Fig. 5.

The internalization step of EVT occurs on the vacuole. a, b Depletion of Vps23 prevents the turnover of pre-existing Pho8. In these cells, expression of GFP-Pho8 was controlled by the tet-off promoter, and Vps23 was tagged with the AID degron. Six hours after dilution to OD₆₀₀=0.2, chemicals to shut-off Pho8 expression (1 μg/ml doxycycline) (Dox) or deplete Vps23 (500 uM IAA) were added as indicated. Cells were then incubated for another 8 h. a Translocation of GFP signal monitored by fluorescent microscopy. Scale bar, 5 μm. b Turnover of GFP-Pho8 and Vps23-AID monitored by immuno-blot. c Control experiment showing that depletion of Vps23 prevents vacuolar sorting of newly synthesized Cps1. Cells expressing GFP-Cps1 under the control of GAL1 promoter and Vps23 tagged with AID were cultured as in A, except for using a raffinose-containing medium (SMR+CA). Cells were then treated with or without IAA for 2.5 h. Finally, galactose was added to induce Cps1 expression for 1 h. Processing of GFP-Cps1 was monitored by immuno-blot. d Depletion of Vps23 does not cause the relocation of pre-existing Pho8 to endosomes. Cells expressing mRubby3-Pho8 under the control of the tet-off promoter, GFP-Cps1 under the CUP1 promoter and Vps23 tagged with AID were treated as in a, except that copper sulfate was added together with doxycycline. After 8 h, cells were imaged by fluorescent microscopy. Scale bar, 5 μm