Figure 1.

Overexpression of a high affinity hemoprotein, Cyt b₅₆₂, attenuates labile heme and the activity of heme-regulated transcription factor, Hap1. A, molecular model and design principles of the heme sensor, HS1. Model derived from the X-ray structures of mKATE (Protein Data Bank code 3BXB) and CG6 (Protein Data Bank code 3U8P). B, cells expressing the heme sensor, HS1-M7A, and an allele of Cyt b₅₆₂ on a galactose (GAL)-inducible promoter (pGAL-Cyt b₅₆₂) or empty vector (pGAL-EV) were cultured in 2% RAF with or without 1.0% GAL or 500 μm SA for 16 h in SCE medium. After growth, HS1-M7A sensor eGFP (excitation 488 nm, emission 510 nm) and mKATE2 (excitation 588 nm, emission 620 nm) fluorescence emission ratios were recorded. C, Hap1 activity was measured in cells expressing pGAL-Cyt b₅₆₂ or pGAL-EV and cultured in 2% RAF with or without 1.0% GAL or 500 μm SA for 16 h in SC medium using a transcriptional reporter consisting of an allele of eGFP driven by the CYC1 promoter (pCYC1-eGFP), a Hap1 target gene. All data represent the mean ± S.D. (error bars) of triplicate cultures, and the statistical significance was assessed using a two-sample t test. *, p < 0.01; **, p < 0.001.