Rane protein Tom6 [11].Fusion AssayCells of opposing mating type (expressing green or red fluorescent proteins) were grown separately (12?6 h, log phase) in YPGALA, transferred to YPGA, mixed and incubated under agitation for 2 h. Cells were then sedimented, incubated for up to 4 hours at 30uC, fixed, and analyzed by microscopy (detailed description in 25033180 Supp. Fig. S1). Zygotes were identified by their characteristic shape (phase contrast) and 
by the presence of red and green fluorescent proteins. No condition or mutant led to any Table 1. Genotypes and sources of yeast strains.Indolactam V chemical information strain MR6 – WT MR10 – Datp6 RKY25 – atp6-L247R MR14 – atp6-L183R MR6 – r0 Datp12 NB371 NB374 CSDcox2 DmgmNuclear genotype ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2?, his3?1,15, ura3?, leu2?,112, trp1?, atp12::LEU2 Mat a ade2?01, ura3?2, leu2D, arg8DURA3, kar1? Mat a ade2?01,ura3?2, arg8DURA3, kar1? ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 mgm1::kanMXmtDNA r+WT r+atp6::ARG8m r+atp6-L247R r+atp6-L183R r0 r+WT r+cox2::ARG8m r+cox2::ARG8m r+cox2::ARG8m rReference [2] [2] [4] [3] [2] [37] N.Bonnefoy N.Bonnefoy This study [12]Each strain was made available with mating type a and a by inducible HO expression. The CSDcox2 strain used to study mitochondrial fusion was generated by cytoduction of the mtDNA of a cox2::ARG8m strain (NB371 or NB374) into a MR6 r0 strain. The crosses produced cytoductants harbouring the nuclear genotype of MR6 and Dcox2 mtDNA. The cox2::ARG8m construct used to generate NB371 and NB374 comes from strain HMD22 [38]. doi:10.1371/order 1948-33-0 journal.pone.0049639.tMitochondrial DNA Mutations Mitochondrial FusionThe content of reactive oxygen species (ROS) was estimated using (blue-fluorescent) dihydroethidium (DHE), which is converted to red-fluorescent ethidium by superoxide, as described in [25]. Cells (DO600 ,1?) were incubated with 200 nM rh123 and 20 mM DHE (agitation, 28uC, 10 min). Cellular fluorescence was analyzed with an accuri C6 flow cytometer: 13 ml/min; 209000 events; FL1 533/30 nm for rh123; FL3.670 nm for ethidium. Preliminary experiments of singly labeled cells revealed negligible fluorescence bleed through between the FL1/rh123 and the FL3/ ethidium channels.Results Mitochondrial Fusion in Yeast ZygotesTo investigate mitochondrial fusion, we used assays based on the mating of haploid cells and on the exchange of matrix fluorescent proteins between fusing mitochondria (see [11] and Supp. Fig. S1). Cells of opposing mating types were grown in galactose (to induce fluorescent protein expression), transferred to glucose (to repress fluorescent 
protein expression), mixed, cultured under agitation for 2 hours and sedimented (to favor zygote formation). Sedimented cells were analyzed immediately (t = 0) or after one to four hours. The proportion of zygotes rose from 5?10 upon sedimentation (t = 0) to 30?0 (t = 4 h). For a quantitative analysis of fusion efficacy (Fig. 1A, B), zygotes were scored as total fusion (T: all mitochondria doubly labeled), no fusion (N: no mitochondria doubly labeled) or partial fusion (P: doubly and singly labeled mitochondria). It is important to note that, because new zygotes are being formed throughout the assay.Rane protein Tom6 [11].Fusion AssayCells of opposing mating type (expressing green or red fluorescent proteins) were grown separately (12?6 h, log phase) in YPGALA, transferred to YPGA, mixed and incubated under agitation for 2 h. Cells were then sedimented, incubated for up to 4 hours at 30uC, fixed, and analyzed by microscopy (detailed description in 25033180 Supp. Fig. S1). Zygotes were identified by their characteristic shape (phase contrast) and by the presence of red and green fluorescent proteins. No condition or mutant led to any Table 1. Genotypes and sources of yeast strains.Strain MR6 – WT MR10 – Datp6 RKY25 – atp6-L247R MR14 – atp6-L183R MR6 – r0 Datp12 NB371 NB374 CSDcox2 DmgmNuclear genotype ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1-1 CAN+arg8::HIS3 ade2?, his3?1,15, ura3?, leu2?,112, trp1?, atp12::LEU2 Mat a ade2?01, ura3?2, leu2D, arg8DURA3, kar1? Mat a ade2?01,ura3?2, arg8DURA3, kar1? ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 ade2-1, his3?1,15, ura3-1, leu2?, trp1? CAN+arg8::HIS3 mgm1::kanMXmtDNA r+WT r+atp6::ARG8m r+atp6-L247R r+atp6-L183R r0 r+WT r+cox2::ARG8m r+cox2::ARG8m r+cox2::ARG8m rReference [2] [2] [4] [3] [2] [37] N.Bonnefoy N.Bonnefoy This study [12]Each strain was made available with mating type a and a by inducible HO expression. The CSDcox2 strain used to study mitochondrial fusion was generated by cytoduction of the mtDNA of a cox2::ARG8m strain (NB371 or NB374) into a MR6 r0 strain. The crosses produced cytoductants harbouring the nuclear genotype of MR6 and Dcox2 mtDNA. The cox2::ARG8m construct used to generate NB371 and NB374 comes from strain HMD22 [38]. doi:10.1371/journal.pone.0049639.tMitochondrial DNA Mutations Mitochondrial FusionThe content of reactive oxygen species (ROS) was estimated using (blue-fluorescent) dihydroethidium (DHE), which is converted to red-fluorescent ethidium by superoxide, as described in [25]. Cells (DO600 ,1?) were incubated with 200 nM rh123 and 20 mM DHE (agitation, 28uC, 10 min). Cellular fluorescence was analyzed with an accuri C6 flow cytometer: 13 ml/min; 209000 events; FL1 533/30 nm for rh123; FL3.670 nm for ethidium. Preliminary experiments of singly labeled cells revealed negligible fluorescence bleed through between the FL1/rh123 and the FL3/ ethidium channels.Results Mitochondrial Fusion in Yeast ZygotesTo investigate mitochondrial fusion, we used assays based on the mating of haploid cells and on the exchange of matrix fluorescent proteins between fusing mitochondria (see [11] and Supp. Fig. S1). Cells of opposing mating types were grown in galactose (to induce fluorescent protein expression), transferred to glucose (to repress fluorescent protein expression), mixed, cultured under agitation for 2 hours and sedimented (to favor zygote formation). Sedimented cells were analyzed immediately (t = 0) or after one to four hours. The proportion of zygotes rose from 5?10 upon sedimentation (t = 0) to 30?0 (t = 4 h). For a quantitative analysis of fusion efficacy (Fig. 1A, B), zygotes were scored as total fusion (T: all mitochondria doubly labeled), no fusion (N: no mitochondria doubly labeled) or partial fusion (P: doubly and singly labeled mitochondria). It is important to note that, because new zygotes are being formed throughout the assay.