Extended Data Fig. 2: Redox metabolism of wild-type, naive and evolved petites.
From: The metabolic growth limitations of petite cells lacking the mitochondrial genome
a. Wild-type (⍴+) and petite (⍴0) BY4741 or BY4741 Δzwf1 yeast containing empty vector or plasmid-encoded ATP3, ATP3-6 or ATP3-7 were cultured in SC without histidine (SC-H) medium to mid-exponential phase, harvested, and spotted in serial dilutions onto freshly prepared SC-H agar plates ±the oxidant diamide (1.2 mM). Suppressor mutations (ATP3-6, ATP3-7) benefit growth in both BY4741 and BY4741 Δzwf1 background (left panel). Naive petites are slightly more sensitive to diamide compared to evolved petites and wild-type (right panel). In a Δzwf1 background, all strains are hypersensitive to diamide. Shown is one representative experiment n = 3 independent replicates. b.-d. Wild-type (⍴+) and petite (⍴0) YSBN11 strains containing empty vector or plasmid-encoded ATP3, ATP3-6 or ATP3-7 were cultured in SC-H medium to mid-exponential phase and reduced glutathione (GSH) and oxidized glutathione (GSSG) were quantified by LC-MS/MS. In comparison to untreated wild-type controls, treatment with H2O2 (0.5 mM) reduces GSH and increases GSSG levels (positive control). Instead, GSSG and GSH levels are largely unaffected in petites compared to wild-type. Statistics based on two-sided, unpaired t-tests of n = 3 biological replicates comparing wild type to other genotypes. Mean values ± SD. e. Wild-type (⍴+) and petite (⍴0) YSBN11 yeast containing empty vector or plasmid-encoded ATP3-6 were cultured in SM medium, harvested in mid-log phase and re-suspended in medium ± 2 mM tert-BOOH. Superoxide levels were quantified by DHE oxidation after 5 min using fluorescence microscopy. 3 % of wild-type cells, 1 % of naive petite cells and 2 % of evolved petites show DHE fluorescence, while 31 % of wild-type cells challenged with tert-BOOH exhibit fluorescence. Statistics based on two-sided, unpaired t-tests of the following number of biological replicates comparing wild type to other genotypes ⍴+, n = 6 replicates, 13260 cells; ⍴0, n = 11 replicates, 14873 cells; ⍴0 ATP3-6, n = 7 replicates, 17721 cells; ⍴+ test-BOOH, n = 6 replicates, 17721 cells. Mean values ± SEM are shown.
