Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2

Journal Pre-proof Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2 Kseniia V. Galkina, Anna N. Zyrina, Sergey A. Golyshev, Nataliia D. Kashko, Olga V. Markova, Svyatoslav S. Sokolov, Fedor F. Severin, Dmitry A. Knorre

PII:

S0171-9335(20)30010-8

DOI:

https://doi.org/10.1016/j.ejcb.2020.151071

Reference:

EJCB 151071

To appear in:

European Journal of Cell Biology

Received Date:

2 June 2019

Revised Date:

30 January 2020

Accepted Date:

30 January 2020

Please cite this article as: Galkina KV, Zyrina AN, Golyshev SA, Kashko ND, Markova OV, Sokolov SS, Severin FF, Knorre DA, Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2, European Journal of Cell Biology (2020), doi: https://doi.org/10.1016/j.ejcb.2020.151071

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Mitochondrial dynamics in yeast with repressed adenine nucleotide translocator AAC2

Kseniia V. Galkina1,2, Anna N. Zyrina2, Sergey A. Golyshev2, Nataliia

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D. Kashko1, Olga V. Markova2, Svyatoslav S. Sokolov2, Fedor F. Severin2, Dmitry A. Knorre2,3

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Leninskiye Gory 1–73, Moscow, 119991, Russia. 2

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Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University,

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Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

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Leninskiye Gory 1–40, Moscow, 119991, Russia. 3

Institute of Molecular Medicine, Sechenov First Moscow State Medical University, Moscow,

highlights

clonal yeast populations are extremely heterogeneous in mitochondrial network

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

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119991, Russia.

structure 

AAC2 repression increases mitochondrial ΔΨ and induces mitochondrial

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fragmentation

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AAC2 repression prevents further protonophore-induced mitochondrial fragmentation

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AAC2 is dispensable for mitochondrial fusion in yeast

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Abstract The mitochondrial network structure dynamically adapts to cellular metabolic challenges. Mitochondrial depolarisation, particularly, induces fragmentation of the network. This fragmentation may be a result of either a direct regulation of the mitochondrial fusion machinery by transmembrane potential or an indirect effect of metabolic remodelling. Activities of ATP synthase and adenine nucleotide translocator (ANT) link the mitochondrial transmembrane potential with the cytosolic NTP/NDP ratio. Given that mitochondrial fusion

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requires cytosolic GTP, a decrease in the NTP/NDP ratio might also account for protonophoreinduced mitochondrial fragmentation. For evaluating the contributions of direct and indirect mechanisms to mitochondrial remodelling, we assessed the morphology of the mitochondrial

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network in yeast cells with inhibited ANT. We showed that the repression of AAC2 (PET9), a major ANT gene in yeast, increases mitochondrial transmembrane potential. However, the

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mitochondrial network in this strain was fragmented. Meanwhile, AAC2 repression did not prevent mitochondrial fusion in zygotes; nor did it inhibit mitochondrial hyperfusion induced by

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Dnm1p inhibitor mdivi-1. These results suggest that the inhibition of ANT, rather than preventing mitochondrial fusion, facilitates mitochondrial fission. The protonophores were not

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able to induce additional mitochondrial fragmentation in an AAC2-repressed strain and in yeast cells with inhibited ATP synthase. Importantly, treatment with the ATP synthase inhibitor oligomycin A also induced mitochondrial fragmentation and hyperpolarization. Taken together,

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our data suggest that ATP/ADP translocation plays a crucial role in shaping of the

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mitochondrial network and exemplify that an increase in mitochondrial membrane potential does not necessarily oppose mitochondrial fragmentation.

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Keywords:

adenine nucleotide translocator,

AAC2,

mitochondrial dynamics,

transmembrane potential, mitochondrial fusion, mitochondrial dysfunction

Introduction Mitochondria are dynamic organelles capable of both dividing and fusing with each

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other (Chen and Chan, 2009; Eisner et al., 2014; Okamoto and Shaw, 2005). The frequencies at which these fission and fusion events occur determine the lengths of the individual mitochondria (Cagalinec et al., 2013). Simultaneously, the metabolic state of the cells and

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mitochondrial dynamics are linked (Koopman et al., 2005; Rossignol et al., 2004). For example, the inhibition of mitochondrial enzymes or dissipation of transmembrane potential

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decreases the connectivity of the mitochondrial network (Jones et al., 2017; MacVicar and Lane, 2014; Pletjushkina et al., 2006; Sauvanet et al., 2010; Zhang et al., 2014). In mouse

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embryonic fibroblasts, the substitution by acetoacetate of glucose in the incubation medium upregulates oxidative metabolism and increases the elongation of mitochondria (Mishra et al.,

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2014). However, causal relationships between the changes of mitochondrial network structure and the changes in cellular bioenergetics remain unclear.

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On the one hand, because mitochondrial fusion can upregulate oxidative phosphorylation (OxPhos) remodelling of the mitochondrial network affects cell metabolism.

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One of the possible mechanisms of this regulatory link is metabolic complementation between different mitochondria. For instance, it was shown that in cybrids, cytoduction of Rho+ (wild type mtDNA) and Rho- (mutant mtDNA) cells enabled rapid joining of the mitochondrial network and energization of mitochondria with Rho- mtDNA (Yang et al., 2015). In addition, mitochondrial dynamics are an essential component of mitochondrial quality control (Knorre, 2020; Sebastián et al., 2016; Twig et al., 2008a; Westermann, 2010a), thereby affecting

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metabolism indirectly. The disruption of mitochondrial fusion has been demonstrated to repress cellular energetics (Chen et al., 2010; Song et al., 2017). On the other hand, metabolism regulates mitochondrial dynamics (Mishra and Chan, 2016). For instance, the functioning of mitochondrial fusion and fission machinery requires GTP hydrolysis (Cao et al., 2017; Francy et al., 2015; Kalia et al., 2018; Meeusen et al., 2004; Meglei and McQuibban, 2009). Mitofusins, which are sensitive to the redox status of the cell (that is, the ratio of oxidised and reduced glutathione) mediate the fusion of the outer mitochondrial membrane (Anton et al., 2011). The oxidised glutathione dimer (GSSG) promotes the formation of disulphide bonds

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and the dimerisation of mitofusins—the first step in the fusion of the outer membranes (Shutt et al., 2012). Finally, that artificial dissipation of the transmembrane potential inhibits the fusion of the inner membranes (Legros et al., 2002; Meeusen et al., 2004). It follows that the

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mitochondrial transmembrane potential (ΔΨ) regulates the fusion of the inner membranes.

The mechanism directly linking the transmembrane potential and the ability of the

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mitochondria to fuse lies in the alternative processing of OPA1 isoforms (Mgm1 in yeast) (Herlan et al., 2003)). Depolarisation of the mitochondria destabilises the long OPA1 isoform

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that is required for mitochondrial fusion (Ishihara et al., 2006; Song et al., 2007). In yeast, depletion of ATP also inhibits Mgm1 translocation, thereby inducing an imbalance between

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long and short isoforms of Mgm1, which then inhibits fusion. At the same time, the first step of Mgm1 insertion in the inner membrane requires ΔΨ (Herlan et al., 2004).

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Transmembrane potential can also indirectly regulate mitochondrial fusion via multiple non-exclusive mechanisms. The principal bioenergetic factors of the cell are coupled with each

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other. Mitochondrial transmembrane potential depends on both the NADH/NAD+ ratio and the activity of the respiratory chain. Next, inside the mitochondrial matrix, ATP synthase provides coupling between the transmembrane potential and the ATP/ADP ratio. Finally, due to the activity of the adenine nucleotide translocator (ANT), this mitochondrial ATP/ADP ratio is coupled to the ATP/ADP ratio in the cytosol (Klingenberg, 1980; Kunji et al., 2016). Experiments with isolated phosphorylating mitochondria showed that ANT activity maintains the ratio of (ATP/ADP)external/(ATP/ADP)mitochondrial at a level of approximately five (Heldt et al.,

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1972; Klingenberg, 2008). Importantly, the ANT activity is electrogenic, and the direction of the reaction can be reversed by depolarisation of the mitochondria (Klingenberg, 1980; Metelkin et al., 2009). Meanwhile, the activity of nucleoside diphosphate kinases maintains the ratios of various nucleotide triphosphates at similar levels. Therefore, in the same compartment, the GTP/GDP ratio is coupled to the ATP/ADP ratio (Lascu and Gonin, 2000). The dissipation of transmembrane potential can therefore inhibit mitochondrial dynamics either directly—by preventing OPA1/Mgm1 processing—or indirectly—by affecting cellular energetics and the availability of NTP in the cytosol. To evaluate the relative contributions of

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the direct and the indirect mechanisms and to critically re-appraise the role of mitochondrial transmembrane potential, using baker’s yeast as a model, we studied the effects of protonophores on the mitochondrial network structure in yeast with inhibited adenine

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nucleotide translocation and ATP synthase.

We report here that AAC2 repression both induced mitochondrial fragmentation and

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prevented the further effect of the protonophore FCCP. Our results point to the major ANT playing a crucial role in yeast mitochondrial dynamics and representing a connecting link

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between cell metabolism and mitochondrial network structure.

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Methods

Strains, reagents and growth conditions

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The yeast strains used in this study—derivatives of W303 (MAT a ade2-101 his3-11 trp1-1 ura3-52 can1-100 leu2-3, 112, GAL, psi+)—are listed in Table S1. We grew cells

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overnight at 30°C with rotary shaking (250 rpm) to a density of 2 × 106 cells/ml in rich growth medium yeast peptone D-glucose (YPD), yeast peptone glycerol (YPGly), yeast peptone raffinose (YPRaf), yeast peptone galactose (YPGal), yeast peptone raffinose galactose (YPRafGal), synthetic medium yeast nitrogen base raffinose galactose without histidine (YNBRafGal -His) and raffinose without uracil (YNBRaf -Ura) according to (Sherman, 2002). We obtained yeast extract from BD, bactoagar and bactopeptone from Amresco, D-glucose

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from Helicon, raffinose from Chimmed, glutaraldehyde, Spi-pon 812 epoxy resin and sodium cacodylate from SPI and the other chemicals from Sigma-Aldrich.

PGAL-AAC2 strain construction To generate a strain with repressible AAC2, we substituted the native AAC2 promoter with a gene cassette containing the PGAL promoter and a marker gene (Figure 1A). To produce the cassette, we used the PCR-based approach described in Longtine et al. (1998) using pFA6a-HIS3MX6-PGAL1 plasmid as matrix DNA. The primers are listed in Supplementary

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Table S2. The same procedure and pFA6a-GFP-KANMX4 plasmid were used to tag the AAC2 C-terminus with GFP (Figure 1A). Because C-terminus tagging of transmembrane proteins could affect their incorporation into membranes, we did not rely on protein localization

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information obtained with this construct. However, we used it for evaluation of AAC2

Fluorescent microscopy

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expression in the PGAL-AAC2 strain.

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We used an Olympus BX51 microscope with the following filter sets: U-MNIBA3 filter for GFP (excitation wavelength λ = 470 nm–495 nm; dichroic mirror λ = 505 nm; emission

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wavelength 510 nm–550 nm), U-MNU2 filter for DAPI (excitation λ = 360 nm–370 nm; dichroic mirror λ = 400 nm; emission wavelength >420 nm); U-MNG2 filter for tetramethylrhodamine

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ethyl ester, TMRE (excitation wavelength λ = 530 nm–550 nm, beamsplitter filter λ = 570 nm; emission λ > 590 nm) to analyse fluorescence in the yeast cells. We took photographs with a

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DP30BW CCD camera with the shutter opening synchronised with the start of the exposure. We visualised the mitochondrial network using mitochondria-targeted GFP

(Westermann and Neupert, 2000) or mitochondrial isocitrate dehydrogenase fused with GFP (Karavaeva et al., 2017). Importantly, we automatically contrasted the photographs with IDH1GFP-expressing yeast, thus ensuring that the figures with Idh1-GFP fluorescence provided within this manuscript illustrate only the mitochondrial network structure and cannot be used to discuss accumulation levels of Idh1-GFP.

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To visualise mitochondrial DNA, we fixed yeast cells with 70% ethanol and stained them with DAPI (30 min, 0.1 µg/ml). We visualised mitochondrial potential with TMRE (10 min, 200 nM). After 50 min of incubation with the inhibitors (or the solvent), we stained the yeast cells with TMRE.

Isolation of mitochondria, respirometry We isolated mitochondria from the control (W303-1A) and PGAL-AAC2 strains using the protocol described in (Bazhenova et al., 1998). Briefly, we first acquired yeast protoplasts by

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digesting the cells with zymolyase (2.5 mg/g of yeast wet weight) preincubated with dithiothreitol (DTT, 10 mg/g of yeast dry weight). We then transferred protoplasts to the hypotonic medium (mannitol, 0.3 M; EDTA, 1 mM; Tris-HCl, 1 mM; BSA, 4 mg/ml, pH = 7.2)

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to remove the plasma membrane. We isolated the mitochondrial fraction by differential centrifugation. We then measured the protein concentration with a Pierce™ BCA Protein

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Assay Kit (Thermo Fisher, cat. Number 23225) according to the manufacturer’s instructions and assessed the respiration of isolated mitochondria with Clark-type oxygen electrode

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(Strathkelvin Instruments 782, United Kingdom) at 25°C. The incubation medium was 0.6 M mannitol, 10 mM Tris-HCl, 2 mM potassium phosphate (pH 7.4) and 15 mM pyruvate-malate

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(4:1)

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Transmembrane potential quantification We photographed cells expressing Idh1-GFP and stained with TMRE (200 nM) using

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first U-MNG2 and then U-MNIBA3 filters (wavelengths described above). We photographed the cells and then measured the intensity of the TMRE signal using ImageJ software. We selected five random circles (d = 5 px) in mitochondrial areas according to fluorescence in the GFP-channel. We then measured the average TMRE signal intensity in these selections for each cell and subtracted the background signal intensity.

Analysis of mitochondrial morphology

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To assess mitochondrial morphology, we used yeast cells grown overnight in YPraffinose or YP-glucose (indicated in the figure legends). We inoculated yeast cells to ensure that at the beginning of the experiments cell density was in the range of 4 × 10 6–8 × 106 cells/ml, which corresponds to the exponential phase of growth. To test the effects of mitochondrial inhibitors and uncouplers, we added the designated concentrations of inhibitors or solvent as controls. We incubated the cells for 60 min and took photographs with an Olympus BX51 microscope (see above in Fluorescent microscopy section). We selected a concentration of inhibitors that was sufficient to prevent growth on a non-fermentable carbon

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source (Figure S1). Nonetheless, given that changes in mitochondrial morphology could occur with time, we limited the time interval for microscopical analysis to 60 min and randomised the sequence of probes to prevent contribution of the time factor. As a result, the cells were

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exposed to inhibitors for a maximum of 2 h.

We assigned each photograph of an individual cell expressing Idh1-GFP or

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mitochondria-targeted GFP to one of three mitochondrial morphology classes: fragmented, normal or elongated [see sample photographs in (Azbarova et al., 2017)]. Importantly, in each

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case the experimenter who assigned each cell photograph did not know from which sample that cell had come. We pooled the photographs from several separate day experiments for

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the analysis, however treated pools for each figure separately. Thus it is possible to compare the results within a single figure but not between different figures. These results allowed us to

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generate stack bars illustrating the proportion of cells with different types of mitochondrial

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network structure.

Transmission Electron Microscopy For transmission electron microscopy we pelleted the cells and removed the culture

medium and fixed the pellets with 2.5% glutaraldehyde in 100 mM sodium cacodylate for 1 h at +4°C. After thorough washing in 100 mM sodium cacodylate, we placed the cells into spheroplasting medium containing 1 M sorbitol, 1 mM calcium chloride, 10 mM DTT and 100 mM sodium cacodylate. We incubated the cells for 10 min at 37°C under continuous agitation

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before adding 10 units of lyticase for the next 45 min. We removed the spheroplasting medium and replaced it with the solution containing 1% of osmium tetroxide and 1% of potassium ferrocyanide for 30 min at +4° C. We replaced the osmium tetroxide solution with the series of ethanol solutions with increasing concentrations. At this stage we additionally contrasted the samples with 2% uranyl acetate in 70% ethanol overnight at +4°C. We transferred the cells from 96% ethanol into acetone, following this by infiltration of the mixtures of acetone and epoxy resin with increasing concentrations of the resin. Finally, we infiltrated the cells with freshly made pure resin (Spi-

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pon 812), which we polymerised at +70°C for 72 h.

We sectioned the resin blocks using an Ultracut E ultramicrotome (Reichert-Jung, Austria) equipped with a diamond knife. We mounted sections with a nominal thickness of 80

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nm on formvar-coated copper slot-grids and post-stained them with 2% aqueous uranyl acetate for 40 min and with lead citrate for 3 min, according to (Venable and Coggeshall,

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1965).

We examined and photographed the samples using a JEM-1400 electron microscope

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(Jeol, Japan) running at 80 KV and equipped with a Quemesa digital camera (OSIS,

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Germany).

Mitochondrial fusion assay

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To study mitochondrial fusion, we generated yeast zygotes. We grew the cells of a tested strain (MATa) and Rho0 Idh1-GFP (MATα) cells overnight in YPD medium to 4×106

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cells/ml. We then mixed the strains in equal proportions and left them at room temperature for yeast mating and zygote formation. After 6 h we added DAPI to a final concentration of 20 µg/ml and left the mixture at room temperature for 10–15 min. Then we calculated the percentages of zygotes (that had already formed the first bud to ensure that mating had occurred) with non-fused mitochondrial networks using fluorescent microscopy. We scored the fusion of mitochondrial networks of the parental cells in the zygotes as positive in cases of full colocalization of Idh1-GFP and DAPI signals.

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In the second approach, we grew strains containing pCM188-mitoRFP (MATa) overnight in YNBRaf -Ura medium up to 4×106 cells/ml. Tested strains with Idh1-GFP (MATα) were grown overnight in a rich raffinose medium to the same density. Than we centrifuged cells, dissolved them in YPD medium, mixed the strains in equal proportions and left them at room temperature to mate and form zygotes. We added cycloheximide after 5 h of incubation to a final concentration of 50 µg/ml to prevent synthesis of fluorescent protein in the formed zygotes. After 1 h of incubation with cycloheximide, we calculated the percentage of zygotes with non-fused mitochondrial networks using fluorescent microscopy. FCCP (or an equal

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volume of ethanol) was added to the mixture to a final concentration of 1 μM and the mixture was then left at room temperature for 6 h for zygote formation. Fusion of mitochondrial networks of the parental cells in zygotes was scored as positive in cases of full colocalization

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of GFP and RFP signals.

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Suppressivity

In testing mitochondrial fusion, we also measured the displacement of the wild type

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mtDNA with Rho- mtDNA. To estimate the suppressivity — i.e. the ability of Rho- mtDNA to displace the wild type one — we followed the protocol described in (Karavaeva et al., 2017).

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In brief, we crossed MATα Rho- strains with a MATa Rho+ PGAL-AAC2 strain or with the wild type MATa Rho+ strain. The mating strains carried different genome-encoded selectable

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markers (URA3 and HIS3, respectively), allowing the selection for diploid cells. We grew cells in a rich medium to early exponential phase and mixed them in equal proportions to a final

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density of 2 × 105 cells/ml. After a 20 h incubation at room temperature, we diluted the mixtures 1000 times with sterile distilled water and plated them on solid double-selective medium YNB -Ura -His. The medium contained 0.1% glucose and 2% glycerol in order to distinguish grande and petite colonies. After 3 days, we counted the numbers of colonies and determined the suppressivity as the percentage of petite descendants in the total diploid progeny.

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Results AAC2 repression phenotypes To decouple NTP/NDP ratios in the mitochondrial matrix and cytoplasm, we repressed the major ATP/ADP translocator, AAC2 (also known as PET9). We set a chromosomal copy of AAC2 under the control of a galactose-inducible PGAL promoter (see Tables S1,S2 and methods section). The PGAL-AAC2 strain was not able to grow on a non-fermentable carbon source (glycerol, Figure 1B). Yeast cells, upon the deletion of AAC2, cannot survive in the

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absence of functional mtDNA (Chen and Clark-Walker, 1999)—a phenotype usually referred to as ‘petite-negative’. To test whether the inability of the PGAL-AAC2 strain to utilize glycerol as a carbon source is associated with a nuclear-encoded gene, we demonstrated that the

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PGAL-AAC2 strain retains its mtDNA (Figure 1C). Moreover, crossing PGAL-AAC2 cells with Rho0 cells harbouring wild-type copies of the AAC2 gene restored their ability to grow on a

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non-fermentable carbon source (Figure 1B). This confirmed that the PGAL-AAC2 strain preserved a functional (wild-type) mtDNA genome.

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To prove that the PGAL promoter is functional in our strain we tagged its AAC2 with GFP. While we did not detect any GFP signal when the cells were grown in rich media

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supplemented with glucose or raffinose, with galactose we were able to induce accumulation of Aac2-GFP (Figure 1D). Finally, we isolated mitochondria from wild type and AAC2repressed cells. We found that the repression of AAC2 inhibited ADP-stimulated oxygen

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consumption (Figure 1E). At the same time, FCCP was able to increase its oxygen

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consumption rate by mitochondria isolated from AAC2-repressed cells (Figure 1E). The latter result proved that the respiratory chain in AAC2-repressed cells is inhibited by the transmembrane potential, and proton shuttling by the uncoupler removes this inhibition. This observation, in addition to the petite-negative status of the aac2 strain that has been known for a long time (Kovácová et al., 1968), confirmed that mitochondria of the PGAL-AAC2 strain still maintained their mitochondrial transmembrane potential.

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Next, using expressed and repressed AAC2, we assessed the mitochondrial transmembrane potential in intact yeast cells. We incubated raffinose-grown wild type and PGAL-AAC2 yeast cells with fluorescent dye TMRE. In accordance with basic bioenergetic principles, we found that in the wild type cells protonophore FCCP prevented the mitochondrial TMRE staining (Figure 2). Inhibition of respiratory chain with myxothiazol also prevented TMRE staining. Conversely, the ATP synthase inhibitor oligomycin A increased transmembrane potential. This indicates that ATP synthase synthesises rather than hydrolyses ATP when yeast grow on the poorly fermentable carbon source raffinose.

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Importantly, all three tested compounds were able to prevent yeast growth on glycerol, a nonfermentable carbon source (Figure S1). At the same time, we detected an increase in mitochondrial transmembrane potential in yeast cells with repressed AAC2 gene. Addition of

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oligomycin A did not cause further increase in TMRE staining of AAC2-repressed cells (Figure 2). Indeed, inhibition of ANT is expected to increase ATP concentration in the mitochondrial

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matrix of actively respiring coupled mitochondria. Increased ATP concentration, in turn, should inhibit ATP-synthesis by the excess of the product. For this reason oligomycin A did not

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provide additional increase in ΔΨ upon the repression of AAC2. We obtained similar results with OxPhos inhibitors treated yeast cells while using another fluorescent dye, namely

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Rhodamine 6G (R6G) (Figure S2). Notably, R6G signal did not significantly change upon the AAC2 repression, while showing an increase in variance (Figure S2). However, it has been

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shown earlier that R6G can inhibit respiratory chain at the level of ANTs (Gear, 1974). We suggest that in our experiments with R6G, the dye indeed partially inhibited AAC2 and masked

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the effect of AAC2 repression on ΔΨ level.

Mitochondrial dynamics in cells with inhibited adenine nucleotide translocation To test whether inhibition of adenine nucleotide translocation affects mitochondrial

dynamics we analysed the mitochondrial network structure in both wild type cells and cells with repressed AAC2. We visualised mitochondria using a mitochondrial matrix protein isocitrate dehydrogenase Idh1 tagged with GFP. Yeast strains show high individual cell

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heterogeneity by their mitochondrial network structure (Figure 3A). This heterogeneity can have several causes—for instance, amplified stochastic process within the cells or replicative age-dependent heterogeneity (Azbarova et al., 2017; Knorre et al., 2018). In our further analysis we proposed that a change in mitochondrial fission or fusion frequencies should affect the proportion of cells with fragmented or hyperfused mitochondria. We found the repression of AAC2 to increase the fragmentation of the mitochondrial network (Figure 3B,C). Importantly, in some cases, we found that AAC2-repressed cells harboured aggregated mitochondria at one of the poles of the cells (Figure 3B). Such cells,

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during our blind mitochondrial network analysis, could be annotated as cells with hyperfused mitochondria. Indeed, conventional fluorescent microscopy does not distinguish the hyperfusion phenotype and the aggregation of tightly tethered small mitochondria. We

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therefore, using electron microscopy, analysed the ultrastructure of PGAL-AAC2 yeast cells grown in the absence of PGAL inducer. We found that—in contrast to the control Δdnm1 strain—

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PGAL-AAC2 contains aggregated non-connected mitochondria (Figures 4, S3, S4). Thus, although our semi-quantitative analysis detected an increase in mitochondrial fragmentation

the PGAL-AAC2 cells.

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(Figure 3) it may possibly have underestimated the proportion of fragmented mitochondria in

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Protonophore FCCP decreased mitochondrial connectivity in wild type cells (Figure 3B,C), although this effect appeared to be less marked than that in cells of higher eukaryotes.

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We did not detect changes in PGAL-AAC2 cells treated with FCCP (Figure 3B,C). The effect of the protonophores on the mitochondrial network morphology was well pronounced when the

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cells were grown on the poorly fermentable carbon source, raffinose, but not in glucose (Figure 3D,E). These results indicate that, when glycolysis is a major source of ATP in the cell, decoupling of the nucleotide pools in the cytosol and in the matrix does not affect the mitochondrial morphology. In raffinose-grown yeast, however, oxidative phosphorylation does also contribute to ATP production (see discussion above and Figure 2). As a result, both protonophore-induced mitochondrial depolarisation and the inhibition of adenine translocation

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decrease the phosphoryl potential in the cytosol and might in this way affect mitochondrial dynamics.

AAC2 is dispensable for mitochondrial fusion Fragmentation of the mitochondrial network can be either a result of inhibited mitochondrial fusion or an increased frequency of mitochondrial fission. We tested the ability of mitochondria from AAC2-repressed cells to fuse by a mitochondria complementation assay (Hermann et al., 1998). We used two modifications of the protocol: (1) To test the ability of

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AAC2-deficient mitochondria to fuse with AAC2-proficient (wild type) mitochondria we crossed PGAL-AAC2 MATa cells with Rho0 MATα ones expressing Idh1-GFP. In zygotes with medial buds, we analysed the degrees of DAPI and GFP signal overlays. As mitochondrial DNA came

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only from petite-negative PGAL-AAC2 cells and Rho0 cells were the only source of Idh1-GFP (Figure 5A), we used the degree of DAPI and Idh1-GFP signals overlay as a measure for the

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fusion efficiency. We used Rho+ X Rho0 IDH1-GFP and Rho- X Rho0 IDH1-GFP crossings as a control for this experiment. (2) To test the ability of AAC2-deficient mitochondria to fuse

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with each other, we transformed PGAL-AAC2 cells with mitochondria-targeted RFP. Crossing of PGAL-AAC2 Idh1-GFP mat α X PGAL-AAC2 MATa cells mainly produced zygotes with

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intermixed mitochondrial networks (Figure 5B). Surprisingly, both approaches showed that AAC2 repression did not prevent mitochondrial fusion (Figure 5A-D). Addition of FCCP did not

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prevent mitochondrial fusion in zygotes, however, however we found that combination of AAC2 repression and uncoupling of mitochondria did partially inhibit the intercomplementation

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of mitochondrial matrix (Figure 5D). Next, we reasoned that the inability of mitochondria to fuse with each other should in turn prevent the clonal expansion of mitochondrial DNA (see the discussion in (Karavaeva et al., 2017)). To test this, we crossed three different Rho- MATα strains with a PGAL-AAC2 strain and calculated the percentage of petite diploids which were formed as a result of these crossings. Meanwhile, in accordance with the results of PGAL-AAC2 X Rho0 crossings, we observed that the inhibition of ANT did not abolish the ability of RhomtDNA to displace Rho+ mtDNA from PGAL-AAC2 mitochondria (Figure 5E).

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Given that we did not detect an effect on mitochondrial fusion, we suggested that repression of AAC2 promotes mitochondrial fission. To test this hypothesis, we produced a double Δdnm1 PGAL-AAC2 IDH1-GFP mutant strain (see Table S1); however, this strain had an obvious defect in mitochondrial import. In most cells, mitochondrially targeted protein Idh1GFP had pronounced cytosolic, but not mitochondrial, localization (Figure S5). Nevertheless, some cells showed clusters of fragmented mitochondria or hyperfused mitochondria (Figure S5). Given the high heterogeneity of the observed phenotypes and the defects in mitochondrial import in the majority of the cells we did not draw conclusions from the data obtained with this

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strain. Thus, we measured the steady-state mitochondrial morphology in yeast cells treated with an inhibitor of Dnm1, namely mdivi-1 (Cassidy-Stone et al., 2008). It has recently been shown that mdivi-1 is a poor inhibitor of Drp1, a mammalian Dnm1 homologue. Moreover,

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mdivi-1 has an off-target effect on respiratory chain complex I (Bordt et al., 2017). However, the same study showed that mdivi-1 inhibits GTP hydrolysis by yeast protein. Thus, given that

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bakers’ yeast has no proton-translocating respiratory chain complex I, mdivi-1 still can be applied in yeast as a Dnm1 inhibitor. We found that mdivi-1 induces mitochondrial hyperfusion

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in both the control and the PGAL-AAC2 cells (Figure 6). At the same time, supplementation of FCCP partially abrogated the effect of Mdivi-1. The result of mdivi-1 treatment, in agreement

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with the experiments on zygotes, demonstrated the inhibition of adenine nucleotide translocation to be dispensable for mitochondrial fusion in yeast.

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Protonophore effects on mitochondrial dynamics in yeast with inhibited ATP synthase

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& respiratory chain

Inhibition of adenine nucleotide translocation disrupts the coupling between the

membrane potential and the NTP/NDP ratio in the cytosol (see Figure S6), at the same time inducing mitochondrial fragmentation (Figures 3). The repression of AAC2 did not show the addition of the effect on mitochondrial structure with acute FCCP treatment (Figure 3), implying that, in the absence of the coupling between the NTP/NDP ratio and the potential, the dissipation of the transmembrane potential does not affect mitochondrial dynamics. Inhibition

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of the mitochondrial ATP synthase can also disrupt the coupling of the transmembrane potential and the NTP/NDP ratio, thus also leading to a similar phenotype. To test this, we treated a wild type yeast strain with the ATP synthase inhibitor oligomycin A. We found that oligomycin, similar to AAC2 repression, both induced mitochondrial fragmentation and prevented any further effects of the protonophore FCCP (Figure 7A). Conversely, an inhibitor of the respiratory chain myxothiazol produced a much weaker effect on the mitochondrial network structure (Figure 7A) and did not prevent protonophore-induced fragmentation. The lack of an effect after adding myxothiazol may be explained by its failure to decouple the

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NTP/NDP ratio and the potential.

Finally, we tested the effect of mitochondrial protonophores on yeast Rho0 cells. Rho0 cells, as mitochondrial DNA encodes respiratory chain and ATP synthase subunits, are

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incapable of both respiration and ATP-synthesis. Surprisingly, Rho0 cells harboured net-like mitochondria and underwent protonophore-induced fragmentation (Figure 7B). Our protocol

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of generation of Rho0 cells included the use of ethidium bromide treatment, which could induce mutations in the nuclear genome. Thus, we used a Rho- yeast strain with a

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spontaneous deletion in mtDNA in the same assay and (Figure 7B) demonstrated that it displayed the same phenotype.

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From the purely bioenergetic point of view, Rho0 and Rho- cells appear to be identical to wild type cells treated with a mixture of oligomycin and myxothiazol—i.e. they lack both

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respiratory and ATP synthase activity. The mitochondrial morphology, however, is different in the two cases. We speculate that the difference could be explained by the time duration of the

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mitochondrial abnormalities. While we assayed the effect of oligomycin for the maximum of 2 h after its addition, Rho0/Rho- cells grow for many generations after the loss of functional mtDNA. It is possible that they may acquire some genetic or epigenetic suppressors of mitochondrial fragmentation during this growth period.

Discussion

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The yeast Saccharomyces cerevisiae is a well-established model for studying mitochondrial dynamics; the primary components of yeast fusion and fission machinery are homologous to those of mammalian proteins (Westermann, 2010a, 2010b). In contrast to mammalian cells, however, baker’s yeast can tolerate mitochondrial dysfunction and proliferate without OxPhos (Contamine and Picard, 2000). Nevertheless, the inhibition of mitochondrial energetics in yeast still affects its mitochondrial dynamics. Deletion or mutation of mitochondrial genes has been shown to inhibit mitochondrial fusion (Sauvanet et al., 2012). NaN3, an inhibitor of cytochrome oxidase and ATP synthase, has also been demonstrated to

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induce mitochondrial fragmentation in yeast (Klecker et al., 2015). Protonophore FCCP prevented the fusion of yeast mitochondria in vitro (Meeusen et al., 2004) but in yeast zygotes did not prevent the fusion of mitochondria (Karavaeva et al., 2017).

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Here, we assessed the roles of mitochondrial membrane potential in the yeast mitochondrial network structure. Mitochondrial ΔΨ is the factor that drives mitochondrial

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fusion, in this way ensuring segregation of the dysfunctional mitochondria with low ΔΨ from the network (Twig et al., 2008b). However, we showed that, as well as the addition of

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protonophore FCCP, the chronic inhibition of nucleotide translocation or acute inhibition of ATP synthase with oligomycin induced mitochondrial fragmentation. Notably, the inhibition of

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ATP synthase and the repression of adenine nucleotide translocator AAC2 rather than decreasing ΔΨ, on the contrary, elevated it.

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At the same time, the combination of AAC2 repression and the dissipation of the membrane potential did not show any additive effect on the steady-state fragmentation of the

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mitochondrial network (Figure 3). Possibly, each interference produced the maximal possible effect, and the mitochondrial network could therefore not fragment any further due to physical constraints of the membrane curvature. However, some conditions (such as high ethanol concentration or amiodarone treatment) may induce far more pronounced mitochondrial fragmentation in yeast (Kitagaki et al., 2007; Pozniakovsky et al., 2005). In our experiments, we noted that 82% of the wild type yeast cells treated with 15% (v/v) ethanol (n = 159) had a fragmented mitochondrial network (not shown in figures). We thus speculate that

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mitochondrial network fragmentation in AAC2-repressed and FCCP-treated yeasts is primarily the result of the mild changes in mitochondrial fusion and fission frequencies rather than that of non-specific network rupture. On the other hand, it should be noted that AAC2 repression enhanced rather than diminished the effect of FCCP in mitochondrial complementation assay in the zygotes (Figure 5D). This discrepancy between the steady state morphology analysis and mitochondrial complementation assays is consistent with the idea that mitochondrial fusion and fission mechanisms have different dependencies on cellular bioenergetic factors. Given that the repression of AAC2 and inhibition of ATP synthase increase ΔΨ (Figure

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2) but induce mitochondrial fragmentation (Figures 4,7) we propose that in yeast mitochondrial membrane potential is not a decisive factor in the regulation of mitochondrial dynamics. Moreover, myxothiazol which inhibits respiratory chain and decreases mitochondrial

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transmembrane potential (Figure 2) did not induce mitochondrial network fission in yeast grown in poorly fermentable carbon source (Figure 7). It should also be mentioned that

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ATP/ADP exchange is electrogenic and ANT activity is finely regulated by the transmembrane potential (Klingenberg, 1980). Moreover, the direct activity of ANT (ATP export from

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mitochondria/ADP import) can be significantly inhibited or even reversed by a moderate decrease of mitochondrial ΔΨ from 180 mV to approximately 100 mV (Chinopoulos et al.,

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2009; Metelkin et al., 2009). This makes it difficult to distinguish the direct effect of mitochondrial transmembrane potential on mitochondrial dynamics from the indirect one

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mediated by ANT activity and NTP levels in different compartments. The primary goal in this study was to separate the direct and indirect effects of

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transmembrane potential on mitochondrial dynamics by studying the effect of uncoupling in yeast cells with inhibited ANT. Our hypothesis was that the effect of uncouplers on mitochondrial dynamics is to a certain extent mediated by ANT carriers. If true, the inhibition of ANT would abolish the effect of the uncouplers in yeast cells. However, it appeared that the inhibition of ANT by itself significantly affects mitochondrial dynamics, most likely by facilitating mitochondrial fission. Thus, we are not able neither to confirm nor to reject our original hypothesis. Nevertheless, the results of our survey showed that in many cases the connectivity

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of mitochondrial network is negatively related to mitochondrial transmembrane potential. Our observations exemplify that the relationship between the transmembrane potential and the mitochondrial dynamics is not straightforward and should be interpreted with caution. Other bioenergetic parameters of the cell should be always taken into consideration. At the same time, our data are consistent with the idea that the ATP/ADP ratio in the cytoplasm or intermembrane space determines the degree of mitochondrial fragmentation. Our results obtained on yeast cannot be directly applied to higher eukaryotes. Although the mitochondrial fusion proteins (for instance, Fzo1/Mfn1, Mgm1/OPA1) are homologues,

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there is a substantial difference in the regulation mechanisms (Wagener, 2016). While yeast MGM1 gene does not harbour any introns, for example, the OPA1 gene has a complex exonintron structure and produces at least eight alternative splice isoforms (Del Dotto et al., 2018).

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Despite such differences, previous studies have demonstrated that the inhibition of ATP synthase induces mitochondrial fragmentation in certain mammalian cell lines (Gautier et al.,

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2012; Pletjushkina et al., 2006). ANT activity may therefore also contribute to the shaping of mitochondrial network morphology in animal cells.

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Treatment of the W303 yeast strain, which is the parental strain of PGAL-AAC2 used in our study, with the membrane-permeable ANT inhibitor bongkrekic acid did not prevent yeast

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growth on a non-fermentable carbon source (Figure S7), whereas another well-known inhibitor, carboxy-atractilazide, is not membrane permeable. Thus, in our study, we assessed

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mitochondrial network structure during long-term repression of the AAC2 gene produced by genetic manipulation. However, in some cases, prolonged mitochondrial dysfunction can be

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manifested in unexpected ways. For example, it has been shown that prolonged inhibition of complex I in human embryonic kidney cells with rotenone inhibits respiration but unexpectedly increases transmembrane potential (Forkink et al., 2014). The increased transmembrane potential in this study was explained by reduced ATP synthase and forward ANT activities in the cells, both emerging as an indirect consequence of rotenone treatment. Moreover, a cell line with a mutation in the mitochondria-encoded subunit of ATP synthase ATP6 showed decreased transmembrane potential (Szczepanowska et al., 2004), whereas one would

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expect that inhibition of ATP synthase should increase ΔΨ. Meanwhile, recently it has been shown that inactivation or deletion of AAC2 disregulates mitochondrial translation and can disturb the proportion of mitochondrially encoded respiratory chain and ATP synthase subunits (Ogunbona et al., 2018). Therefore, the results obtained under chronic repression of ANT should be considered with caution, although in our experiments with yeasts the acute effects of oligomycin and chronic repression of ANT were in agreement with each other. To summarise, we have shown that yeast cells harbour mitochondria with network structures that are highly variable. However, the degree of fragmentation of individual

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mitochondrial filaments appeared to be uncorrelated with the transmembrane potential. For instance, an oligomycin-induced increase in ΔΨ facilitated both fragmentation of the mitochondrial network and protonophore-induced mitochondrial depolarisation (Figures

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2,3,4,7). Some other interventions known to decrease ΔΨ—such as inhibition of the respiratory chain with myxothiazol or depletion of mitochondrial DNA—did not induce drastic

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changes in mitochondrial morphology. At the same time, we found that the repression of the major nucleotide translocator which presumably decoupled the mitochondrial and cytosolic

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pools of NTPs did not significantly change ΔΨ while it did, however, induce fragmentation. Therefore, the balance of mitochondrial fusion and fission—at least in our experimental

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model—seems to be dependent on nucleotide triphosphate availability rather than on mitochondrial ΔΨ. Our results show that a mitochondrial inner membrane transporter can link

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the functional state to the spatial organisation of the mitochondrial network.

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Conflict of interest statement Nothing to declare.

Acknowledgements We are very grateful to the reviewers for their valuable comments, we have used some of their reasoning in the discussion of our data. The study was supported by RFBR grants 16-04-

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01381-А and 19-04-00782-A. Part of this study was performed using the equipment obtained under MSU development program PNR 5.13. This work was also supported by Moscow State University Grant for Leading Scientific Schools «Depository of the Living Systems» in frame

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of the MSU Development Program.

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Figure legends

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Figure 1. AAC2 repression inhibits ANT activity in yeast cells. (A) Scheme of AAC2 promoter replacement with PGAL promoter followed by C-terminus GFP tagging. HIS+ and

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KanMX are marker genes. (B) Verification of the petite-negative status of the PGAL-AAC2 phenotype. We crossed IDH1-GFP PGAL-AAC2 cells with W303-1A Rho0 cells to produce

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heterozygous diploids (left sector). We transferred the diploid and parental haploid cells to the solid YPGly medium. The ability of the diploids cells to grow on a non-fermentable carbon

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source indicated that IDH1-GFP PGAL-AAC2 strains preserved full-length mtDNA. (C) DAPI staining of PGAL-AAC2 revealed the presence of mtDNA under the conditions of PGAL-AAC2

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repression. We used Rho0 and Rho+ cells as negative and positive controls. (D) Localisation and the accumulation levels of Aac2-GFP in PGAL-AAC2-GFP yeast cells under the conditions of PGAL repression (dextrose—D, raffinose—Raf) and expression (raffinose with galactose— RafGal). (E) Oxygen consumption rates by mitochondria isolated from PGAL-AAC2 and WT (W303-1A) yeast cells. Additions were as follows: CATR (1 µM Carboxyatractyloside), ADP (150 µM), FCCP (200 nM). Numbers near the curves are nmol O2/min × mg protein.

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Figure 2. AAC2 repression increases mitochondrial membrane potential. (A) A representative photograph of yeast cells with mitochondria expressing Idh1-GFP. To assess

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mitochondrial membrane potential we treated yeast cells with TMRE (200 nM). Mitochondrial inhibitors (5 µM FCCP; 5 µg/ml oligomycin or 7 µM myxothiazol) affect the levels of the

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mitochondrial membrane potential. (B) Quantification of the results. We conducted the experiments on a rich medium with a poorly fermentable carbon source raffinose. ** P < 10-4

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for comparisons with mock treated control strain, unpaired Mann–Whitney test with Bonferroni adjustment. # #P < 10-6 for comparisons with mock treated PGAL-AAC2 strain, unpaired Mann– Whitney test with Bonferroni adjustment.

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Figure 3. The repression of AAC2 causes mitochondrial fragmentation. (A) Wild type yeast cells showing significant heterogeneity in mitochondrial network structure in control

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conditions; (B) Protonophore FCCP (5 µM) increases fragmentation in mitochondrial network of yeast in raffinose (C) Quantification for B. (D) Protonophore FCCP (5 µM) did not affect

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mitochondrial morphology in yeast cells grown on glucose; (E) Quantification for D. P-values of Pearson's Chi-squared test with Yates' continuity correction are indicated when significant.

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Figure 4. Ultrastructure of yeast cell with repressed AAC2.

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Ultrastructure of yeast cell with repressed PGAL-AAC2 gene (left) grown in YPRaf. Δdnm1 cell

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(right) is shown as an illustration of increased mitochondrial fusion. Scale bar — 1 µm

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Figure 5. Repression of AAC2 does not prevent the fusion of mitochondria in zygotes. (A) Representative photograph of zygotes produced by the fusion of PGAL-AAC2 and Rho0

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cells. Lower panel: the fusion results in intermixing of Idh1-GFP (a mitochondrial marker of the

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Rho0 cells) and DAPI (mtDNA intercalating agent, a marker of PGAL-AAC2 cells). (B) Representative photograph of zygotes produced by fusion of PGAL-AAC2 IDH1-GFP and PGAL-

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AAC2 mitoRFP Rho+ cells. Lower panel: The fusion results in intermixing of Idh1-GFP and mitoRFP. Green and red are pseudo-colours. (C) and (D) Quantification of the results

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indicating the number of analysed zygotes with fused and unfused mitochondria for (A) and (B), correspondingly. FCCP was added to a final concentration equal to 1 µM. P-values of Pearson's Chi-squared test with Yates' continuity correction and Bonferroni adjustment are

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indicated when significant. (E) AAC2 repression does not prevent the displacement of Rho+

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mtDNA with Rho- and hypersupressive (HS) Rho- mtDNA in the diploids.

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Figure 6. Mdivi-1 induces mitochondrial fusion in the AAC2-repressed strain. (A)

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Representative microphotographs of yeast mitochondria expressing Idh1-GFP in the WT and PGAL-AAC2 strains. We treated yeast cells with a solvent (0.5% v/v ethanol + 0,6% DMSO,

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Control); mdivi-1 (60 µM) and mdivi-1 + FCCP (5 µM). We grew yeast cells in YPRaf medium; the incubation time with mdivi-1 was 1 h. (B) Quantification of the yeast cells with various mitochondrial network morphologies. P-values of Pearson's Chi-squared test with Yates' continuity correction are indicated when significant.

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Figure 7. Protonophore-induced changes in mitochondrial network morphology in yeast cells with dysfunctional mitochondria. (A) Effect of protonophore FCCP (5 µM) on mitochondrial morphology in yeast treated with ATP synthase inhibitor oligomycin (5 µg/ml) or

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respiration inhibitor myxothiazol (7 µM). We grew yeast cells in YPRaf medium; incubation time with inhibitors was 1 h. We visualised the mitochondrial network using Idh1-GFP.

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Representative figures and a stacked barplot for quantification. * corresponds to P value < 10according to Pearson's Chi-squared test with Yates' continuity correction (B) Effect of FCCP

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(5 µM) on yeast cells with (Rho+) or without (Rho- and Rho0) functional mtDNA. We grew yeast cells in YPRafGal medium; incubation time with inhibitors was 1 h. We visualised the

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mitochondrial network using mitoGFP. Representative figures and a stacked barplot for quantification. The numbers at the right side of the stacked barplot indicate the number of

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analysed individual cells.

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