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Snx4/41/42, the retromer, Ere1, and Ere2 are involved in the recycling of Can1. (A) Can1 localization in wild-type cells under steady conditions (left) and following treatment with cycloheximide (+ CHX, right). Wild-type cells expressing Can1-GFP were grown to mid-log phase, subjected to cycloheximide for 4 h to induce Can1 internalization, and analyzed by fluorescence microscopy. The plasma membrane (PM) and vacuoles are indicated. (B) The recycling of Can1 in ESCRT-mutant cells is mediated by Ere1, Ere2, Snx41, and the retromer complex. Mutant vps23Δ, snx41Δ vps23Δ, snx3Δ vps23Δ, ere1Δ vps23Δ, and ere2Δ vps23Δ cells expressing Can1-GFP were grown to mid-log phase, subjected to cycloheximide for 4 h to induce Can1 internalization, and analyzed by fluorescence microscopy. The PM and endosomes (E) are indicated. (C) The ratio of Can1-GFP fluorescence at endosomes and the PM in vps23Δ and ere1Δ vps23Δ, ere1Δ vps23Δ, snx41Δ vps23Δ, and snx3Δ vps23Δ double-mutant cells as described in Figure 1B; N = 25 for each cell type. See Materials and Methods for additional details. (D) Loss of Ere1 or Ere2 rescues the canavanine-hypersensitive phenotype in ESCRT-mutant cells. Serial dilutions of wild-type, snf7Δ, ere1Δ snf7Δ, ere2Δ snf7Δ, and snx41Δ snf7Δ cells were grown on synthetic media plates with or without 0.6 μg/ml canavanine for 2 d at 26°C.