Cristae-dependent quality control of the mitochondrial genome

Christopher Jakubke; Rodaria Roussou; Andreas Maiser; Christina Schug; Felix Thoma; Raven Bunk; David Hörl; Heinrich Leonhardt; Peter Walter; Till Klecker; Christof Osman

doi:10.1126/sciadv.abi8886

. 2021 Sep 1;7(36):eabi8886. doi: 10.1126/sciadv.abi8886

Cristae-dependent quality control of the mitochondrial genome

Christopher Jakubke ^1,^2,^†, Rodaria Roussou ^1,^2,^†,^✉, Andreas Maiser ¹, Christina Schug ³, Felix Thoma ^1,², Raven Bunk ¹, David Hörl ¹, Heinrich Leonhardt ¹, Peter Walter ^4,⁵, Till Klecker ³, Christof Osman ^1,^*

PMCID: PMC8442932 PMID: 34516914

Cristae limit diffusion of mtDNA-encoded proteins and facilitate clearance of mutant mtDNA in a fission-independent manner.

Abstract

Mitochondrial genomes (mtDNA) encode essential subunits of the mitochondrial respiratory chain. Mutations in mtDNA can cause a shortage in cellular energy supply, which can lead to numerous mitochondrial diseases. How cells secure mtDNA integrity over generations has remained unanswered. Here, we show that the single-celled yeast Saccharomyces cerevisiae can intracellularly distinguish between functional and defective mtDNA and promote generation of daughter cells with increasingly healthy mtDNA content. Purifying selection for functional mtDNA occurs in a continuous mitochondrial network and does not require mitochondrial fission but necessitates stable mitochondrial subdomains that depend on intact cristae morphology. Our findings support a model in which cristae-dependent proximity between mtDNA and the proteins it encodes creates a spatial “sphere of influence,” which links a lack of functional fitness to clearance of defective mtDNA.

INTRODUCTION

Mitochondria contain their own genome, known as mitochondrial DNA (mtDNA), which in most organisms encodes core subunits of the respiratory chain and the adenosine triphosphate (ATP) synthase as well as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) required for mitochondrial protein translation. Multiple copies of mtDNA are distributed throughout the mitochondrial network. Mutations in mtDNA can have detrimental consequences for mitochondrial function and can lead to a multitude of mitochondrial diseases, which have a prevalence of ∼20 cases per 100,000 individuals (1). How cells maintain the integrity of mtDNA over generations, despite high mitochondrial mutation rates, has remained unclear. Studies in Drosophila melanogaster and mice have revealed that mutant copies of mtDNA are removed in the female germline in a process known as purifying selection (2–4). Mitochondrial fission has been proposed to contribute to this process in D. melanogaster (5, 6). Generation of small mitochondrial fragments containing only one or a few mitochondrial genomes is believed to separate mtDNA copies from one another to prevent complementation of mutant mtDNA by gene products of intact mtDNA. Subsequently, mitochondrial fragments containing mutated mtDNA are proposed to be detected on the basis of a decreased membrane potential or ATP content and removed by mitophagy. In addition, it has been found that compromised protein import efficiency into damaged mitochondria leads to a decrease in mtDNA replication and hence disfavors propagation of mutated mtDNA copies (3, 7). Despite this progress, many questions remain about the cellular mechanisms that facilitate selection against mutant mtDNA. By exploiting the possibility to genetically manipulate mtDNA in Saccharomyces cerevisiae, we establish budding yeast as a model system to study mtDNA quality control.

RESULTS

A pedigree analysis reveals mtDNA quality control in S. cerevisiae

First, we asked whether the single-celled S. cerevisiae intracellularly distinguishes between intact and mutant mtDNA and supports generation of young progeny with a healthy mtDNA content. We devised an approach to genetically follow segregation of wild-type (WT) and mutant mtDNA copies from heteroplasmic single yeast cells. Two yeast strains of opposing mating types were used for this approach. The first strain is of mating type a and harbors WT mtDNA and a deletion of the nuclear-encoded ARG8 gene that encodes the mitochondrially localized Arg8 protein and is required for synthesis of arginine. Therefore, this strain is able to grow on medium containing nonfermentable carbon sources but not on medium lacking arginine. The second strain is of mating type alpha and harbors a deletion of the nuclear-encoded ARG8, but contains mtDNA in which the COB gene is replaced by the ARG8 gene (Δcob::ARG8) (8). This latter strain can thus grow in the absence of arginine but not on nonfermentable carbon sources due to the deletion of the mtDNA-encoded COB gene. The open reading frames (ORFs) of COB and ARG8 are 1158 and 1294 base pairs (bp) in size, respectively. Replacement of the COB gene, therefore, results in a minor increase of the overall size of the mtDNA of less than 0.2%, which is unlikely to confer a replicative disadvantage on the Δcob::ARG8 mtDNA due to larger size. Both strains contained comparable amounts of mtDNA as determined by quantitative real-time polymerase chain reaction (PCR; fig. S1A).

Both yeast strains were mated to obtain heteroplasmic diploid zygotes containing WT and Δcob::ARG8 mtDNA. Microdissection was used to transfer single zygotes to a cell-free area on an agar plate containing rich medium and glucose as a fermentable carbon source, which supports growth of cells containing WT, Δcob::ARG8, or both mtDNA species. Once the first daughter cell had budded from the zygote, it was moved to a new location on the agar plate. Growth of this former daughter cell was again monitored, until the second-generation daughter cell had budded, which was again transferred to a new position. This procedure was repeated for up to five generations, and isolated cells were incubated to allow growth of colonies (Fig. 1, A and B). The mtDNA genotype in such colonies was inferred by their ability to grow on medium containing a nonfermentable carbon source or medium lacking arginine, indicative of the presence of WT or Δcob::ARG8 mtDNA, respectively (Fig. 1B and fig. S1B). The presence or absence of mtDNA was further corroborated by PCR analysis of selected lineages (fig. S1C). Of note, this growth-based assay cannot distinguish whether mtDNA variants are entirely absent or present in insufficient amounts to support growth on the respective selective medium. Previous analyses in S. cerevisiae have revealed rapid segregation of mtDNA variants containing neutral genetic markers within less than 10 and often within one generation (9). We similarly observe rapid segregation; notably, however, we detected a strong bias for the WT mtDNA copy. More than 60% of the colonies derived from the first-generation daughter contained exclusively the WT mtDNA, while only about ∼15% of the colonies contained exclusively the Δcob::ARG8 mtDNA and ~25% remained in a heteroplasmic state (Fig. 1C). A further segregation toward WT mtDNA was observed in following generations. More than 80% of colonies derived from daughters of the fifth generation contained only the WT mtDNA (Fig. 1C). Only 3% of the colonies of the fifth generation were heteroplasmic, indicating virtually complete segregation of both mtDNA types. Very similar results were obtained in pedigree analyses, where the mating types of the WT and the Δcob::ARG8 mtDNA containing strains had been switched, indicating that selection for WT mtDNA over Δcob::ARG8 mtDNA is independent of the mating type (fig. S2A). Furthermore, we did not observe selection against mtDNA variants with an ARG8 gene inserted into a noncoding region of mtDNA, indicating that the ARG8 gene does not impart a strong selective disadvantage on the mtDNA (fig. S2B).

Fig. 1. — (A) Schematic illustration of the pedigree analysis. Two Δ*arg8* yeast strains harboring WT or Δ*cob::ARG8* mtDNA were mated. Zygotes were isolated, and daughter cells from up to five consecutive generations were separated and placed on free spots on the agar plate by microdissection. (B) Growth pattern of the pedigree analysis after microdissection. Colonies were initially grown on rich medium containing glucose. The mtDNA genotype was inferred from the ability of colonies to grow on synthetic arginine lacking medium (Δ*cob::ARG8* mtDNA) or on medium containing the nonfermentable carbon source glycerol (WT mtDNA). Asterisks indicate cell material that was carried over by replica plating and failed to produce obvious colonies upon further incubation. For further illustrative explanation and confirmation of the pedigree analysis, refer to fig. S1 (B to D). (C) Pedigree analysis of WT cells. Striped bars indicate percentage of heteroplasmic cells containing WT and Δ*cob::ARG8* mtDNA. Gray or red bars indicate percentage of homoplasmic cells containing WT or Δ*cob::ARG8* mtDNA, respectively. (D) Inheritance of either intact or mutated mtDNA. Mating events between two cells either with WT-LacO (GFP, P₁) or Δ*cob*::*ARG8*-TetO (mRuby3, P₂) mtDNA. Both cells expressed a nuclear-encoded, matrix-targeted TagBFP. The percentage of either GFP or mRuby3 spots in the daughter cells relative to total number of the respective mtDNA variant have been plotted. Big circles represent the mean values from individual experiments. **P<0.01, t test. (E) Mitochondrial morphology during mating. Mating events between two cells containing either WT or Δ*cob*::*ARG8* mtDNA. Cells expressed either matrix-targeted mKate2 or mNeonGreen (NG). Mating events were monitored by live-cell microscopy. Time point before mixing of the matrix contents has been defined as T0. (F and G) Pedigree analysis of Δ*dnm1* or Δ*atg32* cells. Scale bars, 10 μm (D and E).

We developed a microscopic approach to visualize selection of WT over Δcob mtDNA during budding of daughter cells from zygotes. To this end, we used our previously generated strain that harbors tandem LacO repeats in a noncoding region of mtDNA upstream of COX2 and expresses a nuclear-encoded LacI-3×GFP (green fluorescent protein) fusion protein harboring an N-terminal mitochondrial targeting sequence (10). In a separate strain, we replaced the mtDNA-encoded COB gene with tandem TetO repeats and introduced a gene encoding a mitochondrially targeted Tet-repressor fused to three repeats of the fluorescent protein mRuby3 into the nuclear genome. This strain contained very similar mtDNA levels compared to a reference strain, indicating that the TetO-TetR system does not lead to major defects in mtDNA maintenance (fig. S3A). In the strains used for this analysis, LacI-3×GFP and TetR-3×mRuby3 bind to the LacO and TetO repeats, respectively. To visualize mitochondria, both strains express the mitochondrially targeted blue fluorescent protein TagBFP. Both strains were mated, and zygotes were analyzed by fluorescence microscopy. We detected mutant mtDNA in the daughter cell. Quantitative image analysis, however, revealed that relative amounts of WT LacO-mtDNA were more abundant in the daughter cell than the Δcob TetO-mtDNA (Fig. 1D and fig. S3B). While we cannot entirely rule out that the LacO-LacI or the TetO-TetR systems affect the mtDNA selection process or lead to biased results in this imaging approach, these data support the conclusion that S. cerevisiae cells can distinguish between WT and mutant mtDNA and promote generation of progeny with predominantly healthy mtDNA content.

Selection against mutant mtDNA occurs in a continuous mitochondrial network

Like in higher eukaryotes, mitochondria form a continuous tubular network in S. cerevisiae that is constantly rearranged by fusion and fission events. A simple explanation for rapid segregation of WT and mutant mtDNA could be that dysfunctional mitochondria do not fuse with healthy mitochondria to form a continuous network upon mating. In such a scenario, mutant and WT copies of mtDNA would not mix but be kept in separated mitochondrial compartments, which could facilitate efficient segregation. To examine this possibility, we used live-cell microscopy to monitor mitochondrial fusion during mating of two yeast strains expressing either mNeonGreen (NG) or mKate2 targeted to the mitochondrial matrix. We first examined mating events between two cells, both containing WT mtDNA. In line with previous observations, we observed fusion of mitochondria in newly formed zygotes (11–13). Fusion was characterized by quick and mostly complete equilibration of mKate2 and NG within 2 min throughout virtually all parts of the mitochondrial network in zygotes (fig. S4A). Similarly, we observed rapid fusion of mitochondrial networks in mating events between cells containing either WT or Δcob::ARG8 mtDNA (Fig. 1E, fig. S4B, and movie S1). Detection of both fluorescent proteins throughout the mitochondrial network was the result of fusion rather than ongoing translation, because unfused fragments could be distinguished during the mitochondrial fusion process in zygotes (arrowheads in Fig. 1E). Despite content mixing of mitochondria, our pedigree analysis reveals that yeast zygotes can still distinguish between mutant and WT mtDNA to produce daughter cells that predominantly contain WT mtDNA.

Mitochondrial fission or Atg32-mediated mitophagy is not required for selection against mutant mtDNA

We asked whether mitochondrial fission could facilitate selection against mutant mtDNA, as has been proposed for D. melanogaster (5, 6). We did not observe increased mitochondrial fragmentation after mating between cells containing WT mtDNA or mating between cells containing either WT or Δcob::ARG8 mtDNA. Thus, excessive mitochondrial fragmentation does not occur during selection against mutant mtDNA in yeast zygotes (Fig. 1E; fig. S4, A and B; and movie S1).

To further assess the role of mitochondrial fission in selection against mutant mtDNA, we performed pedigree analyses of crosses between cells lacking the fission protein Dnm1 and harboring either WT or mutant Δcob::ARG8 mtDNA. Notably, the absence of mitochondrial fission did not compromise rapid selection for WT mtDNA in a colony produced by the first daughter cell of heteroplasmic yeast zygotes (Fig. 1F). Furthermore, live-cell microscopy of mating events between two Δdnm1 strains harboring either WT or mutant mtDNA and expressing matrix-targeted mKate2 or NG, respectively, revealed that mitochondria from both parental cells rapidly fused in zygotes (fig. S4C and movie S2). We also tested the importance of the mitophagy receptor Atg32 (14, 15) for selection against mutant mtDNA. Δatg32 cells behaved similar to WT cells in pedigree analyses and exhibited complete mitochondrial fusion during the mating process (Fig. 1G, fig. S4D, and movie S3). Thus, neither mitochondrial fission nor Atg32-mediated mitophagy is essential for selection against mutant mtDNA during mating events of S. cerevisiae cells. Notably, selection for WT mtDNA in generations 2 to 5 appeared less prominent in pedigree analyses of Δdnm1 or Δatg32 cells compared to matings between WT cells. This observation could indicate that selection toward WT mtDNA in daughter cells of zygotes is independent of fission and mitophagy, whereas further selection in dividing diploids may be supported by these processes.

The mtDNA-encoded protein Atp6 exhibits severely limited diffusion rates

The fact that selection occurs in a continuous network that shares WT and mutant mtDNA presents a conundrum. How can cells distinguish between WT and mutant mtDNA if mitochondrial content equilibrates in a continuous network? We hypothesized that selection can only occur when subdomains are maintained within the mitochondrial network, whose functionality is determined by nearby copies of mtDNA. In such a scenario, it is important that mtDNA-encoded respiratory chain subunits do not exhibit rapid diffusion, because this would allow complementation of dysfunctional subdomains by gene products from WT mtDNA. To examine diffusion of an mtDNA-encoded protein, we modified mtDNA and fused an NG-tag to the C terminus of the mtDNA-encoded ATP6 gene (fig. S5A). Yeast strains exclusively expressed the NG-tagged variant of Atp6 and did neither show growth defects on medium containing fermentable or nonfermentable carbon sources at 30° or 37°C nor exhibit increased formation of so-called petite cells that are respiratory deficient or display altered mtDNA or Atp6 protein levels (Fig. 2A and fig. S5, B to D). These observations indicate full functionality of the NG-tagged Atp6.

Fig. 2. — (A) Growth analyses of strains expressing NG-tagged Atp6. Serial dilutions of indicated strains were spotted on rich medium containing glucose or glycerol as carbon sources and incubated at 30° or 37°C. (B) Structured illumination microscopy of DAPI-stained cells expressing Atp6-NG and matrix-targeted mScarlet. Line graphs of pixel intensities along indicated lines are shown. a.u., arbitrary units. (C) Schematic illustration of the mating experiment of the Atp6-NG–tagged strain with a strain expressing the matrix labeling Su9-mKate2. (D to F) Diffusion of mitochondrial membrane proteins in fused mitochondria of zygotes. Cells expressing matrix-targeted mKate2 were mated with cells expressing mtDNA-encoded Atp6-NG (D), nuclear-encoded Fis1-NG (E), or Pam16-NG (F). Scale bars, 5 μm (B) and 10 μm (D to F).

Fluorescence microscopy of diploid cells harboring the ATP6-NG mtDNA and expressing a nuclear-encoded matrix-targeted mScarlet revealed that, in contrast to the uniformly distributed mScarlet signal, Atp6-NG exhibited a patchy distribution in the mitochondrial network (fig. S5E). This distribution has also been observed previously using nuclear-encoded GFP-tagged variants of ATP synthase subunits, and it has been proposed that these patches report on mitochondrial cristae, in which the ATP synthase is enriched (16). We used structured illumination microscopy of 4′,6-diamidino-2-phenylindole (DAPI)–stained cells to query the relationship between Atp6-NG and mtDNA. Atp6-NG foci were spatially linked to mtDNA and were detected in the immediate surroundings of DAPI spots rather than directly colocalizing with them (Fig. 2B). This finding demonstrates that the ATP synthase localizes close to mtDNA but is largely excluded from areas occupied by mtDNA itself. This observation is in line with recent super-resolution data in HeLa cells that showed a lack of cristae in close proximity to mtDNA (17).

To further test whether Atp6-NG remains close to the copy of mtDNA by which it is encoded, we examined diffusion of Atp6-NG in living cells during a mating experiment. Cells harboring mtDNA encoding Atp6-NG were mated with cells expressing a nuclear-encoded matrix-targeted mKate2 and harboring nonmodified mtDNA. Equilibration of both fluorescent signals throughout the mitochondrial network of zygotes was studied over time by live-cell microscopy (Fig. 2C). In line with our previous observation, soluble mKate2 rapidly equilibrated throughout the mitochondrial network upon fusion of parental mitochondria. Notably, the Atp6-NG signal remained localized to mitochondria from the cell of its origin (Fig. 2D, fig. S6A, and movie S4). Only very small Atp6-NG amounts were present in the mitochondrial network of the mating partner ∼80 min after mitochondrial fusion, when a daughter bud had already emerged at midpoint between both parental cells and had been invaded by mitochondria. To quantify equilibration of Atp6-NG in late zygotes harboring a large medial bud, we first segmented the mitochondrial network based on the mitochondrial mKate2 signal and assigned mitochondrial parts to either the parental Atp6-NG (P₁), the parental mKate2 (P₂), or the daughter cell. The Atp6-NG signal was then determined along the mitochondrial network parts that had been assigned to P₁, P₂, and daughter cell. The Atp6-NG signal was further normalized to the length of the subnetwork localized to the respective cells. This analysis revealed that on average only approximately 20% of the Atp6-NG was detected in the mitochondrial network of the other parental cell (Fig. 3B). In contrast, no difference in the mKate2 signal in both parental cells was apparent in this analysis, indicating complete mitochondrial fusion and full equilibration of this soluble matrix protein (fig. S7A). We cannot distinguish between preexisting and newly synthesized Atp6-NG in this assay but conclude that proteins from neither group efficiently populate the mitochondrial network of the parental cell that did not contain the Atp6-NG mtDNA.

Fig. 3. — (A) Zygotes derived from mating events between WT or indicated mutant cells. In each mating event, one cell contained *ATP6*-NG mtDNA (P₁) and the other cell contained WT mtDNA and expressed matrix-targeted mKate2 (P₂). D, daughter cell. (B) The Atp6-NG signal was quantified in both parental cells of zygotes and normalized to the mitochondrial network length. The ratio of Atp6-NG signals in P₂ cells to P₁ cells of the same zygote is plotted; each point represents one zygote; **P< 0.01, t test. (C) Mating events between WT, Δ*atp20*, or Δ*atp21* cells. Mating cells harbored either Atp6-NG mtDNA and nuclear-encoded Su9-TagBFP (P₁) or Atp6-mKate2 (P₂) mtDNA (D, daughter cell). (D) Mating events between WT, Δ*atp21*, and Δ*mic60* cells. Parental cell P₁ contained WT mtDNA and expressed matrix-targeted mKate2, whereas parental cell P₂ contained mtDNA with integrated LacO repeats (D, daughter cell). mtDNA-LacO spots in P₁, P₂, and D cells were counted and plotted as percentage of total number of spots in the zygote. Each data point in the plot represents one zygote. (E) Electron microscopic analysis of mitochondrial ultrastructure in WT and Δ*atp20*Δ*atp21* cells. (F) Quantification of electron microscopy data shown in (E). Scale bars, 10 μm (A, C, and D) and 250 nm (E).

To examine diffusion of another protein component of the oxidative phosphorylation (OXPHOS) complexes, we created a strain expressing an NG-tagged version of the nuclear-encoded protein Cox4, which is a subunit of complex IV. Cells expressing Cox4-NG cells were mated with cells expressing matrix-targeted mKate2. Of note, upon mating of these cells, continued cytosolic synthesis of Cox4-NG will lead to import of Cox4 into all parts of the mitochondrial network at later time points. Despite this complication and similar to our results obtained with Atp6-NG, Cox4-NG exhibited strongly reduced diffusion throughout the mitochondrial network of zygotes compared to the soluble matrix–targeted mKate2 and remained largely restricted to the cell of its origin during the duration of the microscopy experiment (fig. S6B).

Next, we asked whether limited diffusion of Atp6-NG or Cox4-NG was general to transmembrane (TM) proteins of mitochondrial membranes. We mated cells expressing matrix-targeted mKate2 with cells either expressing NG fused to the TM-domain of the outer membrane protein Fis1 or a functional Pam16-NG fusion protein (Fig. 2, E and F, and fig. S6, C and D). Pam16 is a subunit of the translocase of the inner mitochondrial membrane, which localizes predominantly to the inner boundary membrane (18). Time-lapse microscopy of such mating events revealed that diffusion of Fis1-TM-NG and Pam16-NG was slightly delayed compared to the soluble matrix protein mKate2, but both proteins equilibrated much faster than Atp6-NG throughout mitochondrial networks and no difference in signal intensity in both parental cells could be observed ∼10 min after fusion of mitochondria in zygotes (fig. S6, E and F, and movies S5 and S6). In summary, mtDNA-encoded Atp6-NG and nuclear-encoded Cox4-NG, which are both subunits of respiratory chain complexes consisting of nuclear- and mtDNA-encoded subunits, are severely limited in equilibration throughout mitochondrial tubules compared to proteins of the inner boundary or the outer mitochondrial membranes.

Components important for cristae morphology affect diffusion rates of Atp6-NG

We hypothesized that components involved in maintenance of cristae morphology could be important for hindering diffusion of OXPHOS subunits and particularly mtDNA-encoded proteins within the mitochondrial network. Mic60 and Mic10 are components of the mitochondrial contact site (MICOS) complex, which stabilizes cristae junctions (19–21). Lack of Mic60 leads to cristae that are detached from the inner mitochondrial membrane but maintain their sheet-like morphology in the matrix. Atp20 and Atp21 are crucial for dimerization of the ATP synthase, which, in turn, stabilizes strongly bent cristae rims (22). Lack of the dimeric ATP synthase has been described to lead to an onion-like morphology of mitochondria, where uncontrolled proliferation of cristae membranes leads to a multilayered appearance (23). To examine the role of cristae in limiting equilibration of OXPHOS proteins, we quantified Atp6-NG mobility in our mating assay in Δatp20, Δatp21, Δatp20Δatp21, Δmic10, or Δmic60 strains. Both parental cells contained the respective deletion in these crosses. In all mating events, the nuclear-encoded mKate2 equilibrated across the mitochondrial network (fig. S7A). We could observe a slightly less complete equilibration of mKate2 in zygotes lacking the dimeric ATP synthase, which is likely explained by septae that may create separate matrix compartments (24) and thereby may decrease efficiency of matrix content exchange. Δmic10 and Δmic60 cells exhibited increased diffusion of Atp6-NG between mitochondrial networks of parental cells compared to WT cells in our mating assay, which was characterized by a greater amount of Atp6-NG detected in the mating partner harboring the WT mtDNA (∼35% compared to ∼25% in WT cells; Fig. 3, A and B). Cells lacking Atp20, Atp21, or both proteins showed a strongly increased diffusion of Atp6-NG across the mitochondrial network in zygotes. In many cases, the source cell that contained ATP6-NG-mtDNA was hardly discernible from the mKate2 source cell in the zygotes. On average, ∼45% of Atp6-NG was detected in the mating partner harboring the WT mtDNA in the absence of Atp20, Atp21, or both proteins (Fig. 3, A and B). In contrast to limited diffusion within the mitochondrial network formed by parental cells, Atp6-NG was readily detected in the budding daughter cells. In daughters from zygotes, the Atp6-NG signal was at ∼70% compared to the ATP6-NG signal from the ATP6-NG parental cell in all mutants, with the exception of Δmic60 cells, where Atp6-NG levels only amounted to ∼60% (fig. S7B). The reason why less Atp6-NG is found in the daughter cells of matings between Δmic60 cells is currently unclear. In summary, we observe increased diffusion of Atp6-NG in mutants lacking intact cristae morphology across existing mitochondrial networks of yeast zygotes. Because Δmic10 or Δmic60 mutants still have the dimeric form of the ATP synthase (25, 26), our results strongly suggest that Atp6-NG mobility is increased due to compromised cristae morphology rather than conversion of the ATP synthase into the monomeric form in cells lacking Atp20 or Atp21.

We developed an additional assay to test the generation of mtDNA autonomous domains and the role of cristae therein. Cells expressing mtDNA-encoded Atp6-NG and nuclear-encoded matrix-targeted TagBFP were mated with cells expressing mtDNA-encoded Atp6-mKate2, and the patterns produced by the differently tagged Atp6 proteins were examined in zygotes. The Atp6-mKate2 strain grew normally on medium containing fermentable or nonfermentable carbon sources, and no increase in petite formation was observed, indicating that the mKate2-tagged variant of Atp6 was functional (fig. S7C). In line with our previous observations, the Atp6 proteins remained localized to their respective origin cells in zygotes in crosses between WT cells (Fig. 3C). Notably, we often observed an alternating, mostly nonoverlapping pattern of green and red signals along the TagBFP-stained mitochondrial network in the daughter cell produced by the zygote (Fig. 3C, zoom, and fig. S7D). Thus, Atp6-NG and Atp6-mKate2 occupy separate domains in the mitochondrial network and exhibit limited mixing. This result indicates that mtDNA copies encoding Atp6-NG or Atp6-mKate2 maintain semi-autonomous mitochondrial subdomains. The alternating pattern in the daughter cell further suggests a complex procedure in which mitochondrial domains from both parental cells are sorted into the daughter cell. In mating events between Δatp20 and Δatp21 cells expressing either Atp6-NG or Atp6-mKate2, both fluorescently tagged Atp6 variants showed a stronger colocalization in daughter cells and separate domains were hardly discernible (Fig. 3C and fig. S7D). These observations show that cristae are required for maintenance of inner membrane domains and prevent extensive mixing between gene products of different mtDNA copies.

mtDNA diffusion is limited in WT, Δatp21 and Δmic60 cells

Cristae could support mtDNA-mediated formation of mitochondrial subdomains via at least two mutually nonexclusive mechanisms: (i) Cristae could limit mobility of mtDNA within the mitochondrial matrix by forming physical barriers and thereby promote local synthesis of mtDNA-encoded proteins. (ii) Cristae could corral mtDNA-encoded proteins by preventing their diffusion through cristae junctions into the inner boundary membrane. To determine the importance of cristae in restricting mtDNA mobility, we used our recently developed mtDNA LacO-LacI-GFP system (10) to specifically mark mtDNA in one of the parental cells in a mating experiment (P₂ cell). The other parental strain used in the mating assay contained WT mtDNA lacking the LacO repeats and expressed the matrix-targeted mKate2 protein (P₁ cell). In mating events between two WT cells, two Δatp21, or Δmic60 cells, LacO-marked mtDNA was evident in the daughter cells produced by zygotes, indicating efficient transport of mtDNA into daughter cells. Of note, we observed a slight increase of the percentage of LacO mtDNA spots in the daughter cell in matings between Δatp21 cells compared to matings between WT or Δmic60 cells (Fig. 3D). The underlying reason for this observation remains to be determined. LacO-marked mtDNA did not accumulate in the P₁ parental cell even at late zygotic stages in any of the matings (Fig. 3D). Thus, mobility of mtDNA remains limited in the mitochondrial network of parental cells in matings between Δatp21 or Δmic60 cells that contain strongly compromised cristae architecture. We conclude that WT-like cristae architecture plays a crucial role in limiting equilibration of the Atp6-NG protein, rather than diffusion of mtDNA.

To find a potential explanation for the rapid equilibration of Atp6-NG in the absence of dimer-specific ATP synthase subunits, we carefully examined WT, Δatp20, Δatp21, and Δatp20Δatp21 cells by electron microscopy and also performed serial sectioning to obtain three-dimensional (3D) information about the mitochondrial ultrastructure. Mitochondria of WT cells showed cristae with the typical perpendicular orientation to the mitochondrial tubular axis. In mutant cells lacking dimeric ATP synthase, however, inner membrane structure was markedly altered and virtually no WT-like cristae were apparent. Instead, mitochondria exhibited onion- and balloon-like inner membrane profiles, as previously described. In accordance with previous work, we frequently observed strongly elongated cristae-like structures that traversed the whole mitochondrion (Fig. 3, E and F, and fig. S8) (22, 23,27).

These observations provide a possible explanation for the inability of cells lacking dimeric ATP synthase to constrain diffusion of Atp6-NG. While respiratory chain complexes get trapped in cristae in WT cells, they are free to diffuse along the altered cristae membranes in Δatp20, Δatp21, and Δatp20Δatp21 mutants.

Cristae morphology is required for mtDNA quality control

Having determined that mtDNA-encoded subunits exhibit a severely reduced mobility in the inner mitochondrial membrane that is dependent on normal cristae biogenesis, we asked whether mutants defective in cristae biogenesis would have problems to distinguish between WT and mutant mtDNA. We applied our pedigree analysis to follow inheritance of WT over Δcob::ARG8 mtDNA from heteroplasmic zygotes in the absences of ATP20, ATP21, MIC10, or MIC60. Deletion of these genes did not lead to greatly altered mtDNA levels in strains containing WT or Δcob::ARG8 mtDNA (fig. S9A). In particular, mtDNA levels of the mating partners of each pedigree mating pair did not differ significantly to one another. Notably, clearance of the mutant mtDNA was significantly delayed in the absence of any of these proteins. While 60% of lineages for the WT were already homoplasmic for WT mtDNA in the first generation, this value dropped to 40% in cristae mutants. Moreover, while only ∼20% of cells of the first generation in WT cells still maintained a heteroplasmic state, ∼40% of such cells were detected in the first generation of matings between cristae mutants. After five generations, a significantly smaller proportion of the lineages were homoplasmic for WT mtDNA in the cristae mutants (Fig. 4, A to F). Thus, the ability to choose WT over mutant mtDNA is severely compromised in mutants with defective cristae architecture. In line with these pedigree results and in accordance with previous work, Δatp20, Δatp21, Δatp20Δatp21, Δmic10, or Δmic60 strains harboring exclusively WT mtDNA exhibit increased formation of petites, revealing a reduced ability to maintain a healthy mtDNA population in cells (fig. S9B) (23).

Fig. 4. — (A to F) Pedigree analyses of strains lacking indicated genes. WT pedigree (A) is identical to Fig. 1C, shown to allow comparison to mutant analyses in (B) to (F). χ² test has been applied as statistical hypothesis tests to compare first- and fifth-generation data of mutant strains to the WT. (G) Model for quality control of mtDNA in a fused mitochondrial network. mtDNA copies in WT cells supply only the cristae in their immediate surrounding with mtDNA-encoded proteins. Respiratory chain complex containing mtDNA-encoded subunits has limited diffusion capability and is trapped within cristae, which leads to a sphere of influence of mtDNA. Limited diffusion of respiratory chain complexes is compromised in cells with compromised cristae structure.

DISCUSSION

Our data reveal that the unicellular S. cerevisiae is able to promote generation of progeny with a healthy mtDNA population from heteroplasmic zygotes that contain WT and mutant mtDNA. So far, our demonstration of the purifying selection in yeast is limited to a model mutant mtDNA variant that entirely lacks the COB gene. It will be interesting to examine in future studies how yeast cells deal with deletions of other mtDNA-encoded genes or milder point mutations. Unexpectedly, we show that the purifying selection occurs in a continuous mitochondrial network and does not require mitochondrial fission. This finding raised the question how cells can detect mutant mtDNA copies among WT copies in a mitochondrial network, where soluble matrix proteins rapidly equilibrate and potentially even out physiological differences and thus conceal mutant copies.

A possible solution for this problem is provided by our finding that diffusion of OXPHOS components, namely, the mtDNA-encoded protein Atp6-NG and the nuclear-encoded Cox4-NG, is severely limited in WT cells compared to proteins localized to the inner boundary membrane, the outer membrane, or the matrix. Of particular interest in this respect is the observation that the mtDNA-encoded Atp6-NG forms subdomains that remain in the vicinity of the mtDNA by which it is encoded. These findings are in line with analyses in HeLa cells that have similarly demonstrated reduced mobility of OXPHOS proteins along the longitudinal axis of mitochondrial tubules (28, 29). A previously proposed plausible explanation for the restricted mobility is that OXPHOS proteins, likely aided through their assembly into complexes and supercomplexes (30), get trapped within cristae membranes and are prevented from diffusion past cristae junctions into the inner boundary membrane (18, 31). In strong support of this hypothesis, we find that the mtDNA-encoded Atp6-NG exhibits increased equilibration across the mitochondrial network in mutants with defective cristae architecture, while movement of the mtDNA from which it originates remains restricted. Notably, our pedigree analysis revealed that mutants with defective cristae biogenesis are incapable of distinguishing effectively between WT and mutant mtDNA to support progeny with functional mtDNA.

Together, our findings are in line with a previously proposed model in which each mtDNA copy has a “sphere of influence” and remains spatially linked to its gene products (32, 33). Our data support the hypothesis that such a sphere of influence is dependent on normal cristae architecture (Fig. 4G). According to this model, mutations in mtDNA would lead to mitochondrial subdomains with physiological defects that could flag such areas and facilitate purging of the linked mtDNA copy. In line with this idea, it has recently been shown that individual cristae within the same mitochondrial tubule can maintain independent membrane potentials (34). Defective cristae biogenesis would lead to defective subdomain formation and a weakened link between mtDNA and its gene products, which, in turn, would blur physiological differences caused by mtDNA mutations (Fig. 4G). As a consequence, detection and purging of mutated mtDNA would be hindered.

For such a system to work, not only the proteins but also their mRNAs need to remain close to the mtDNA copy from which they originate. In matings between two WT cells containing either regular or ATP6-NG mtDNA, we observe very low levels of Atp6-NG protein in parts of the mitochondrial network that does not contain the ATP6-NG mtDNA, even more than 80 min after mitochondria of both parental cells have fused (fig. S6A). This result indicates that little synthesis of Atp6-NG occurs in these parts of the network within the time frame of this analysis. It is, however, possible that biogenesis of OXPHOS complexes is generally low in the mitochondrial network of parental cells because of little OXPHOS turnover. In this case, we would see little synthesis of Atp6-NG even if its mRNA would be present. Not much is known from the literature about the mobility of mitochondrial mRNAs. It was recently proposed that mitochondrial transcription and translation may occur in a coupled manner in S. cerevisiae (35). Taking into account that insertion of mtDNA-encoded proteins into the mitochondrial inner membrane occurs cotranslationally (36, 37) and, at least in part, at cristae membranes (38), all steps of mitochondrial gene expression from transcription to insertion of proteins into cristae membranes could be spatially linked. Nevertheless, characterization of mRNA mobility in mitochondria awaits further studies.

We demonstrate that yeast cells can distinguish between mutant and WT mtDNA and propose that cristae-dependent subdomains are an important prerequisite for efficient purging of mutant mtDNA. It remains an outstanding and exciting question how cells mechanistically detect and remove mutant mtDNA. Plausible physiological parameters that may serve as signals on dysfunctional mitochondria are a reduced membrane potential (39), decreased ATP levels (5), or altered redox states (40). What processes may remove mutant mtDNA from cells? Our microscopic analyses suggest that mutant mtDNA transfer to daughter cells of heteroplasmic zygotes may be reduced, but not entirely prevented (Fig. 1D). Preferential transport of fit mitochondria into daughter cells has been demonstrated in yeast previously (40). In combination with our pedigree analysis, where a large percentage of first-generation colonies appears homoplasmic (Fig. 1C), we suggest that further rounds of selection against mutant mtDNA likely occur in the daughter cells that eventually entirely purge mutant mtDNA. While we find that mitochondrial fission or Atg32-mediated mitophagy is not absolutely essential for selection against mutant mtDNA, especially during production of the first daughter cell of heteroplasmic zygotes, these processes may contribute to efficient clearance during further growth and cell divisions. Furthermore, WT mtDNA could also be preferentially replicated as suggested for D. melanogaster (3, 7) or mutant mtDNA could be selectively degraded within mitochondria by nucleases. It was demonstrated in yeast that the mtDNA polymerase Mip1 degrades mtDNA through its exonuclease domain upon prolonged starvation (41). It remains to be determined whether such a mechanism could also be involved in clearance of mutant mtDNA. Together, selection against mutant mtDNA could entail a combination of mechanisms that may include selective transport of mitochondria containing healthy mtDNA to daughter cells, mitophagic removal of mitochondrial fragments containing mutant mtDNA, selective replication of healthy mtDNA, or selective degradation of mutant mtDNA by nucleases. The discovery of a purifying selection in S. cerevisiae combined with the tools we developed in this study will be of great value in elucidating molecular mechanisms responsible for clearance of mutant mtDNA.

MATERIALS AND METHODS

Yeast strains and plasmids

All yeast strains are derived from W303 background. Strain information can be found in table S1. Deletions of genes and C-terminal tagging of nuclear-encoded genes were performed in haploid strains using homologous recombination as described previously (42). For the experiment presented in fig. S3A, the mating type of strain yCO354 had to be switched from Mat alpha to Mat a. Mating type switching was performed by transient (90 min) galactose-induced expression of the HO endonuclease from the plasmid pJH132 (43). Subsequently, a Mat a strain that had lost the plasmid pJH132 was isolated. Primer and plasmid information can be found in tables S2 and S3, respectively.

Pedigree analysis

Approximately 1.85 × 10⁷ cells from early post-log phase [optical density at 600 nm (OD₆₀₀) ~ 1.5] growing cultures of strains with opposing mating types were combined in a 1.5-ml reaction tube, vortexed, centrifuged at 3000 rpm for 3 min, and resuspended in 50 μl of rich medium containing glucose [yeast extract peptone dextrose (YPD)]. Subsequently, the cell suspension was spotted onto a YPD plate and incubated for 2 to 3 hours to allow mating of strains. A small amount of cells was scraped off the plate and was resuspended in 200 μl of YPD medium. From this mated cell suspension, 30 μl was spread as a line on a YPD plate. Individual zygotes were identified on the basis of their characteristic shape and transferred to free areas on the agar plate. Cell growth was monitored, and daughter cells were separated from zygotes or mother cells and transferred to free areas on the agar plate. This procedure was repeated for five generations (Fig. 1, A and B). After incubation for 2 days at 30°C, the grown colonies were replica-plated onto plates containing synthetic defined medium lacking arginine (SD-Arg) and plates containing rich medium and glycerol as a carbon source [yeast extract peptone glycerol (YPG)]. Those plates were again incubated for 24 to 48 hours, and growth was scored. Of note, colonies were scored as “growing” for SD-Arg or YPG even when only parts of the replicated colony displayed growth. Growth rates were not considered in this experiment. The presence or absence of growth was verified by restreaking of cell material from selected colonies onto YPG or SD-Arg plates (fig. S1B). In addition, a mating-type test was performed to confirm that the initially picked cells were zygotes. Lineages were only considered when the starting zygotes were able to grow on YPG and SD-Arg plates, indicating a heteroplasmic state. Of note, we sporadically observed that mtDNA species were lost in one generation but reappeared at a later generation (e.g., replicate 2 in Fig. 1B). We attribute this observation to cases in which all copies of one particular mtDNA species are passed on to the daughter cell. In such a scenario, a mother cell lacking this mtDNA species is produced, whereas this species will be present in the daughter cell (fig. S1D).

Live-cell microscopy

For all live-cell microscopy experiments, yeast cells were imaged in ibidi 8-well μ-Slides (ibidi GmbH, Gräfelfing). For immobilization of yeast cells during microscopy, wells were coated with concanavalin A, a lectin that can bind the cell wall of yeast cells. In brief, wells were filled with 200 μl of concanavalin A (0.5 μg/ml) and incubated for 30 min. Concanavalin A was removed, and wells were dried for 30 min at room temperature. For single-frame images and videos of mating events, 1000 μl from two cultures with strains of opposing mating types was combined in a reaction tube, vortexed thoroughly, and centrifuged for 3 min at 5000 rpm. The pellet was resuspended in 50 μl of YPD medium and spotted onto a YPD plate to allow cells to mate. Plates were incubated for 3 hours for single-frame images and for 1.5 hours for videos. The premated cells were scraped off and resuspended in 400 μl of sterile-filtered 1× phosphate-buffered saline (PBS) buffer, and 200 μl was transferred into a concanavalin A–coated well of ibidi 8-well μ-Slide. The slides were centrifuged for 2 min at 2000 rpm to promote adherence of the cells to the bottom of the well. Wells were washed twice with 400 μl of filtered synthetic complete (SC) medium to remove floating cells and eventually resuspended in SC medium for imaging. Microscopy was performed at 30°C on a Nikon Ti2-Eclipse microscope equipped with a CFI Apochromat TIRF 100×/1.49 numerical aperture (NA) oil objective and a TwinCam LS dual-camera splitter attached to two Photometrics Prime 95B 25-mm cameras. The dual-camera setup enabled simultaneous imaging of red and green fluorophores. Specifications of filters and dichroics can be made available upon request. For time-lapse microscopy, cells were imaged every 2 or 7 min for indicated total periods of time. For quantification of the imaging presented in Fig. 1D, zygotes were only considered when patchy structures were apparent for GFP and mRuby3 signals. Cells harboring the TetO repeats often showed a diffuse red staining of mitochondria, which is likely explained by recombination of the TetO repeats.

Image processing and analysis

All images, except those obtained by structured illumination microscopy, were postprocessed by deconvolution with the Huygens software (Scientific Volume Imaging). Fluorescent channels acquired simultaneously on two different cameras were aligned using a custom-built Python script. Alignment parameters were obtained from simultaneous imaging in bright-field mode. For quantification of Atp6-NG levels presented in Fig. 3A, mitochondria were first segmented in 3D based on the matrix-mKate2 signal using the mitograph software (44). Binary masks for parental and daughter cells were manually created in Fiji by drawing outlines on the bright-field image. Masks were used in a next step to assign coordinates of the mitochondrial network to the respective cells. Then, Atp6-NG intensities were determined and summed up along the mitochondrial network of parental or daughter cells using custom-built Python scripts. To account for differences in the mitochondrial amount in different cells, the Atp6-NG signal was normalized to the mitochondrial network length present in the respective cell.

Quantification of colocalization of Atp6-NG and Atp6-mKate2, presented in fig. S7D, was performed as follows. The mitochondrial network was first segmented in 3D based on the matrix-TagBFP signal using the mitograph software. Manually created binary masks were used to assign parts of the mitochondrial network to parental or daughter cells. Fluorescent intensities of Atp6-NG and Atp6-mKate2 were then determined for pixels along the mitochondrial network. Next, Manders and Pearson correlation coefficients were determined between both signals. For determination of the Manders correlation coefficient, signals along the mitochondrial network were thresholded beforehand with Yen et al.’s method (45).

Genetic manipulation of mtDNA

Strains harboring mtDNA in which the COB gene was replaced with an arginine marker and a nonrecombinable TetO array were generated as follows. First, a synthesized TetO array, in which 21 TetO repeats are separated by spacers of varying length and sequence, was inserted after the stop codon of the ARG8 gene in the plasmid pCOB-ST5 (8). This cloning step resulted in the plasmid pCO307, which thus contains an insert in which sequences homologous to the up- and downstream regions of the COB gene flank the ORF of ARG8 followed by the TetO array. pCO307 was introduced into the kar1-1 strain αDFS160 ρ⁰ by biolistic transformation with the PDS-1000/He particle delivery system (Bio-Rad Laboratories), and transformants were selected by their ability to rescue the cox2-62 mutation of the strain NB40-3C (46). pCO307 was then cytoducted into a Δarg8 W303 WT strain, which resulted in the deletion of the COB gene by ARG8-TetO through homologous recombination. Cells containing the Δcob::ARG8-TetO-mtDNA were selected on the basis of their arginine prototrophy. Last, a construct (pCO407) consisting of the Cup1 promoter driving expression of an ORF in which the Su9 mitochondrial targeting sequence was fused to the TetR gene, which, in turn, was followed by three copies of the red fluorescent protein mRuby3, was chromosomally integrated into the HO locus (strain yCO460).

Strains harboring mtDNA in which the ATP6 is tagged with either NG or mKate2 were generated as follows. First, synthesized gene fragments encoding NG and mKate2 compatible with the mitochondrial genetic code (Twist Bioscience, San Francisco) were fused to the C terminus of the coding region of ATP6, which was amplified from genomic DNA. ATP6-NG or ATP6-mKate2 was flanked with an 806-bp region homologous to the upstream region of ATP6 and a 60-bp region homologous to the downstream region of ATP6. The entire fragments were then cloned into the Xho I site of the plasmid pPT24 (47), which resulted in either plasmid pCO444 or pCJ013 for ATP6-NG and ATP6-mKate2, respectively. pCO444 and pCJ013 were introduced into the kar1-1 strain αDFS160 ρ⁰ by biolistic transformation, and transformants were selected by their ability to rescue the cox2 mutation of NB40-3C (46). pCO444 and pCJ013 were then cytoducted into the strain MR10 (48). Colonies in which ATP6-NG (yCJ043) or ATP6-mKate2 (yCJ120) had successfully integrated into the ATP6 locus were identified by their ability to grow on a non-fermentable carbon source. A strain containing ATP6-NG-mtDNA and mating type alpha was obtained by mating yCJ043 with a ρ⁰ W303 WT strain followed by sporulation and tetrad dissection (yCJ084).

Primer sequences used for cloning and plasmid maps can be made available upon request.

DAPI staining and structured illumination microscopy

Coverslips (no. 1.5) were coated with 50 μl of concanavalin A (0.5 μg/ml) and air-dried for 1 hour. Residual concanavalin A was removed, and coverslips were dried for 45 min before fixation. OD₆₀₀ = 0.5 of log phase yeast cells were harvested at 5000 rpm for 3 min and washed using 1× PBS + 0.02% Tween 20. Cells were resuspended in 20 μl of 1× PBS + 0.02% Tween 20. Subsequently, all cells were placed on the concanavalin A–coated coverslips and incubated for 45 min to let cells settle on the coverslip. Then, cells were washed for 2 min with filtered SC medium. Cells were fixed using a 4% formaldehyde solution + DAPI (1 μg/ml) in filtered SC medium for 30 min. Cells were washed once more for 2 min using filtered SC medium. Twenty microliters of MOVIOL 4-88 (Roth) was added to the coverslips, and microscope slides were put onto the coverslips. Fixed immobilized cells were used for super-resolution imaging by structured illumination microscopy. The acquisition was performed on a 3D SIM DeltaVision OMX V3 microscope (General Electric) equipped with a 10× 1.4 NA oil immersion objective UPlanSApo (Olympus), 405-, 488-, and 593-nm diode lasers, and Cascade II electron-multiplying charge-coupled device (CCD) cameras (Photometrics). After acquisition with an appropriate refractive index oil, raw data were first reconstructed and corrected for color shifts using the provided software softWoRx 6.0 Beta 19 (unreleased). In a second step, a custom-made macro in Fiji (49) finalized the channel alignment and established composite TIFF (tag image file format) stacks, which were used for image analysis.

Electron microscopy

For electron microscopy, all strains were pregrown in YPG medium to select for functional mtDNA. Subsequently, cells were grown to log phase in YPD or rich medium containing galactose medium, as indicated. Sample preparation for electron microscopy was essentially performed as previously described (50) with two minor changes: The fixation with glutaraldehyde was performed for 1 hour, and all centrifugation steps were carried out at 1610g for 5 min. Ultrathin sectioning was performed using a Leica Ultracut UCT (Leica Microsystems, Wetzlar, Germany) ultramicrotome and an ultra 35° diamond knife (Diatome, Nidau, Switzerland). Ultrathin 50- to 70-nm sections were placed on Pioloform-coated copper slot grids (Plano, Wetzlar, Germany) and poststained for 15 min with uranyl acetate and 3 min with lead citrate, as previously described (50). Electron microscopy was performed using a JEOL JEM-1400 Plus transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV. Images were taken with a JEOL Ruby CCD camera (3296 × 2472 pixels) and the TEM Center software Ver.1.7.12.1984 (JEOL, Tokyo, Japan).

Miscellaneous

Western blot analyses were performed with isolated mitochondria. Fifty micrograms of isolated mitochondria was preheated on 95°C for 5 min in 1× SDS loading buffer and separated on a 12% SDS gel. After transfer to polyvinylidene difluoride membranes, membranes were incubated with the following primary antibodies in 5% milk and tris-buffered saline: mouse anti-NG (1:1000; Chromotek GmbH), rabbit anti-aconitase1 (1:1000), and rabbit anti-Atp6 (1:1000). Quantitative real-time PCR experiments to determine mtDNA levels were performed as described previously (51). For the petite analysis, cells were grown overnight at 30°C, then freshly diluted to an OD₆₀₀ = 0.2, and grown for another 3 hours. A total of 200 cells were plated onto YPG plates containing 0.1% glucose. Plates were incubated for 3 days at 30°C. Only cells proficient in respiratory growth are able to continue growth after all glucose has been consumed.

Acknowledgments

We thank T. Kautzleben and N. Lebedeva for technical assistance, L. Heiderscheid and M. Kroker for help with the experimental work, and S. Geimer for help with electron microscopy. OMX microscopy was performed at the Center for Advanced Light Microscopy (CALM). We thank M. Schuldiner and members of the Osman laboratory for critically reading the manuscript. We are grateful for stimulating discussions within the “Mito-Club” throughout the course of this project. We thank Martin Ott and Jean Velours for providing reagents. Funding: C.O., C.J., R.R., and F.T. are supported by a grant from the European Research Council (ERCStG-714739 IlluMitoDNA). T.K. is supported by the Elitenetzwerk Bayern through the” Biological Physics” program and by a grant from the Deutsche Forschungsgemeinschaft (DFG, project number 459304237). A.M., R.B., D.H., and H.L. are supported by the SPP 2202 Priority Program of the Deutsche Forschungsgemeinschaft (DFG, project number 422857584). Author contributions: C.O. and C.J. designed the project. C.O., C.J., R.R., A.M., F.T., C.S., and T.K. performed experiments and/or analyzed experimental data. R.B. and D.H. developed a software to automatically detect yeast zygotes in microscopic images. C.O., C.J., R.R., and T.K. wrote the manuscript, with contributions from all coauthors. H.L. supervised super-resolution microscopy experiments and development of zygote detection software. P.W. supervised early stages of the project. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S9

Tables S1 to S3

Legends for movies S1 to S6

sciadv.abi8886_sm.pdf^{(12.9MB, pdf)}

Other Supplementary Material for this manuscript includes the following:

Movies S1 to S6

sciadv.abi8886_movies_s1_to_s6.zip^{(1.4MB, zip)}

View/request a protocol for this paper from Bio-protocol.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S9

Tables S1 to S3

Legends for movies S1 to S6

sciadv.abi8886_sm.pdf^{(12.9MB, pdf)}

Movies S1 to S6

sciadv.abi8886_movies_s1_to_s6.zip^{(1.4MB, zip)}

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PERMALINK

Cristae-dependent quality control of the mitochondrial genome

Christopher Jakubke

Rodaria Roussou

Andreas Maiser

Christina Schug

Felix Thoma

Raven Bunk

David Hörl

Heinrich Leonhardt

Peter Walter

Till Klecker

Christof Osman

Roles

Abstract

INTRODUCTION

RESULTS

A pedigree analysis reveals mtDNA quality control in S. cerevisiae

Fig. 1. S. cerevisiae cells distinguish between WT and mutant mtDNA.

Selection against mutant mtDNA occurs in a continuous mitochondrial network

Mitochondrial fission or Atg32-mediated mitophagy is not required for selection against mutant mtDNA

The mtDNA-encoded protein Atp6 exhibits severely limited diffusion rates

Fig. 2. mtDNA-encoded Atp6-NG exhibits limited diffusion.

Fig. 3. Intact cristae morphology limits diffusion of mtDNA-encoded Atp6-NG.

Components important for cristae morphology affect diffusion rates of Atp6-NG

mtDNA diffusion is limited in WT, Δatp21 and Δmic60 cells

Cristae morphology is required for mtDNA quality control

Fig. 4. mtDNA quality control depends on normal cristae morphology.

DISCUSSION

MATERIALS AND METHODS

Yeast strains and plasmids

Pedigree analysis

Live-cell microscopy

Image processing and analysis

Genetic manipulation of mtDNA

DAPI staining and structured illumination microscopy

Electron microscopy

Miscellaneous

Acknowledgments

Supplementary Materials

This PDF file includes:

Other Supplementary Material for this manuscript includes the following:

REFERENCES AND NOTES

Associated Data

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