Figure 4.

Modeling of the c.695G>A and c.855T>G Mutated MECR Alleles in the Respiratory-Deficient Yeast BJ1991etr1Δ mtFAS Defective Strain

(A and B) Analysis of rescue of respiratory growth of the etr1Δ mutant by the MECR c.695G>A and c.855T>G allele variants, respectively. Yeast cells transformed with plasmids carrying the indicated constructs were grown on liquid media, normalized according to cell density, and serial diluted (1×, 1/10×, 1/100×, and 1/1,000×). For each mutation two independent clones transformed with mutation plasmids were tested. Equal volumes of cell suspensions were spotted on synthetic media containing only a non-fermentable carbon source (glycerol or lactate) or plates containing the fermentable carbon source glucose as growth control, and grown for several days. Growth on lactate or glycerol indicates respiratory competence.

(C) Western blotting analyses of yeast extracts from BJ1991etr1Δ cells carrying YEp195-ETR1(yETR1), YEp352 (empty), YEp352-MTS-HsMECR (HsMECR), YEp352-MTS-HsMECR-c.695G>A (HsMECR-c.695G>A), or YEp352-MTS-HsMECR-c.885T>G (HsMECR-c.885T>G). Whole yeast cell extract was separated by SDS-page, transferred to a solid support, and probed for the relevant antigens. Antisera used for protein detection are indicated. Lat1: E2 subunit of yeast pyruvate dehydrogenase; Kgd2: E2 subunit of yeast α-ketoglutarate dehydrogenase. Actin: Loading control. An additional loading control (Ponceau S staining of the blotting membrane) can be found in the Supplemental Data.