FIGURE 6:
Coordinated structure formation of Prs5p and Ade4p controls pathway flux. (A) Prs3p, Prs5p, and Ade4p structures disassemble in response to the presence of fresh glucose. Cells expressing GFP-tagged purine biosynthetic enzymes were grown in YPD for 5 d, except for the ADE4::GFP strain (1 d), and then shifted into the indicated media, incubated for 30 min at 30°C, and visualized immediately. Protein levels were determined by Western blot analysis and were normalized to no- treatment samples (indicated below blots). (B) Prs5p and Ade4p have distinct triggers for structure formation. Yeast cells expressing GFP-tagged purine biosynthetic enzymes were grown to log phase in complete SD media, shifted into the indicated media for 30 min at 30°C, and counted immediately. (C) Deletion of downstream enzymes of Ade4p leads to increased structure formation of Ade4p. Wild-type and mutant cells expressing Ade4p-GFP were grown in YPD for 1 d at 30°C and scored for structure formation. Protein levels were determined by Western blot analysis and were normalized to the wild-type strain (indicated below blots). (D) Loss of feedback inhibition increases focus formation by Ade4p. Cells expressing wild-type Ade4p-GFP and Ade4p(K333Q)-GFP were grown to log phase in YPD and cells were scored for frequency of structure formation. Protein levels were determined by Western blot analysis and were normalized to the wild-type strain (indicated below blots). Data are represented as means of at least three independent experiments; error bars indicate SEM. (E) Model for the coordinating activity of Prs5p and Ade4p with regulated structure assembly statuses is illustrated.