidenced by their poorer have orthologous functions in other budding yeasts. Genetics growth on a nonfermentable substrate (Clark-Walker 154: 999–1012. et al. 2000). Accumulation of ADP and lowering of the Cox, G. B., R. J. Devenish, F. Gibson, S. M. Howitt and P. Nagley, 1992 The structure and assembly of ATP synthase, pp. 283–315 ATP/ADP ratio would stimulate respiration (Nicholls in Molecular Mechanisms in Bioenergetics, edited by L. Ernster. and Ferguson 1992; Arnold and Kadenbach 1999; Elsevier Science, Amsterdam. Kadenbach and Arnold 1999), thereby preventing the Dupont, C. H., J. P. Mazat and B. Guerin, 1985 The role of adenine nucleotide translocation in the energization of the inner mem- accumulation of electrons in the respiratory chain and brane of mitochondria isolated from ␳ϩ and ␳0 strains of Saccharo- the formation of detrimental reactive oxygen species myces cerevisiae. Biochem. Biophys. Res. Commun. 132: 1116–1123. (Skulachev 1996; Finkel and Holbrook 2000). Both Esposito, L., S. Melov,A.Panov,B.A.Cottrel and D. C. Wallace, 1999 Mitochondrial disease in mouse results in increased oxida- an intact respiratory chain and a functional F1F0-ATP tive stress. Proc. Natl. Acad. Sci. USA 96: 4820–4825. synthase complex would be required to relieve hyperpo- Ferguson, L. R., and R. C. von Borstel, 1992 Induction of the cytoplasmic ‘petite’ mutation by chemical and physical agents in larization by mutant F1 in agreement with experimental ␳0 Saccharomyces cerevisiae. Mutat. Res. 265: 103–148. results. Thus the suppression of both and AAC-disrup- Finkel, T., and N. J. Holbrook, 2000 Oxidants, oxidative stress and tion lethalities is based on the two functions of F1.In the biology of ageing. Nature 408: 239–247. Foury, F., T. Roganti, N. Lecrenier and B. Purnelle, 1998 The the forward direction, in association with F0, mutant F1 complete sequence of the mitochondrial genome of Saccharomyces is partially uncoupled, whereas in the reverse reaction cerevisiae. FEBS Lett. 440: 325–331. it has a greater affinity for ATP. Both activities would Hardy, C. M., C. L. Galeotti and G. D. Clark-Walker, 1989 Dele- result in a higher level of ADP in mitochondria com- tions and rearrangements in Kluyveromyces lactis mitochondrial DNA. Curr. Genet. 16: 419–427. pared to wild type, stimulating respiration in one case Kadenbach, B., and S. Arnold, 1999 A second mechanism of respi- and promoting electrogenic exchange in the other in- ratory control. FEBS Lett. 447: 131–134. stance. Klingenberg, M., 1984 The ADP/ATP carrier in mitochondrial membranes, pp. 511–553 in The Enzymes of Biological Membranes, We thank Cesira Galeotti for plasmids containing the AAC wild- Vol. 4, edited by A. N. Martouosi. Plenum Press, New York. type and disrupted genes of K. lactis and Lijun Ouyang for skilled Klingenberg, M., and H. Rottenberg, 1977 Relation between the technical assistance. gradient of the ATP/ADP ratio and the membrane potential across the mitochondrial membrane. Eur. J. Biochem. 73: 125– 130. Laris, P. C., 1977 Evidence for the electrogenic nature of the ATP- LITERATURE CITED ADP exchange system in rat liver mitochondria. Biochim. Bio- phys. Acta 459: 110–118. Arnold, S., and B. Kadenbach, 1999 The intramitochondrial ATP/ Law, R. H. P., S. Manon, R. J. Devenish and P. Nagley, 1995 ATP ADP-ratio controls cytochrome c oxidase activity allosterically. synthase from Saccharomyces cerevisiae. Methods Enzymol. 260: FEBS Lett. 443: 105–108. 133–163. Attardi, G., and G. Schatz, 1988 Biogenesis of mitochondria. Mitchell, P., 1979 Keilin’s respiratory chain concept and its chemi- Annu. Rev. Cell Biol. 4: 289–333. osmotic consequences. Science 206: 1148–1159. Boyer, P. D., 1997 The ATP synthase—a splendid molecular ma- Murray, G. L., W. G. Bao, H. Fukuhara, X. M. Zuo, G. D. Clark- chine. Annu. Rev. Biochem. 66: 717–749. Walker et al., 2000 Disruption of the MRP-L23 gene encoding Chen, X. J., and G. D. Clark-Walker, 1993 Mutations in MGI genes the mitochondrial ribosomal protein L23 is lethal for Kluyvero- convert Kluyveromyces lactis into a petite-positive yeast. Genetics myces lactis but not for Saccharomyces cerevisiae. Curr. Genet. 37: 133: 517–525. 87–93. Chen, X. J., and G. D. Clark-Walker, 1995 Specific mutations in Nicholls, D. G., and S. J. Ferguson, 1992 Bioenergetics 2. Academic ␣ ␥ - and -subunits of F1-ATPase affect mitochondrial genome in- Press, London. tegrity in the petite-negative yeast Kluyveromyces lactis.EMBOJ. Norais, N., D. Prome and J. Velours, 1991 ATP synthase of yeast 14: 3277–3286. mitochondria. Characterization of subunit d and sequence analy- Chen, X. J., and G. D. Clark-Walker, 1996 The mitochondrial sis of the structural gene ATP7. J. Biol. Chem. 266: 16541–16549. genome integrity gene, MGI1,ofKluyveromyces lactis encodes the Poyton, R. O., and J. E. McEwen, 1996 Crosstalk between nuclear beta-subunit of F1-ATPase. Genetics 144: 1445–1454. and mitochondrial genomes. Annu. Rev. Biochem. 65: 563–607. Chen, X. J., and G. D. Clark-Walker, 1999 The petite mutation Rothstein, R. J., 1983 One-step gene disruption in yeast. Methods in yeasts: 50 years on. Int. Rev. Cytol. 194: 197–238. Enzymol. 101: 202–211. Chen, X. J., P. M. Hansbro and G. D. Clark-Walker, 1998 Suppres- Saraste, M., 1999 Oxidative phosphorylation at the fin de sie`cle. ␳0 sion of lethality by mitochondrial ATP synthase F1 mutations Science 283: 1488–1493. in Kluyveromyces lactis occurs in the absence of F0. Mol. Gen. Genet. Skulachev, V. P., 1996 Role of uncoupled and non-coupled oxida- 259: 457–467. tions in maintenance of safely low levels of oxygen and its one- Clark-Walker, G. D., and X. J. Chen, 1996 A vital function for electron reductants. Q. Rev. Biophys. 29: 169–202. mitochondrial DNA in the petite-negative yeast Kluyveromyces Slater, E. C., 1973 The mechanism of action of the respiratory lactis. Mol. Gen. Genet. 252: 746–750. inhibitor, antimycin. Biochim. Biophys. Acta 301: 129–154. Clark-Walker, G. D., F. Francois, X. J. Chen, M. R. Vieira Da Trezeguet, V., I. Zeman, C. David, G. J. Lauquin and J. Kolarov, Silva and M. L. Claisse, 1997 Mitochondrial ATP synthase 1999 Expression of the ADP/ATP carrier encoding genes in subunit 9 is not required for viability of the petite-negative yeast aerobic yeasts; phenotype of an ADP/ATP carrier deletion mu- Kluyveromyces lactis. Curr. Genet. 31: 488–493. tant of Schizosaccharomyces pombe. Biochim. Biophys. Acta 1410: Clark-Walker, G. D., P. M. Hansbro, F. Gibson and X. J. Chen, 229–236. 2000 Mutant residues suppressing ␳0-lethality in Kluyveromyces Uh, M., D. Jones and D. M. Mueller, 1990 The gene coding for lactis occur at contact sites between subunits of F1-ATPase. Bio- the yeast oligomycin sensitivity-conferring protein. J. Biol. Chem. chim. Biophys. Acta 1478: 125–137. 265: 19047–19052. 938 G. D. Clark-Walker and X. J. Chen

Vander Heiden, M. G., N. S. Chandel, P. T. Schumacker and C. B. plements the Saccharomyces cerevisiae op1 mutation. Curr. Genet. Thompson, 1999 Bcl-xL prevents cell death following growth 27: 229–233. factor withdrawal by facilitating mitochondrial ATP/ADP ex- Viola, A. M., T. Lodi and I. Ferrero, 1999 A Klaac null mutant change. Mol. Cell 3: 159–167. of Kluyveromyces lactis is complemented by a single copy of the Velours, J., P. Durrens, M. Aigle and B. Guerin, 1988 ATP4, the Saccharomyces cerevisiae AAC1 gene. Curr. Genet. 36: 29–36. structural gene for yeast F0F1 ATPase subunit 4. Eur. J. Biochem. Weber, J., and A. E. Senior, 1997 Catalytic mechanism of F1-ATPase. 170: 637–642. Biochim. Biophys. Acta 1319: 19–58. Viola, A. M., C. L. Galeotti, P. Goffrini, A. Ficarelli and I. Fer- rero, 1995 A Kluyveromyces lactis gene homologue to AAC2 com- Communicating editor: B. J. Andrews