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Aconitase Couples Metabolic Regulation to Mitochondrial DNA Maintenance

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Science  04 Feb 2005:
Vol. 307, Issue 5710, pp. 714-717
DOI: 10.1126/science.1106391

Abstract

Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.

Mitochondrial DNA (mtDNA) nucleoids have been purified from several organisms (15). In addition to DNA packaging proteins, which are required for mtDNA maintenance (6, 7), nucleoids also contain proteins whose functions are ostensibly unrelated to mtDNA activities (1, 5, 8, 9). We previously identified 11 proteins that are associated with mtDNA (1) (Table 1) and have now identified 11 more (whose gene names are indicated in bold in Table 1). These proteins can be grouped into four functional categories: (I) mtDNA transactions with no other known functions in mitochondria; (II) protein import and mitochondrial biogenesis; (III) the citric acid cycle and upstream glycolytic steps; and (IV) amino acid metabolism.

Table 1.

Mitochondrial nucleoid proteins identified by in organello formaldehyde cross-linking (supporting online text). ATP, adenosine triphosphate KGDC, α-ketoglutarate dehydrogenase complex; PHDC, pyruvate dehydrogenase complex; BCADC, branched-chain amino acids dehydrogenase complex. Genes encoding proteins identified in this study are boldfaced.

Gene Protein Primary functions
Category I
ABF2 Abf2p mtDNA packaging, ρ+ genome maintenance
MGM101 Mgm101p Maintenance of ρ+ and ori-less mtDNA, mtDNA repair
RIM1 Single-stranded DNA binding protein mtDNA replication
RPO41 DNA-directed RNA polymerase Mitochondrial transcription, ρ+ mtDNA maintenance
SLS1 Sls1p Mitochondrial translation, ρ+ mtDNA maintenance
Category II
HSP60 mtHsp60p Mitochondrial chaperonin
HSP10 mtHsp10p Mitochondrial chaperonin
SSC1 mtHsp70p Protein import
ATP1 α-Subunit of F1-ATPase ATP synthesis, protein import
Category III
ACO1 Mitochondrial aconitase Citric acid cycle
ALD4 Aldehyde dehydrogenase Ethanol metabolism
IDH1 NAD+-dependent isocitrate dehydrogenase, subunit 1 Citric acid cycle
IDP1 NADP+-dependent isocitrate dehydrogenase Oxidative decarboxylation of isocitrate
KGD1 2-oxoglutarate dehydrogenase, E1 component of KGDC Citric acid cycle
KGD2 2-oxoglutarate dehydrogenase, E2 component of KGDC Citric acid cycle
LPD1 Dihydrolipoamide dehydrogenase, E3 component of PDHC, KGDC, and BCADC Citric acid cycle, catabolism of branched-chain amino acids
LSC1 Succinate-CoA ligase, α subunit Citric acid cycle
PDA1 Pyruvate dehydrogenase, E1 α-subunit of PDHC Oxidation of pyruvate
PDB1 Pyruvate dehydrogenase, E1 β-subunit of PDHC Oxidation of pyruvate
Category IV
ILV5 Acetohydroxyacid reductoisomerase Biosynthesis of Val, Ile, and Leu
ILV6 Acetolactate synthase regulatory subunit Biosynthesis of Val, Ile, and Leu
CHA1 L-serine/L-threonine deaminase Catabolism of hydroxy amino acids

As proteins in category III could potentially connect respiratory and fermentative metabolism to mtDNA maintenance, we examined mtDNA stability in strains in which a selection of these genes were inactivated. Expression of some category III genes is repressed by glucose (10). Therefore, mutant cells were grown in raffinose, a fermentable carbon source that does not repress mitochondrial respiration, and then assayed for the fraction of respiratory-deficient (petite) mutants in the population. mtDNA is relatively stable in kgd1Δ and kgd2Δ cells, less so in the pda1Δ, pdb1Δ, idh1Δ, and lpd1Δ strains, and very unstable in aco1Δ cells (Fig. 1A). mtDNA was previously noted to be unstable when some of these genes were inactivated (11). Further experiments established that ACO1 is essential for mtDNA maintenance. Southern blot analysis of total cellular DNA from nascent meiotic segregants derived from an aco1Δ/ACO1 ρ+ diploid strain showed that the aco1Δ spores lack mtDNA (Fig. 1B).

Fig. 1.

ACO1 is required for mtDNA maintenance. (A) mtDNA stability in cells with null mutations of genes that encode metabolic proteins found in nucleoids. mtDNA stability is expressed as percentage of respiratory-competent (ρ+) colonies after growth for 35 generations in complete raffinose medium. Data are shown as means ± SEM for triplicate experiments. WT, wild type. (B) Southern blot analysis of Cfo I–digested total DNA probed for mtDNA in nascent meiotic segregants containing ACO1 (+) or aco1Δ (–) alleles. Controls are ρ+ and ρ0 cells. i.c., internal control for sample loading. (C) Southern blot analysis showing the loss of mtDNA in hap2Δ rtg1Δ meiotic segregants. (D) Suppression of mtDNA loss from the hap2Δ rtg1Δ double mutants by constitutive expression of a chromosomally integrated copy of ADH1-ACO1 (ACO1*).

Aco1p, citrate synthase (CS), and two subunits of nicotinamide adenine dinucleotide (NAD+)–dependent isocitrate dehydrogenase (collectively, the aconitase metabolon) function to produce α-ketoglutarate, a precursor to glutamate. Expression of the genes encoding these proteins is positively regulated by the glucose-repressible HAP2-5 transcription complex and the transcription factors Rtg1p and Rtg3p (12), which are components of the mitochondria-to-nucleus retrograde (RTG) signaling pathway (13). Aco1p localizes to the mitochondrial matrix, as revealed by fluorescence microscopy of an Aco1p–green fluorescent protein fusion construct (fig. S1). Like other genes in the aconitase metabolon, expression of ACO1 becomes progressively HAP-dependent in response to increased respiratory activity when cells are shifted from glucose to raffinose medium, whereas a combined inactivation of the HAP and RTG systems leads to a 14-fold reduction of ACO1 expression, independent of the carbon source (fig. S2).

To ask whether the HAP and RTG transcription complexes can directly affect mtDNA maintenance, we generated a diploid strain heterozygous for hap2Δ and rtg1Δ. After sporulation and dissection on rich glucose medium, both the rtg1Δ and hap2Δ spores can maintain mtDNA (Fig. 1C), whereas mtDNA was barely detectable in rtg1Δ hap2Δ segregants. However, when the diploids contained an integrated single copy of ACO1 under the control of the constitutive ADH1 promoter, the loss of mtDNA from the rtg1Δ hap2Δ double mutants was largely reversed in meiotic segregants that also received the ADH1-ACO1 allele (Fig. 1D).

To determine whether the mtDNA instability in aco1Δ cells is due to a block in metabolic flux through the aconitase metabolon, we inactivated all three known genes encoding CS: CIT1, CIT2, and CIT3 encoding, respectively, the citric acid cycle CS, a peroxisomal isoform of CS, and a mitochondrial CS that may function in a methyl citrate pathway (14, 15), so that no substrate would be available to aconitase. This resulted in a strong glutamate auxotrophy (Fig. 2A), demonstrating that metabolic flux through the aconitase metabolon had been blocked. Nevertheless, mtDNA was stable in the cit1Δ, cit2Δ, and cit3Δ triple mutant (Fig. 2B).

Fig. 2.

Functional bisection of Aco1p. (A) Growth of wild-type and mutant cells with the indicated citΔ alleles on minimal glucose medium with or without 0.2% glutamate (YNBD, 0.67% yeast nitrogen base, 2% dextrose). (B) Southern blot analysis of mtDNA in meiotic segregants from CIT/citΔ diploid cells. (C) Schematic for generation of diploid strains used for the functional test of the cysteine mutants of ACO1. Chromosome locations are indicated (XII and XV). (D) Southern analysis showing that the aco1C448S but not the aco1C382S and aco1C445S alleles retain mtDNA among the meiotic segregants in a representative tetrad.

We next asked whether aconitase catalytic activity is required for Aco1p function in mtDNA maintenance. Aconitase contains an iron-sulfur center (4Fe-4S) in which 3 of the 4 mol of iron are coordinated with three cysteines, Cys382, Cys445, and Cys448 (16); the fourth iron is coordinated with the substrate, citrate (17). The integrity of this iron sulfur center is essential for aconitase enzymatic activity (18). We changed each of these cysteine residues to a serine and analyzed the mutants to see whether they could still function in mtDNA maintenance. Each mutant allele tagged with URA3 was inserted into the ADE2 locus of an ACO1/aco1Δ::KAN diploid strain (Fig. 2C). After sporulation, Southern blot analysis of meiotic segregants from a representative tetrad from each diploid strain showed that mtDNA was retained in aco1Δ aco1C448S but not in the aco1Δ aco1C382S or aco1Δ aco1C445S segregants (Fig. 2D); however, all three of the segregants are glutamate auxotrophs and respiratory deficient, indicating that they lack aconitase activity. These differences in the ability of the aco1cys mutant alleles to maintain mtDNA are not due to any appreciable differences in the expression of the mutant proteins, because all are expressed at levels comparable to wild-type Aco1p (fig. S3). Further experiments showed, however, that the aco1C382S and aco1C445S mutant alleles can support mtDNA maintenance when overexpressed from the strong ADH1 promoter, although those mutants remain respiratory-deficient and auxotrophic for glutamate (fig. S4).

The requirement of Aco1p for mtDNA maintenance is comparable to that of the mtDNA packaging protein Abf2p, which is essential for mtDNA maintenance in cells grown on glucose medium (6). The only conditions known in which mtDNA in abf2Δ cells is relatively stable is when another DNA packaging protein, such as Escherichia coli HU, is expressed in abf2Δ cells and targeted to mitochondria (19) or when abf2Δ cells are grown on a nonfermentable carbon source (6). This latter result has generally been interpreted to mean that abf2Δ cells can be propagated on medium such as glycerol because of the strong selection for cells that retain mtDNA. However, under these growth conditions, ACO1 expression is elevated (10). This raises the possibility that, under derepressed conditions, Aco1p might be able to substitute for Abf2p in mtDNA maintenance. To test this, we first examined mtDNA stability in abf2Δ cells grown on raffinose, which does not repress the aconitase metabolon. These experiments (Fig. 3A) show that growth on raffinose almost completely suppresses the mtDNA instability seen when abf2Δ cells are grown on glucose. Southern blot analysis of abf2Δ cells grown on glucose versus raffinose medium showed that after growth of the abf2Δ mutant in glucose for six generations, mtDNA was barely detectable in the cell population, whereas cells grown in raffinose for the same number of generations retained a much greater amount of mtDNA (Fig. 3B).

Fig. 3.

Control of mtDNA inheritance by the HAP and RTG pathways. (A) mtDNA stability of abf2Δ cells grown in raffinose (Raf) or glucose (Glu) medium expressed as the percentage of ρ+ colonies on complete glucose medium after a growth in liquid medium for the indicated generations. (B) Southern blot analysis of mtDNA in abf2Δ cells grown for six generations in complete glucose or raffinose medium. Lane 1, ρ+ control; lane 2, abf2Δ; lane 3, ρo control; i.c., internal control. (C) Suppression of mtDNA instability in abf2Δ cells by overexpression of HAP4 from the PGK1 promoter in rich glucose medium. abf2Δ cells were also transformed with an empty vector and with wild-type ABF2 on a plasmid as controls. (D) Suppression of mtDNA instability in abf2Δ cells by constitutive activation of the RTG pathway because of the mks1Δ mutation. (E) Suppression of mtDNA loss from abf2Δ cells by overexpression of ACO1 and its C382S, C445S, and C448S variants from the constitutive ADH1 promoter. mtDNA stability was determined by counting the fraction of ρ+ colonies after plating transformants on complete glucose medium. (F) EB sensitivity of abf2Δ cells with ABF2, ACO1, or an empty vector on complete ethanol medium.

We next determined whether the HAP-RTG targets are responsible for the marked mtDNA stability observed in abf2Δ cells grown under derepressed conditions. We first manipulated the HAP2-5 transcription complex by constitutively expressing HAP4, which activates the expression of respiratory genes, including ACO1, in glucose-repressed cells (20). Accordingly, when HAP4 was expressed from the constitutive PGK1 promoter, the loss of mtDNA from abf2Δ cells grown on glucose was considerably delayed (Fig. 3C).

Next, we inactivated the MKS1 gene encoding a negative regulator of the RTG pathway (2123); this results in constitutive, high levels of expression of RTG target genes, including Aco1p. The mks1Δ mutation almost completely suppressed the loss of mtDNA from abf2Δ cells grown on glucose (Fig. 3D). That the mks1Δ suppressor activity acts through the RTG pathway is shown by the reappearance of mtDNA instability in the abf2Δ mks1Δ double mutant when RTG1 or RTG3 was also inactivated.

To show directly that the suppression of mtDNA instability of abf2Δ cells grown on glucose is related to ACO1 expression, we expressed either the wild-type or one of the three cysteine mutants of Aco1p under control of the constitutive ADH1 promoter in centromeric plasmids. The transformants, maintained on glycerol medium, were examined for mtDNA stability after plating on glucose medium. As expected, most abf2Δ cells transformed with vector alone formed petite colonies, indicating instability of mtDNA (Fig. 3E). However, in cells expressing wild-type Aco1p or its cysteine mutants, the mtDNA instability phenotype was reversed, yielding petite frequencies comparable to that of the control cells expressing ABF2.

One interpretation for the suppression of mtDNA instability in abf2Δ cells by activation of ACO1 is that mtDNA is repackaged into more stable nucleoid structures. To investigate this, we measured the sensitivity of ρ+ cells to ethidium bromide (EB), which, by intercalating between the base pairs of mtDNA, produces petites because of the loss of the ρ+ mtDNA. EB accessibility to DNA has been shown to be a useful probe for detecting differences in the organization of DNA-protein complexes (24). Indeed, by comparison to wild-type ρ+ cells, abf2Δ cells are hypersensitive to EB as determined by growth on ethanol medium (Fig. 3F). This EB hypersensitivity can be suppressed by overexpression of ACO1. We confirmed that, under these growth conditions, expression of ACO1 from the multicopy plasmid increases the steady-state level of Aco1p by threefold compared with that in cells containing the empty vector (fig. S5). Aco1p suppression of EB sensitivity of abf2Δ cells was also observed under glucose repressed conditions (fig. S6). Together, these data suggest that Aco1p may be providing some packaging function for mtDNA.

Our data establish a direct link between control of ACO1 expression by the HAP and RTG pathways and the maintenance of mtDNA. Our finding that Aco1p is required for mtDNA stability independent of aconitase catalytic activity is reminiscent of the bifunctionality of the cytosolic form of mammalian aconitase, also known as iron-responsive element binding protein. That protein switches between an enzymatic and RNA binding form on the basis of the assembly or disassembly of the [4Fe-4S] cluster (25, 26). Whether a similar mechanism applies to Aco1p function in mtDNA maintenance remains to be determined. Our results further suggest that mtDNA nucleoids may exist in different states depending on the metabolic condition of cells. Under glucose repressed conditions, Abf2p is essential for mtDNA maintenance because of its DNA packaging function. In derepressed cells with robust oxidative metabolism, or in response to RTG signals, mtDNA packaging may also involve Aco1p. This raises the possibility that nucleoid remodeling may be part of a strategy for adjusting mtDNA maintenance to the changes in cellular metabolism. Because aconitase is susceptible to oxidative damage during oxidative stress and cell aging (2729), this property could contribute to compromised mtDNA integrity as a result of a decoupling between metabolism and mtDNA transactions.

Supporting Online Material

www.sciencemag.org/cgi/content/full/307/5710/714/DC1

Materials and Methods

SOM Text

Figs. S1 to S6

Table S1

References and Notes

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