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. 2021 Sep 22;22(19):10208. doi: 10.3390/ijms221910208

Figure 4.

Figure 4

Cycloheximide promotes endocytosis of Can1 via TORC1 hyperactivation and/or blockage of transporter recycling. (A) Immunoblots of total protein extracts of wt and bul1/2Δ cells expressing Can1-GFP. Cells were grown on Gal Proline (Pro) medium. Glu was added for 30 min followed by Am for another 30 min. Total protein extracts were probed and analysed as in Figure 2B. The low mobility bands correspond to Ub-Can1-GFP conjugates. Quantifications: The ratio of Ub-Can1-GFP / Total Can1-GFP are plotted in scattered plots from one biological replicate. (B) Immunoblots of total protein extracts of a wild-type strain expressing HA-Npr1 and grown in Pro medium, followed by the addition of Ammonium (Am). Samples with or without rapamycin addition 15 min after Am addition were collected at the indicated time-points and probed with anti-HA and anti-Pgk1 antibodies. High and low exposures are shown. (C) Immunoblots of total protein extracts of a wild-type strain expressing HA-Npr1. Cells were grown on Gal Proline (Pro) or Ammonium (Am) medium, followed by the addition of CHX. Samples with or without rapamycin addition 15 min after CHX addition were collected at the indicated timepoints and probed with anti-HA and anti-Pgk1 antibodies. High and low exposures are shown. (D) Epifluorescence microscopy of Can1-GFP in wt, bul1/2Δ, art1Δ, and art1Δ bul1/2Δ cells in YPT6+ or ypt6Δ background, grown in Gal Pro medium. Glu was added for 1 h followed by addition of Am for 3 h. Quantifications: Plasma membrane (PM) to intracellular GFP fluorescence intensity ratios are plotted (n = 45–101 cells). Quantifications, representations and scale-bars as in Figure 2A. (E) Model for the mechanisms controlling the endocytosis of Can1 upon TORC1 activation. Upon addition of Am (Left) in cells grown on a poor nitrogen source, TORC1 gets activated and leads to phosphorylation of Npr1. The resulting inactivation of Npr1 leads to activation of Art1 and the Bul1/2. However, only the Bul1/2 can recognize the Bul binding sequence (residues 62–69, green triangle) since the Art1-BS remains masked (red hemicycle). Can1 ubiquitylation via Rsp5 leads to endocytosis, but subsequent vacuolar sorting is limited because the permease recycles back to the plasma membrane via the Trans-Golgi Network (TGN), in a Ypt6-dependent way. When CHX is added (Right), on the contrary, TORC1 gets hyperactivated, leading to hyper-phosphorylation of Npr1 and activation of Bul1/2 and Art1. At the same time, recycling of Can1 is somehow inhibited, thus the ubiquitylated Can1 is sorted to the vacuole.