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. Author manuscript; available in PMC: 2016 May 7.
Published in final edited form as: Cell. 2015 May 7;161(4):858–867. doi: 10.1016/j.cell.2015.04.017

Figure 2.

Figure 2

Mcx1 promotes heme biosynthesis at the step of ALA synthesis. (A) Total porphyrin levels were measured by fluorescence in oxalic acid cell extracts (ex. 400 nm, em. 662 nm; p < 0.001 for difference between wt and MCX1 mutants). +ALA indicates supplementation of growth medium with 50 μg/ml ALA. See also Fig. S2. (B) Metabolites involved in the first step of heme biosynthesis (KG, α–ketoglutarate; SA, succinic acid; GLY, glycine; GLX, glyoxylate; SER, serine) were measured in extracts of the indicated yeast strains by LC-MS. P < 0.001 for ALA perturbation in mcx1Δ cells. (C) ALA levels in cell extracts were measured using modified Ehrlich’s reagent. p ≤ 10−5 for ALA reduction in MCX1 and HEM1 mutants. (D) Mcx1 was isolated with α-FLAG antibody-conjugated beads from cells harboring HEM1-3xMYC and MCX1-3xFLAG (wt or Mcx1EQ (EQ)) or untagged Mcx1 (−)) alleles at the genomic loci, and eluted with 3xFLAG peptide. The eluate was analyzed by Western blot for Mcx1 (α-FLAG) and Hem1 (α-Myc). See Extended Experimental Procedures for detailed procedure. (E) Cellular levels of Hem1-3xMyc were analyzed by Western blot, using alkaline cell extracts (von der Haar, 2007). Hem1-3xMyc intensity: in mcx1Δ = 1.1 ± 0.1 relative to wt, p = 0.35 for difference; in hem1-DAmP = 0.3 ± 0.1, p = 0.01. The mitochondrial protein Por1 was probed as a loading control. Error bars represent mean ± SD.