Figure 5. Mislocalization of the bacterialized ferrochelatase enzyme identifies a porphyria-like phenotype in yeast.
(A) Bacterialization of the ultimate yeast gene in the heme biosynthesis pathway results in a distinct pink colony phenotype on YPD agar medium. In contrast, wild type BY4741 strain colonies appear as creamy-white. (B) Acetate-extracted secreted products from the pink Sc-hem15Δ::Ec-hemH strains show strongly enhanced fluorescence at 635 nm (excitation 399 nm), comparable to a protoporphyrin IX standard and unlike a heme standard or extracts from the parental BY4741 strain. The introduction of an MLS to the bacterialized yeast strain (Sc-hem15Δ::Ec-MLS-hemH) significantly reduced protoporphyrin IX secretion, while deletion of the MLS from the native yeast locus in strain Sc-ΔMLS-HEM15 caused several strains to increase protoporphyrin IX secretion.
Figure 5—figure supplement 1. Absorbance (top) and emission (bottom) spectra of extracts obtained from acetate (left) and pyridine (right) extraction of the wild type or bacterialized yeast colonies grown on YPD medium.
Figure 5—figure supplement 2. Deletion of protoporphyrinogen oxidase, Sc-HEM14, in the Sc-hem15Δ::Ec-hemH strain suppressed the porphyria-like pink phenotype.
