We used the scaffold of the self-assembling Superfolder Split-GFP fragments designed by Cabantous and coworkers (Cabantous et al., 2005b; Pédelacq et al., 2006), where the 11 beta strands forming active Superfolder GFP are separated in a fragment encompassing the 10 first beta strands (GFP_β1-10) and a smaller one consisting of the remaining beta strand (GFP_β11). Seven amino acid (aa) residues of GFP_β1-10 and three of GFP_β11 were replaced in order to increase the stability and the self-assembly of both fragments (Figure 1—figure supplement 1). To increase the fluorescent signal and facilitate observation of low-abundant proteins, we concatenated and fused three β11 strands (GFP_{β11-chaplet; β11ch}) linked by GTGGGSGGGSTS spacers (see Materials and methods for DNA sequence, Figure 1—figure supplement 1, as in Kamiyama et al., 2016; Figure 1A).

Our objective was to integrate the gene encoding the GFP_β1-10 fragment into the mtDNA so that it will only be translated inside the mitochondrial matrix, while the GFP_β11ch fragment is fused to the nuclear-encoded protein of interest and thus translated by cytosolic ribosomes (Figure 1A). To achieve this, we constructed a strain (RKY112) in which the coding sequence of the ATP6 gene has been replaced by ARG8m (atp6::ARG8m), and where ATP6 is integrated at the mitochondrial COX2 locus under the control of the 5’ and 3’ UTRs of COX2 gene (Supplementary file 1; Table 1; Figure 1—figure supplement 2A–C; see Materials and methods section for details). The RKY112 strain grew well on respiratory carbon source as wild type yeast (MR6) (Figure 1B), produced ATP effectively (Figure 1C), and expressed normally Atp6 and all the other mitochondria-encoded proteins (Figure 1D). We next integrated at the atp6::ARG8m locus of RKY112 strain mtDNA, the sequence encoding GFP_β1-10 (Figure 1A; Figure 1—figure supplement 2). To this end, we first introduced into the ρ⁰ mitochondria (i.e. totally lacking mtDNA) of DFS160 strain, a plasmid carrying the GFP_β1-10 sequence flanked by 5’ and 3’ UTR sequences of the native ATP6 locus (pRK67, see Materials and methods for DNA sequence), yielding the RKY172 strain (bearing a non-functional synthetic ρ^-S mtDNA, Figure 1—figure supplement 2C). This strain was crossed to RKY112 to enable replacement of ARG8m with GFP_β1-10. The desired recombinant clones, called RKY176, were identified by virtue of their incapacity to grow in media lacking arginine due to the loss of ARG8m and their capacity to grow in respiratory media (Figure 1B). Integration of GFP_β1-10 in mtDNA was confirmed by PCR (Figure 1—figure supplement 2E, Supplementary file 2) and Western blot with anti-GFP antibodies (Figure 2C). Finally, the BiG Mito-Split-GFP strain (Table 1) was obtained by restoring the nuclear ADE2 locus in order to eliminate interfering fluorescence emission of the vacuole due to accumulation of a pink adenine precursor (Fisher, 1969; Kim et al., 2002).

The BiG Mito-Split-GFP system was first tested with Pam16 which localizes in the matrix at the periphery of the mitochondrial inner membrane and Atp4, an integral membrane protein with domains exposed to the matrix (Kozany et al., 2004; Velours et al., 1988; Figure 2A). The BiG Mito-Split-GFP host strain was transformed with centromeric plasmids expressing either Pam16_β11ch or Atp4_β11ch bearing the GFP_β11ch tag at their C-terminus under the constitutive GPD promoter. Expression of Pam16_β11ch and Atp4_β11ch resulted in strong GFP signal emissions that colocalized with MitoTracker Red CMXRos-stained mitochondria, whereas no fluorescence was detected with the corresponding empty plasmid (Figure 2B; Figure 2—figure supplement 1A). These observations confirmed that the GFP_β1-10 polypeptide is well expressed from the mtDNA, stably and correctly folded, allowing reconstitution of an active GFP upon association with the mitochondrial GFP_β11ch-tagged protein. So far, the positive controls we used for the proof of concept of the BiG Mito-Split-GFP approach are proteins more or less abundant: Atp4 (30000–40000 copies/cell) and Pam16 (3000 copies/cell) (Morgenstern et al., 2017; Vögtle et al., 2017). We will report soon, in BioRxiv, tests with other proteins with a known mitochondrial location and varying abundance to better estimate the sensitivity of the BiG Mito-Split-GFP system, including the GatF subunit of the GatFAB tRNA-dependent amidotransferase chromosomally expressed from its own promoter. This is a mitochondrial protein that has been reported to be present at only 40–80 copies (Vögtle et al., 2017).

We next tested the BiG Mito-Split-GFP system with a GFP_β11ch-tagged version of Pgk1, which is commonly used as negative cytosolic marker protein to probe the purity of mitochondrial preparations. Pgk1_β11ch and endogenous Pgk1 were well detected by Western blot of total protein extracts probed with anti-Pgk1 antibodies (Figure 2C). No GFP fluorescence was observed with Pgk1_β11ch (Figure 2B; Figure 2—figure supplement 1A) despite its good expression (Figure 2C). This is an interesting observation considering that Pgk1 localizes at the external surface of mitochondria (Cobine et al., 2004; Kritsiligkou et al., 2017; Levchenko et al., 2016). This provides the proof that the BiG Mito-Split-GFP system does not yield any unspecific fluorescence with cytosolic proteins even when they are externally associated to the organelle (see also Source data 4). Another negative control (His3) that further confirms the absence of false positive signal will be provided soon in BioRxiv. In conclusion, these data show that any GFP_β11ch-tagged protein that localizes inside the mitochondrial matrix or at matrix side periphery of the inner membrane triggers GFP reconstitution and fluorescence emission, making this emission a robust in vivo readout for the mitochondrial importability of proteins of nuclear genetic origin.

We next tested whether the BiG Mito-Split-GFP system also allows visualization of the mitochondrial echoform of a protein located in both the cytosol and the organelle. We chose the cytosolic glutamyl-tRNA synthetase (cERS) encoded by the GUS1 gene as a proof of concept. As we have shown, cERS is an essential and abundant protein of the cytosolic translation machinery, and a small fraction (15%) is located in mitochondria where it is required for mitochondrial protein synthesis and ATP synthase biogenesis (Frechin et al., 2009; Frechin et al., 2014). After transformation of the BiG Mito-Split-GFP strain with plasmids expressing a GFP_β11ch-tagged version of cERS under the control of either the GPD promoter (pGPD) or its own promoter (pGUS1), a GFP signal was observed only in mitochondria (Figure 2B; Figure 2—figure supplement 1A). We also generated a stable BiG Mito-Split-GFP strain in which the gene encoding cERS_β11ch was chromosomally expressed under the dependence of its own promoter at the TRP1 locus (Supplementary file 3, Figure 2—figure supplement 1B). Again, GFP fluorescence was strictly confined to mitochondria (Figure 2B, Figure 2—figure supplement 1A). These observations demonstrate that the BiG Mito-Split-GFP system enables a specific detection in vivo of the mitochondrial pool of cERS (_mtecERS), without any interference by the cytosolic echoform, which is not possible when cERS is tagged with regular GFP (Frechin et al., 2009). We also expressed Pam16_β11ch and Pgk1_β11ch under the dependence of the GPD promoter at the TRP1 locus. Again, as shown with the plasmid-borne strategy, Pam16_β11ch expression resulted in a specific mitochondrial fluorescence, while Pgk1_β11ch gave no fluorescence (Figure 2—figure supplement 1B).

Using high-resolution Airyscan confocal microscopy, a typical 3D mitochondrial network was reconstituted from the fluorescence induced by the expression of Pam16_β11ch, Atp4_β11ch and cERS_β11ch in the BiG Mito-Split-GFP strain whereas, as expected, no fluorescent at all was detected with Pgk1_β11ch (Figure 2D), which further illustrates the mitochondrial detection specificity of this system. These data were corroborated by flow cytometry analyses of the BiG Mito-Split-GFP strain stably expressing Pam16_β11ch and Pgk1_β11ch (Figure 2E). These data will soon be completed (in BioRxiv) with flow cytometry experiments aiming to know if the BiG Mito-Split-GFP system could be used in systematic screens for proteins with a mitochondrial localization.

We next evaluated whether the BiG Mito-Split-GFP approach represents a significant technical advance compared to the existing MTS-based Split-GFP methods that are currently used. To this end, we constructed cells (with a wild type mitochondrial genome) that co-express in the cytosol the mitochondrial protein GatF (with its own MTS) fused at its C-terminus with GFP_β1-10 (mtGatF_β1-10) and either cERS_β11ch (dual localized, positive control) or Pgk1_β11ch (cytosolic, negative control) (Figure 2F, left panel). As expected, a strong and specific mitochondrial fluorescent signal was obtained with cERS_β11ch (Figure 2F, right panel). However, Pgk1_β11ch resulted in a mitochondrial signal of similar intensity. This is presumably due to the location at the external surface of mitochondria of a small fraction of the Pgk1 pool that could interact with mtGatF_β1-10 prior to its import into the organelle. These results show that due to the high affinity of both self-assembling Split-GFP fragments, the MTS-based strategy can generate a mitochondrial fluorescence without mitochondrial protein internalization (Figure 2F, right panel). These experiments suggest that compartment-restricted expression of the GFP_β1-10 fragment and GFP_β11ch-tagged proteins increases the reliability of identifying mitochondrial echoforms of dual-localized proteins.

Originally, screening cytosolic aminoacyl-tRNA synthetases (caaRSs) that can additionally relocate to mitochondria was motivated by several inconsistencies concerning this family of enzymes. The first and most documented example concerns cERS (Frechin et al., 2009; Frechin et al., 2014). We showed that the fraction of cERS which is imported (_mtecERS) into mitochondria is essential for the production of mitochondrial Gln-tRNA^Gln by the so-called transamidation pathway (Frechin et al., 2009; Frechin et al., 2014). In the latter, _mtecERS aminoacylates the mitochondrial tRNA^Gln with Glu thereby producing the Glu-tRNA^Gln that is then converted into Gln-tRNA^Gln by the GatFAB amidotransferase (AdT) (Frechin et al., 2009; Frechin et al., 2014). These results argued against the proposal that mitochondrial import of cQRS compensates for the absence of nuclear-encoded mtQRS in yeast (Rinehart et al., 2005). This being said, nothing excludes that cQRS can be imported into mitochondria to fulfill additional tasks beyond translation.

Another puzzling concern is the absence in S. cerevisiae of genes encoding six stricto-senso mtaaRSs: mtARS, mtCRS, mtGRS, mtHRS, mtQRS and mtVRS (Table 2). This suggests that the genes encoding their cytosolic equivalents (_cytecaaRS) might also encode their mitochondrial echoforms (_mtecaaRSs). This has been confirmed for cARS, cGRS1, cHRS, cVRS for which alternative translation/transcription initiation allows the expression of both echoforms (Figure 3D; Chang and Wang, 2004; Chatton et al., 1988; Chen et al., 2012; Natsoulis et al., 1986; Turner et al., 2000).

We therefore applied the BiG Mito-Split-GFP strategy to the S. cerevisiae caaRSs (See supplementary file 4), aiming to discover new mitochondrial echoforms of caaRSs. We successfully expressed in the BiG Mito-Split-GFP strain the full length GFP_β11ch-tagged versions of 18 out of 20 yeast caaRSs or _cyteaaRSs (Figure 3A–C; Figure 3—figure supplement 1, Supplementary files 3 and 4). For unknown reasons, we failed to obtain the full-length GFP_β11ch-tagged versions of cCRS and cPRS despite repeated attempts, but successfully cloned the first hundred N-terminal aa residues of cCRS (N₁₀₀cCRS) (Figure 3C). An unambiguous mitochondrial fluorescent signal was observed with cFRS2_β11ch (the α-subunit of the α₂β₂ cFRS), _cytecHRS_β11ch and N₁₀₀cCRS_β11ch (Figure 3A–C; Figure 3—figure supplement 1). Since the existence of a fully functional mtFRS has been demonstrated (Koerner et al., 1987), it is possible that supernumerary _mtecFRS2 we identified is not necessary for charging mitochondrial tRNA^Phe but exerts some non-canonical functions, in addition to its role in cytosolic protein synthesis. The mitochondrial fluorescence triggered by expression of N₁₀₀cCRS_β11ch suggests that this part of cCRS harbors a MTS, which has recently been proposed (Nishimura et al., 2019, see Discussion). The mitochondrial fluorescence triggered by _cytecHRS_β11ch is more intriguing. The most plausible hypothesis is that the MTS of the _mtecHRS is longer than the one originally characterized. The other possibility is that there is indeed a second mitochondrial echoform of cHRS imported inside mitochondria through a cryptic MTS that has yet to be identified and, like for cFRS2, this new _mtecHRS would then most probably exert a non-canonical function.

As already mentioned, cARS, cGRS1, cHRS and cVRS genes are known to produce both cytosolic and mitochondrial forms of these proteins (Figure 3D). When _mtecARS_β11ch, _mtecGRS1_β11ch, _mtecHRS_β11ch and _mtecVRS_β11ch (echoforms that start with the most upstream methionine initiator codon, Figure 3D) were expressed in the BiG Mito-Split-GFP strain, a mitochondrial GFP staining was, as expected, observed with these four _mtecaaRSs (Figure 3D). Conversely, _cytecARS_β11ch, _cytecGRS1_β11ch and _cytecVRS_β11ch, versions without their MTS) did not produce any detectable GFP signal confirming the MTS-dependency of these cytosolic echoforms for mitochondria localization (Figure 3D; Figure 3—figure supplement 1A). The mitochondrial fluorescence produced by _cytecHRS_β11ch has already been discussed above.

Unlike proteins with a MTS that is cleaved upon import into mitochondria, _mtecERS does not involve any processing (Frechin et al., 2009). Presumably, the mitochondrial targeting residues are located in the N-terminal (N-ter) region of cERS as in precursors of mitochondrial proteins destined to the matrix. To identify them, we tagged with GFP_β11ch three N-ter domains of cERS of varying length that correspond to the first 30 (cERS_β11ch-N1), 70 (cERS_β11ch-N2) and 200 (cERS_β11ch-N3) residues of cERS (Supplementary files 3 and 4; Figure 4A) and we tested their ability to be imported in the mitochondria of the BiG Mito-Split-GFP strain (Figure 4B). All three peptides produced a GFP fluorescence signal that matched the labeling of mitochondria with MitoTracker Red CMXRos (Figure 4B). Consistently, no GFP fluorescence was detected with cERS_β11ch lacking the residues 1–30 or 1–200 (cERS_β11ch-∆N1 and cERS_β11ch-∆N2 respectively) (Figure 4B) despite detection by WB of these truncated proteins in cells (Figure 4C). For unknown reasons, cERS_β11ch-N1 and cERS_β11ch-N2 constructs were not detected by Western blot but gave a proper mitochondrial fluorescence staining (Figure 4B and C). These data narrow down cERS’ MTS to the 30 first aa residues of its N-ter domain; this segment is made of a short β-strand and a 13 aa long α-chain (Simader et al., 2006) likely harboring the import signal. This further illustrates the strength of our technique towards the identification of unconventional MTSs in dual localized proteins.

The BiG Mito-Split-GFP system is based on modifications in the mitochondrial genome for expressing the GFP_β1-10 fragment inside the organelle. Modifying the mitochondrial genome is thus far only possible in S. cerevisiae and Chlamydomonas reinhardtii (Remacle et al., 2006). Owing to the high degree of conservation of mitochondrial protein import systems (Lithgow and Schneider, 2010), we used the yeast BiG Mito-Split-GFP strain to test the mitochondrial importability of proteins from various eukaryotic origins. We first tested two glutamyl-tRNA synthetases from Arabidopsis thaliana, AthcERS and Athmt/chlERS. According to independent MTS prediction tools, AthcERS would be a cytosolic protein with a putative chloroplastic targeting signal (TargetP1.1), whereas Athmt/chlERS is strongly predicted to be located in mitochondria and chloroplast (Figure 5A). cDNAs encoding the AthcERS and Athmt/chlERS proteins were fused to GFP_β11ch (Supplementary files 3 and 4) and the resulting plasmids were transformed into the BiG Mito-Split-GFP strain. Expression of these proteins was confirmed by Western blot (Figure 5C). AthcERS_β11ch did not produce any GFP signal, whereas consistent with its predicted localization Athmt/chlERS_β11ch resulted in a specific mitochondrial fluorescence staining (Figure 5B). These data show that the yeast BiG Mito-Split-GFP system can be used to analyze mitochondrial localization of plant proteins.

We also used the BiG Mito-Split-GFP system to address a yet-unresolved question regarding the presence of mammalian Argonaute protein 2 (Ago2) in mitochondria. This protein mainly localizes to the nucleoplasm and cell junctions where it is required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC) (Hammond et al., 2000). In some studies, Ago2 was suggested to be associated to mitochondria, but it remains unclear whether it localizes at the external surface or inside the organelle (Barrey et al., 2011; Shepherd et al., 2017). Using four different algorithms a potential MTS could not be predicted in Ago2 proteins from human, mouse, Bos taurus, Danio rerio andDrosophila melanogaster, casting doubts on the mitochondrial import of Ago2 (Figure 5D). To help resolve this question, the BiG Mito-Split-GFP yeast strain was transformed with plasmids expressing mouse and human Ago2_β11ch proteins (MmuAgo2_β11ch and HsaAgo2_β11ch, respectively, Supplementary files 3 and 4). Expression of each of these GFP_β11ch-tagged constructs was confirmed by WB, and both generated a solid and specific GFP fluorescence restricted to mitochondria (Figure 5E and F). These observations provide strong evidence that in addition to a cytosolic and nuclear location, Ago2 is also transported into mitochondria and is really a multi-localized protein with a mitochondrial echoform.

ATP6 gene flanked by 75 bp of 5`UTR and 118 bp of 3`UTR of COX2 was synthesized by Genescript and cloned at the EcoRI site of pPT24 plasmid bearing the sequence of COX2 gene along with its UTRs (Thorsness and Fox, 1993), giving pRK49-2. The GFP_β1-10 sequence (optimized for mitochondrial codon usage) encoding the first ten β-strands of GFP flanked by the regulatory sequences of ATP6 gene and BamHI/EcoRI sites was synthesized by Genescript. The BamHI-EcoRI DNA fragment was cloned into pPT24 plasmid, giving the pRK67-2. The sequences of inserts were verified by sequencing.

The GFP_β11ch coding sequence, synthesized by Genescript, was subcloned into the pAG414 pGPD-ccdB vector to generate the pAG414pGPD-ccdB_β11ch. All genes encoding cytosolic or mitochondrial proteins were amplified from genomic DNA using the PrimeSTAR Max polymerase according to the manufacturer instructions (Takara), purified by PCR clean up (Macherey-Nagel) and subcloned either by Gateway (Thermofisher) (Katzen, 2007) or Gibson assembly (NEB) (Gibson et al., 2010; Gibson et al., 2009) according to the manufacturer’s instructions (see Table S2).

The genotypes of strains used in this study are listed in Table 1. The ρ⁺ indicates the wild-type complete mtDNA (when followed by deletion/insertion mutation it means the complete mtDNA with a mutation). The ρ^- synthetic genome (ρ^-S) was obtained by biolistic introduction into mitochondria of ρ⁰ DFS160 strain (devoid of mitochondrial DNA) of the plasmids (pRK49-2 or pRK67-2) bearing indicated genes. The integration of ATP6 gene into the mtDNA under the control of regulatory sequences of COX2 was done using a previously described procedure (Steele et al., 1996). The pRK49-2 plasmid was introduced into mitochondria of DFS160 ρ⁰ strain by ballistic transformation using the Particle Delivery Systems PDS-1000/He (BIO-RAD) as described (Bonnefoy and Fox, 2001), giving the ρ^-S strain RKY89. For the integration of the ATP6 gene at the COX2 locus, we first constructed a ρ⁺ strain (RKY83, Fig. S2A) with a complete deletion of the coding sequence of ATP6 (atp6::ARG8m) and a partial deletion in COX2, cox2-62 (Table 1), by crossing YTMT2 (Matα derivative of strain NB40-3C carrying the cox2-62 mutation (Steele et al., 1996) and MR10 (atp6::ARG8m) (Rak et al., 2007). After crossing, cells were allowed to divide during 20–40 generations to allow mtDNA recombination and mitotic segregation of the double mutation. The double atp6::ARG8m cox2-62 mutant colonies were identified by crossing with the ρ^-S strain SDC30 (Duvezin-Caubet et al., 2003) that carries ATP6 and COX2 which restored the respiratory competence and by crossing with the YTMT2 strain, ρ⁺cox2-62, which did not restored the respiratory competence of the double mutant. Next, the ρ^-S strain RKY89 was crossed with strain RKY83. This cross resulted in the respiratory competent progenies, named RKY112, which were growing on minimal medium without arginine (Table 1, Figure 1B and S2B). The ectopic integration of the ATP6 gene into COX2 locus was verified by PCR using oligonucleotides oAtp6-2, oAtp6-4, o5`UTR2 and o5`UTR1 (Table S1, Fig. S2D). To integrate GFP_β1-10 into ATP6 locus the ρ^-S strain RKY172 was obtained by biolistic transformation of DFS160ρ⁰ with pRK67-2, as described above. RKY172 was crossed with RKY112, heterokaryons were allowed to divide during 20–40 generations to allow mtDNA recombination and mitotic segregation (Fig. S2C). The RKY176 progenies were selected by their respiratory competence and inability to grow on arginine depleted plates. The correct integration of the GFP_β1-10 gene into ATP6 locus was verified by PCR using oligonucleotides oAtp6-1, oAtp6-10, oXFP-pr and oXFP-lw (Table S1, Fig. S2E). Finally, ADE2 WT sequence was amplified from the genomic DNA of a BY strain using oligonucleotides ADE2 Fw and ADE2 Rv (Table S2) and transformed into the RKY176 strain. Red/white colonies were then screened on adenine depleted plates to select ADE2-bearing RK176 strain.

Yeast cell culture media and their composition: complete glucose YP medium (1% Bacto yeast extract, 1% Bacto peptone, 2% glucose, 40 mg/l adenine), complete YP Gal (1% Bacto yeast extract, 1% Bacto peptone, 2% galactose, 40 mg/l adenine), synthetic media composed of 0.67% (w/v) yeast nitrogen base without amino acids (aa), 0.5% (w/v) ammonium sulfate, either 2% (w/v) glucose (SC), galactose (SC Gal) or glycerol (SC Gly) and a mixture of aa and bases from Formedium (Norfolk, UK). Low sulfate medium LSM contained 0.67% (w/v) yeast nitrogen base without aa and ammonium sulphate, 2% galactose and 50 mg/L histidine, tryptophan, leucine, uracil, adenine, and arginine. The solid media contained 2% (w/v) of agar. Every strain was grown at 30°C with rotational shaking to mid-log (OD_{600 nm} = 0.7). SC Gal was filtered on 25 µm filters and not autoclaved before use.

Labeling of mitochondrial translation products was performed using the protocol described by Barrientos et al., 2002. Yeast cells were grown to early exponential phase (10⁷ cells/mL) in 10 mL of liquid YP Gal medium. Cells were harvested by centrifugation and washed twice with LSM medium then suspended in the same medium and incubated for cysteine and methionine starvation for 2 hr at 28°C with shaking. Cells were suspended in 500 µL of LSM medium, and 1 mM cycloheximide was added. After a 5 min incubation at 28°C, 0.5 mCi of [³⁵S]methionine and [³⁵S]cysteine (Amersham Biosciences) was added and cell suspension was further incubated for 20 min at 28°C. Total proteins were isolated by alkaline lysis and suspended in 50 µL of Laemmli buffer. Samples with the same level of incorporated radioactivity were separated by SDS-PAGE in 17.5% (w/v) acrylamide gels (to separate Atp8 and Atp9) or 12% (w/v) acrylamide containing 4 M urea and 25% (v/v) glycerol (to separate Atp6, Cox3, Cox2 and cytochrome b). After migration, the gels were dried and [³⁵S]-radiolabeled proteins were visualized by autoradiography with a PhosphorImager after a one-week exposure. To measure ATP synthase activities in the RKY112 strain, mitochondria were prepared by the enzymatic method as described in Guérin et al., 1979. For the rate of ATP synthesis, the mitochondria (0.15 mg/mL) were placed in a 1 mL thermostatically controlled chamber at 28°C in respiration buffer (0.65 M mannitol, 0.36 mM EGTA, 5 mM Tris-phosphate, 10 mM Tris-maleate pH 6.8) (Rigoulet and Guerin, 1979). The reaction was started by adding 4 mM NADH and 750 µM ADP; 100 µL aliquots were taken every 15 s and the reaction was stopped by adding 3.5% (v/v) perchloric acid and 12.5 mM EDTA. Samples were neutralized to pH 6.5 by KOH and 0.3 M MOPS. ATP was quantified using the Kinase-Glo Max Luminescence Kinase Assay (Promega) and a Beckman Coulter's Paradigm Plate Reader.

5 mL of cells stably expressing Pam16_β11ch and Pgk1_β11ch strains (see Table 1) grown in YPD to confluence were diluted in 4 mL of SC Gal and grown overnight to reach mid-log phase. They were then diluted again in SC Gal and grown for 6 hr. Cells were then centrifuged and resuspended in water, passed for GFP detection on a BD FACS Canto II cytometer and Data analysis was performed using FlowJo.

10 mL of cells grown to mid-log phase were harvested and spin down 5 min at 2000 × g at room temperature (RT). Cells were suspended in 500 µL of deionized water, 50 µL of 1.85 M NaOH was added and the mixture was incubated 10 min on ice. After addition of 50 µL of TCA 100% and 10 min of incubation on ice, the total precipitate was pelleted by centrifugation 15 min at 13000 × g at 4°C. After removing the supernatant, pellets were suspended in 200 µL of Laemmli buffer (1×) supplemented with 20 µL of 1M Tris Base pH 8.

For each strain, 10 µL of total proteins were separated by SDS-PAGE on 8-, 10- or 12% (w/v) polyacrylamide gels prior to electroblotting with a Trans-Blot Turbo system (BIO-RAD) onto PVDF membranes (BIO-RAD, #1704156). Detection was carried out using mouse monoclonal IgG anti-GFP primary antibodies (1:5000; Roche Clone 7.1 and 13.1) + mouse polyclonal for the recognition of GFP_β1-10 (1:5000, Sigma #G1544), and mouse monoclonal IgG1 anti-Pgk1 primary antibodies (1:5000; Molecular Probes Clone 22C5D8). Secondary antibodies were Goat anti-mouse IgG (H+L) HRP-conjugated antibodies (BIO-RAD; #1706516), at a concentration of 1:10000. ECL-plus reagents (BIO-RAD) was used according to the manufacturer’s instructions and immuno-labeled proteins were revealed using a ChemiDoc Touch Imaging System (BIO-RAD). Total load of protein (Loading control) was assessed by UV detection using a ChemiDoc Touch Imaging System (BIO-RAD; Stain-free procedure) and detected by addition of 0.5% (v/v) 2,2,2-Trichloroethanol (Sigma #T54801) to the 30% acrylamide/bis-acrylamide solution.

Cells were incubated overnight in the appropriate media, diluted to an OD_{600 nm} of 0.3 prior to microscopy studies and stained after 6 hr of growth at 30°C. For mitochondria staining, cells were centrifuged 1 min at 1500 × g at room temperature, suspended in 1 mL of SC Gal supplemented with Red-Mitotracker CMXRos at a final concentration of 100 nM (Molecular Probes), and incubated 15 min at rotational shaking at 30°C. Cells were washed three times in one volume of deionized water, and suspended in 100 µL of deionized water for microscopic studies. Epifluorescence images were taken with an AXIO Observer d1 (Carl Zeiss) epifluorescence microscope using a 100 × plan apochromatic objective (Carl Zeiss) and processed with the Image J software. Images for 3D reconstruction were taken using a confocal LSM 780 high resolution module Airyscan with a 63 × 1.4 NA plan apochromatic objective (Carl Zeiss) controlled by the Zen Black 2.3 software (Carl Zeiss). Z-stack reconstruction was performed on the IMARIS 9.1.2 (Bitplane AG) software.