Mutant residues suppressing ρ0-lethality in Kluyveromyces lactis occur at contact sites between subunits of F1-ATPase

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Abstract

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing ρ0-lethality are limited to the ATP1, 2 and 3 genes coding for the α-, β- and γ- subunits of mitochondrial F1-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F1F0-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress ρ0-lethality. Positioning of the seven amino acids subject to mutation on the bovine F1-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F1 in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F1-ATPase in the maintenance of mitochondrial inner membrane potential.

Introduction

Since the initial description of respiratory deficient petite mutants in baker’s yeast, Saccharomyces cerevisiae, 50 years ago [1], researchers have been attempting to uncover the property that allows some yeasts to produce petites while others cannot. In S. cerevisiae, petite mutants, with large deletions or a complete absence of mitochondrial DNA (mtDNA), occur spontaneously or can be induced by DNA-targeting drugs [1], [2], [3], [4]. On the other hand, loss of mtDNA in the petite-negative yeast Kluyveromyces lactis, is lethal (ρ0-lethality) [5], [6]. However, lack of ability to respire, exemplified by disruption of the unique nuclear gene for cytochrome c in K. lactis, does not cause death [7]. That is, K. lactis has sufficient fermentative capacity to grow like respiratory deficient petite mutants of S. cerevisiae, yet it cannot sustain large deletions in mtDNA. Progress has been made towards solving this puzzle by the discovery of chromosomal mutations in K. lactis that can suppress ρ0-lethality. Analysis of seven suppressor mutants has shown that changes occur in ATP1 (formerly MG12), in ATP2 (MGI1) and in ATP3 (MG15) genes that encode the α-, β- and γ-subunits of F1 in mitochondrial F1F0-ATP synthase [8], [9].

In mitochondria, the F1F0-ATP synthase is located in the inner membrane and ATP synthesis occurs when protons pass through the complex from the intermembrane space to the matrix [10], [11]. The active site for ATP synthesis is found in the F1 complex which is attached to F0 on the matrix side. F1 is composed of five subunits with a ratio of 3α, 3β, 1γ, 1δ and 1ϵ.

An understanding of the role played by three F1 subunits in the operation of ATP synthase has been provided by structural analysis of bovine mitochondrial F1 [12]. The α- and β-subunits alternate in a hexameric complex where the catalytic site for ATP synthesis occurs in β-subunits at a contact region with α-subunits. A central space formed by the hexameric array is occupied by amino and carboxy terminal α-helices of the γ-subunit.

The rotation of the γ-subunit within the hexameric array has been directly demonstrated using proteins from a thermophilic bacterium [13] and support the idea that the catalytic sites in the β-subunit are equivalent and operate consecutively due to structural changes induced by rotation of the γ-subunit.

The function of F0 in ATP synthesis is not well understood. Moreover, differences occur in the number of subunits between bacteria and mitochondria [14], [15], [16]. In S. cerevisiae, F0 contains six major structural subunits [17], [18], [19]. Three of the structural subunits, Atp6, 8 and 9, are coded by mitochondrial DNA, while the chromosomal genes, ATP4, 5 and 7, specify subunits b [20], OSCP [21] and d [22]. Biochemical and genetic analysis has implicated subunits 6 and 9 in the transit of protons through the complex, but it is unclear how this transmission is converted to rotation of the γ-subunit in F1 [23].

A possible role of F0 in suppression of ρ0-lethality in K. lactis has been investigated following the suggestion that loss of mtDNA encoded subunits 6, 8 and 9 may create a proton leaky pore that could be blocked by mutations in F1 subunits [8]. However a K. lactis strain containing disruptions in the three nuclear genes, ATP4, 5 and 7, can still form mitochondrial genome deletion mutants in the presence of an atp-suppressor mutation [24]. Hence suppression of ρ0-lethality by mutations in F1 is not due to blockage of a proton leaky pore through a residual F0 complex. An implication from this result is that suppressor mutations would not be expected to occur in genes encoding F0 subunits.

In an attempt to delimit the genes involved in suppression of ρ0-lethality in K. lactis, we have expanded by 5-fold the number of characterised mutants as it seemed possible that additional loci may be found in genes specifying δ- and ϵ-subunits of F1 and in proteins that might interact with this complex. In addition, as initial observations indicated that properties of mutant alleles are influenced by genetic background, a series of isogenic strains have been created that differ only by the introduced mutation. Consequently, alterations in phenotypic properties can be attributed more confidently to changes caused by the mutant alleles. Finally, to gain insight into the mechanism of ρ0-lethality suppression, we examined the location of amino acids subject to mutation in the α-, β- and γ-subunits using the crystallographic structure of bovine F1 [12].

Section snippets

Strains and media

The genotypes and sources of K. lactis strains are listed in Table 1. The media used are GYP (0.5% Bacto yeast extract, 1% Bacto-peptone, 2% glucose), GlyYP (0.5% Bacto yeast extract, 1% Bacto-peptone, 2% glycerol) and GMM (0.6% Difco yeast nitrogen base without amino acids, 2% glucose). For solid medium, 2% Bacto agar was added. GMM medium was supplemented with appropriate auxotrophic requirements at 25 μg/ml for bases and 50 μg/ml for amino acids. Ethidium bromide (EB) medium is GYP plus EB

Isolation of suppressor mutants

Suppressor mutants of PM6-7A were obtained as papillae growing on EB medium at 16 μg/ml. As all isolates lack mtDNA, the first step in their characterisation involved crossing to the wild-type, CK56-16C, followed by sporulation of the resulting zygotes. Haploid colonies, obtained as random spores and containing mtDNA, were examined for growth on glycerol and resistance to ethidium bromide. Strains unable to grow on glycerol (Gly) were subjected to a second round of mating and sporulation to

Discussion

Analysis of 35 ρ0-lethality suppressor mutants, representing a 5-fold increase from previous studies, has uncovered new alleles in ATP1, 2 and 3. A suppressor phenotype has been confirmed for the mutations listed in Table 3, by introduction of each allele on an integrative vector following cloning and sequencing of a PCR product. An added advantage of this procedure is that properties of the mutant allele can be studied in an isogenic background. Hence changes to growth on different substrates

Acknowledgements

We thank Alan Senior for helpful discussions, John Walker for sending the F1 coordinates and Li Jun Ouyang for skilled technical assistance.

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  • Cited by (0)

    1

    Present address: Faculty of Medicine and Health Sciences, University of Newcastle, NSW 2308, Australia.

    2

    John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra City, ACT 2601, Australia.

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