Ere1 is not required for the transport of the retromer cargoes Vps10 and Ftr1. (A) Serial dilutions (fourfold) of wild-type, snx3Δ, ere1Δ, and vps5Δ cells were grown on synthetic media plates with or without 10 μg/ml calcofluor white for 2 d at 26°C. (B) Localization of Vps10-GFP in wild-type, vps5Δ, and ere1Δ cells. Cells were grown in yeast extract/peptone/dextrose (YPD), and vacuoles were labeled with FM4-64. Note: vps5Δ deletion-mutant cells show a fragmented-vacuole phenotype. (C) Localization of Ftr1-GFP in wild-type, vps5Δ, and ere1Δ mutant cells in the absence of exogenous iron.

Ere1 associates with an internalized plasma membrane protein, Can1, and colocalizes with Vps5. (A) ESCRT-mutant (vps23Δ) cells coexpressing an integrated Ere1-13xmyc fusion and Can1-3xHA (from its own promoter on a centromeric plasmid) were treated with cycloheximide for the indicated times and then cross-linked with DSP and DTBP. Can1-3xHA was immunoprecipitated with antibodies specific for HA from solubilized cell lysates. Immunoprecipitates were subsequently analyzed by Western blotting using antibodies against the myc or HA epitopes to detect Ere1 and Can1, respectively. (B) Ere1 and Vps5 colocalize on endosomal compartments. ESCRT-mutant (vps23Δ) cells coexpressing Ere1-RFPmars and Vps5-GFP (top) or coexpressing Ere1-RFPmars and Snx41-GFP (bottom) were examined by confocal microscopy.

Ere1 localizes to endosomal compartments. (A) Representative fluorescence microscopy images showing wild-type cells coexpressing Ere1-GFP and the endosomal marker DsRed-FYVE. Ere1-GFP localized to intracellular puncta (corresponding to endosomes with arrow) in ∼10% of wild-type cells. (B) Ere1 is stabilized on endosomal compartments in ESCRT -mutant cells. Wild-type and vps27Δ mutant cells expressing Ere1-GFP were grown in YPD medium at 30°C. Vacuoles and E compartments (in vps27Δ cells) were labeled with FM4-64. (C) Subcellular fractionation of Ere1-FLAG in lysates from wild-type and vps23Δ mutant cells was analyzed by differential centrifugation and subsequent SDS–PAGE and immunoblotting. Pep12 was used as a marker for membrane fractions (P13 and P100), whereas glucose-6-phosphate dehydrogenase was used as a cytosolic marker (S100). (D) Quantification of Ere1-FLAG in P13 and S100 fractions from wild-type and vps23Δ cell lysates.

Model for Ere1 and Ere2 cargo-specific function in retromer-mediated recycling from endosomes. After stimulation, Can1 is internalized from the plasma membrane (PM) and delivered to early endosomes (EE). On early endosomes, a pool of Can1 is selected and sorted by the Snx4/41/42 complex for recycling to the Golgi and subsequent delivery back to the cell surface. Additional cargoes, such as the SNARE protein Snc1, are recycled from early endosomes in a retromer- and Ere1/Ere2–independent process. The remaining fraction of Can1 that is not recycled by the Snx4/41/42 complex is transported to late endosomes (LE). At late endosomes, Can1 is subject to different fates. First, Can1 is recognized by the Ere1/Ere2 complex for sorting into the retromer-mediated recycling pathway (see Discussion). The Ere1/Ere2 complex is not required for the sorting of additional retromer cargoes, such as Ftr1 and Vps10, into recycling tubules generated at late endosomes. Internalized Can1 that is not recycled by the Snx4/41/42 or retromer pathways is sorted into the ESCRT-mediated MVB pathway and eventually delivered to the vacuole for degradation.

Cells lacking Snx4/41/42, retromer components, Ere1, or Ere2 are resistant to canavanine. (A) Model for Can1 trafficking and the screen for recycling mutants. In wild-type cells, the lifetime of Can1 at the PM is regulated by endocytosis, recycling, and degradation (left). In mutant cells deficient for Can1 recycling, Can1 cell-surface levels are reduced, resulting in reduced uptake of the toxic arginine analogue canavanine (squares). (B) Classification of cvr mutants identified in the canavanine resistance screen. (C) Cells impaired in endosomal recycling are resistant to canavanine. Serial dilutions (fourfold) of wild-type (WT) and deletion mutant cells (as indicated) were grown on synthetic media plates with or without 2 μg/ml canavanine for 2 d at 26°C. (D) Schematic diagrams for Ere1, Ere2, and their human orthologues WDR85 and WDR6, respectively.

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.

The retromer and Snx4/41/42 pathways independently recycle Can1, and Ere1 functions in the retromer pathway. (A) Loss of both retromer and Snx41 function results in additive resistance to canavanine (top), whereas loss of multiple retromer subunits is not additive (bottom). (B) Loss of both Ere1 and Snx41 confers an additive canavanine resistance phenotype, but loss of both Ere1 and the retromer subunit Snx3 is not additive. (A, B) Serial dilutions of wild-type and various mutant cells (as indicated) were grown on synthetic media plates with or without 2 μg/ml canavanine for 2 d at 26°C. (C) Rates of Can1 degradation are increased in cells lacking Snx41, Snx3, or Ere1. Double deletion of ERE1 and SNX41 further increases the rate of Can1 turnover. Wild-type and mutant cells (as indicated) expressing FLAG-tagged Can1 were grown to mid-log phase and treated with cycloheximide to induce Can1 endocytosis. Cell lysates were prepared at the indicated time points and analyzed by immunoblotting to monitor Can1 degradation rates (top; t_1/2 times are shown on the right). G6PDH was used as a protein loading control (bottom).

Ere1 and Ere2 physically interact and colocalize on endosomal compartments. (A) Glycerol velocity gradient sizing analysis of Ere1-FLAG and Ere2-FLAG in membrane fractions (P13 and P100) from wild-type cells. Major peak fractions for known molecular weight standards are indicated. The predicted molecular weight of monomeric Ere1 is ∼46 kDa and that of monomeric Ere2 is ∼114 kDa. (B) Ere2-3xHA specifically coimmunoprecipitates with Ere1-FLAG. Wild-type cells expressing an integrated Ere2-3xHA fusion or coexpressing integrated Ere1-FLAG and Ere2-3xHA fusions were grown to mid-log. Ere1-FLAG was immunoprecipitated from solubilized cell lysates, and the resulting immunopurified material was analyzed by Western blotting using antisera against the FLAG and HA epitopes to detect Ere1 and Ere2, respectively. (C) Ere1 and Ere2 colocalize on endosomal compartments. ESCRT-mutant (vps23Δ) cells coexpressing Ere1-RFPmars and Vps5-GFP were examined by confocal microscopy. (D) Interaction between Ere1 and Can1 is increased in cells lacking Ere2. Cross-linkers were added to vps23Δ mutant and vps23Δ ere2Δ double-mutant cells expressing Ere1-13xmyc alone or coexpressing both Ere1-13xmyc and Can1-3xHA following treatment with cycloheximide for 1.5 h. Can1-3xHA was immunoprecipitated from solubilized cell lysates and analyzed by Western blotting to detect Ere1 and Can1 in the input and immunoprecipitate fractions.