Dual Mutations Reveal Interactions Between Components of Oxidative Phosphorylation in Kluyveromyces Lactis
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Copyright 2001 by the Genetics Society of America 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 phosphoryla- tion 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.