Fig 1: Under non-permissive conditions, Mae1 is a key enzyme responsible for cataplerosis in maf1∆. Idp-dependent reductive carboxylation of glutamine is proposed as an alternative metabolic strategy in rpc128-1007 to maintain viability on the glucose medium. Yeast cells were grown in rich medium (YP) supplemented with either 2% glucose or 2% glycerol at 30 °C. To verify the phenotypic effect of MAF1 deletion, cells grown in YPGly were transferred for 2 h to 37 °C. All enzymatic activities were measured in cell-free yeast extracts (A–F). The reaction rates were monitored by measuring NADH concentration change over time at 340 nm. Protein concentration was assessed according to the Bradford assay. Data are expressed as the mean Vmax obtained from at least three independent biological replicates. NAD+ and NADH levels were measured according to NAD/NADH Assay Kit II (colorimetric, ab221821, Abcam) as stated in manufacturer’s protocol (G–I). NAD+ and NADH concentrations expressed in µM were calculated from standard curve and standardized to the total concentration of proteins expressed in mg. The + standard deviations (SD) are shown. Asterisks (*) indicate a p value of ≤0.05 and double asterisks (**) mark p-values of ≤0.01 by Student’s t-test.