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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.