He mitochondrial ATP6 gene that are pathogenic in humans [3,4]. We demonstrate

He mitochondrial ATP6 gene that are pathogenic in humans [3,4]. We demonstrate that all genetic OXPHOS defects are associated to an 3-Bromopyruvic acid web inhibition of inner but not outer membrane fusion. Fusion inhibition is dominant, and hampers the fusion of mutant mitochondria with wild-type mitochondria. We further show that the inhibition induced by point mutations associated to neurogenic ataxia retinitis pigmentosa (NARP) or maternally inherited Leigh Syndrome (MILS) is of similar extent to that induced by the deletion of mitochondrial OXPHOS genes or by the removal of the entire mtDNA.major defect in mating. For a quantitative analysis, zygotes (n 100/condition and time-point) were scored as total fusion (T: all mitochondria are doubly labeled), no fusion (N: no mitochondria are doubly labeled) or partial fusion (P: doubly and singly labeled mitochondria are observed). Mutant strains were always analyzed in parallel to a wild-type strain.Microscopical and Biochemical AnalysisCell extracts were prepared and analyzed by Western-blot as described [12]. For fluorescence microscopy, sedimented cells were fixed for 20 min by addition of formaldehyde to the culture medium (3.7 final concentration). Fixed cells were spotted onto glass slides and observed in a Zeiss AxioSkop 2 Plus Microscope. For electron microscopy, cells were processed as described [4] and analyzed in the Bordeaux Imaging Center (BIC) of the University of Bordeaux Segalen.Cellular BioenergeticsAll analysis were performed after growing cells under the conditions of a fusion assay (12?6 h exponential growth in YPGALA followed by 1? h in YPGA). Oxygen consumption was measured with a Clark electrode after addition of 143 mM ethanol to cells in YPGA (DO600 ,1?). The degree of coupling between respiration and ATP-synthesis was evaluated by the capacity of the ATP-synthase inhibitor (triethyl tin bromide – TET: 83 mM) or a protonophore (carbonyl cyanide m-chlorophenyl hydrazone cccp: 83 mM) to inhibit or stimulate respiration, respectively. ATP and ADP levels were determined by luminometry [23]. Cells (1 ml, DO600 ,1?) were sedimented, washed with H20 and immediately extracted by vortexing (3615 sec) in 200 ml PE (7 perchloric acid, 25 mM EDTA) with 50?00 ml glass beads. The pH was equilibrated to pH ,6 with KOMO (2 M KOH, 0,5 M MOPS), glass beads and KClO4-precipitate were sedimented by centrifugation and the supernatant was stored at 280uC. The ATP-content was determined by luminometry (ATPlite 1step Perkin Elmer) in an LKB luminometer. For the determination ATP+ADP, all ADP was phosphorylated (30 min, room temperature) with phosphoenolpyruvate (PEP: 5 mM) and pyruvate kinase (PK: 0,1 mg/ml) and the ADP-content was calculated by subtraction. Mitochondrial inner membrane potential DYm was estimated with rhodamine 123 (rh123), which is accumulated by mitochondria in a DYm-dependent manner, as described in [24].Materials and Methods Strains, Media and PlasmidsThe origins and genotypes of the S. cerevisiae strains are listed in Table 1. The media (glucose-containing YPGA; galactosecontaining 16574785 YPGALA; CSM; CSM-U CSM-R-U) are described elsewhere [3,4]. For GSK -3203591 labeling of the mitochondrial matrix we used pYES-mtGFP [21] and pYEF-mtRFP [22], which encode EGFP and DsRed fused to the mitochondrial presequence of subunit 9 of the F0-ATPase of Neurospora crassa. For labeling of the mitochondrial outer membrane, we constructed pYES-GFPOM and pYESRFPOM, which encode EGFP and tdTomato fused to the outer memb.He mitochondrial ATP6 gene that are pathogenic in humans [3,4]. We demonstrate that all genetic OXPHOS defects are associated to an inhibition of inner but not outer membrane fusion. Fusion inhibition is dominant, and hampers the fusion of mutant mitochondria with wild-type mitochondria. We further show that the inhibition induced by point mutations associated to neurogenic ataxia retinitis pigmentosa (NARP) or maternally inherited Leigh Syndrome (MILS) is of similar extent to that induced by the deletion of mitochondrial OXPHOS genes or by the removal of the entire mtDNA.major defect in mating. For a quantitative analysis, zygotes (n 100/condition and time-point) were scored as total fusion (T: all mitochondria are doubly labeled), no fusion (N: no mitochondria are doubly labeled) or partial fusion (P: doubly and singly labeled mitochondria are observed). Mutant strains were always analyzed in parallel to a wild-type strain.Microscopical and Biochemical AnalysisCell extracts were prepared and analyzed by Western-blot as described [12]. For fluorescence microscopy, sedimented cells were fixed for 20 min by addition of formaldehyde to the culture medium (3.7 final concentration). Fixed cells were spotted onto glass slides and observed in a Zeiss AxioSkop 2 Plus Microscope. For electron microscopy, cells were processed as described [4] and analyzed in the Bordeaux Imaging Center (BIC) of the University of Bordeaux Segalen.Cellular BioenergeticsAll analysis were performed after growing cells under the conditions of a fusion assay (12?6 h exponential growth in YPGALA followed by 1? h in YPGA). Oxygen consumption was measured with a Clark electrode after addition of 143 mM ethanol to cells in YPGA (DO600 ,1?). The degree of coupling between respiration and ATP-synthesis was evaluated by the capacity of the ATP-synthase inhibitor (triethyl tin bromide – TET: 83 mM) or a protonophore (carbonyl cyanide m-chlorophenyl hydrazone cccp: 83 mM) to inhibit or stimulate respiration, respectively. ATP and ADP levels were determined by luminometry [23]. Cells (1 ml, DO600 ,1?) were sedimented, washed with H20 and immediately extracted by vortexing (3615 sec) in 200 ml PE (7 perchloric acid, 25 mM EDTA) with 50?00 ml glass beads. The pH was equilibrated to pH ,6 with KOMO (2 M KOH, 0,5 M MOPS), glass beads and KClO4-precipitate were sedimented by centrifugation and the supernatant was stored at 280uC. The ATP-content was determined by luminometry (ATPlite 1step Perkin Elmer) in an LKB luminometer. For the determination ATP+ADP, all ADP was phosphorylated (30 min, room temperature) with phosphoenolpyruvate (PEP: 5 mM) and pyruvate kinase (PK: 0,1 mg/ml) and the ADP-content was calculated by subtraction. Mitochondrial inner membrane potential DYm was estimated with rhodamine 123 (rh123), which is accumulated by mitochondria in a DYm-dependent manner, as described in [24].Materials and Methods Strains, Media and PlasmidsThe origins and genotypes of the S. cerevisiae strains are listed in Table 1. The media (glucose-containing YPGA; galactosecontaining 16574785 YPGALA; CSM; CSM-U CSM-R-U) are described elsewhere [3,4]. For labeling of the mitochondrial matrix we used pYES-mtGFP [21] and pYEF-mtRFP [22], which encode EGFP and DsRed fused to the mitochondrial presequence of subunit 9 of the F0-ATPase of Neurospora crassa. For labeling of the mitochondrial outer membrane, we constructed pYES-GFPOM and pYESRFPOM, which encode EGFP and tdTomato fused to the outer memb.

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