Extended Data Fig. 3: Retrograde response is activated in petites, mitigated in evolved petites, but is not required for suppression of the petite phenotype.
From: The metabolic growth limitations of petite cells lacking the mitochondrial genome
a., b., Wild-type (⍴+) and petite (⍴0) BY4741 strains containing empty vector or plasmid-encoded ATP3-6 and ATP3-7 variants were transformed with a plasmid encoding Pts1-GFP and cultured in SC-HU (histidin, uracil dropout) medium. Cells were harvested in mid-exponential growth phase, fixed with formaldehyde and observed by fluorescence microscopy. Dot-like structures represent peroxisomes (a). Scale bar = 1 μm. Compared to wild-type (10 ± 1) and evolved petites (10 ± 1), naive petites display a significant increase of peroxisomes (17 ± 1) (b). P-values shown based on unpaired, two-sided t-tests, comparing wild type to other genotypes. Mean values ± SEM. Number of cells quantified is indicated by n in the plot. c. Wild-type (⍴+) and petite (⍴0) YSBN11 yeast containing empty vector or plasmid-encoded ATP3-6 were cultured in SM medium. In the mid-exponential growth phase, cells were harvested and subjected to a proteomics workflow. Shown protein fold-changes were normalized to the wild-type. Proteins previously described to be controlled by retrograde response (RTG)1,61,77,78 as well as regulator Rtg2p are significantly upregulated in naive petites (empty plasmid). In comparison, up-regulation in the evolved petite (ATP3-6) is significantly less pronounced. Statistics based on two-sided, unpaired t-tests of n = 3 biological replicates comparing wild type to other genotypes. Mean values ± SD. d. RTG2 or CIT2 are not required for petite suppression. Wild-type (⍴+) BY4741 yeast and strains deleted for RTG2 and CIT2 were transformed with empty plasmid or plasmid encoding for ATP3-6, and mtDNA was depleted. Cells were grown to mid-log growth phase, and equal numbers were spotted in serial dilutions onto SC-H agar. Petites suffer from a growth defect compared to wild-type, which is further amplified in Δrtg2 and Δcit2 strains. Instead, expression of ATP3-6 restores growth irrespective of the genetic background (wild-type, Δrtg2 and Δcit2).
