FIGURE 5:

The inactive Can1(E184Q) mutant is down-regulated upon Arg uptake. (A) Schematic representation of the regulation of the Art1 and Bul α-arrestins by nitrogen availability via the TORC1 pathway. (B) Immunoblots of total protein extracts of a wild-type strain expressing HA-Npr1. Samples with or without rapamycin addition 30 min before Am or Arg addition were collected and probed with anti-HA antibodies. (C) Epifluorescence microscopy analysis of Gal Pro grown gap1Δ can1Δ cells expressing Can1-GFP or Gap1-GFP. Glu was added for 1.5 h and then Arg or Am for 3 h. (D) Strains (with gap1Δ can1Δ mutations) expressing Can1-GFP were grown on Gal Pro. Glu was added for 30 min and then Arg or Am for 15 min. Total protein extracts were probed as in Figure 1C. (E) Epifluorescence microscopy analysis (as in Figure 1A) and (F) immunoblotting (as in Figure 1C) of Can1-GFP and the indicated mutants in Gal Pro grown strains (with a gap1Δ mutation). (G) Epifluorescence microscopy analysis (as in Figure 1A) and (H) immunoblotting of Can1-GFP and the indicated mutants; conditions as in E and F, respectively. (I) Epifluorescence microscopy analysis (as Figure 1A) of the corresponding strains expressing Can1-GFP or Can1(E184Q)-GFP; conditions as in E. (J) Epifluorescence microscopy analysis (as in Figure 1A) of a gap1Δ can1Δ strain expressing Can1(7KR)-mCherry and Can1(E184Q)-GFP.