Figure 3.—

Proline induces the expression of MCH5. (A) Alignment of the conserved Put3 binding sites from the MCH5 promoters of various Saccharomyces species. The figure was generated using weblogo (http://weblogo.berkeley.edu/). (B) Growth of rib5Δ and a corresponding wild-type strain on minimal media containing proline or ammonium as sole nitrogen sources and various concentrations of riboflavin (from top to bottom: 200, 20, 2, or 0.2 mg/liter). Serial dilutions of cell suspensions were spotted and grown for 3 days at 30°. (C) Abundance of Mch5 and a Put1 after growth in proline or ammonium. Western blots were performed with the anti-Mch5 antibody or the anti-HA antibody (which binds to the ZZ-tag of Put1). PUT1-ZZ was expressed from a centromeric plasmid under control of its own promoter. Both strains were from the BY genetic background and prototrophic for riboflavin. (D) The Put3 binding site from the MCH5-promoter confers increased reporter activity in proline-grown cells. The GFP reporter construct containing the Put3 binding site from the MCH5 promoter in a MEL1 minimal promoter was transformed into BY4742 wild-type cells. The cells were continuously grown in media containing either ammonium or nitrogen as sole nitrogen source and GFP fluorescence was determined as described in materials and methods. (E) rib5Δ mch5Δ and rib5Δ put3Δ double mutants show an increased requirement for riboflavin. Serial dilutions of cells of the indicated genotype were spotted on YPD plates to which various amounts of riboflavin were added. The additions were (from top to bottom) 200, 20, 2 mg/liter, or no riboflavin as indicated. The rib5Δ mch5Δ double mutant had the genotype rib5Δ∷HIS3MX6 mch5Δ∷KanMX4; the rib5Δ put3Δ double mutant had the genotype rib5Δ∷KanMX4 put3Δ∷LEU2.