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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.

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