Figure 5.

Artificial exposure of the masked Art1-BS at the N-tail of Can1 renders the permease sensitive to TORC1-activated Art1. (A) Epifluorescence microscopy of wild type Can1-GFP and Can1(E184Q) -GFP in wt, bul1/2Δ, art1Δ, and art1Δ bul1/2Δ strains. Cells were grown in Gal and Am MM. Glu was added for 1 h followed by addition of CHX for 3 h. Quantifications: Plasma membrane (PM) to internal GFP fluorescence intensity ratios are plotted (n = 38–135 cells). Quantifications, representations and scale bar as in Figure 2A. The quantifications for wt Can1 are the same from Figure 2A, since the experiments have been performed in parallel. (B) Epifluorescence microscopy of gap1Δ can1Δ cells expressing the indicated Can1 alleles combining E184Q with Ala substitutions in either the Art1-BS (residues 74–77), or the Bul-BS (residues 66–69) or both. Conditions, quantifications (n = 128–160) and representations as in A. (C) Schematic representation of the results concerning the CHX-induced endocytosis of Can1(E184Q). This allele stabilizes Can1 in an Inward-Facing conformation and constitutively exposes the binding site for Art1 (residues 70–81, green hemicycle). Upon CHX addition, Can1 is recognized not only by the Bul1/2 (Figure 4E), but also by Art1, leading to more efficient ubiquitylation by Rsp5, endocytosis and degradation.