Ist1 is an endosomal protein that requires Vps27 and Vps4 for proper localization. (A) Endogenous Ist1 was C-terminally tagged with GFP by genomic integration. The resultant fusion protein colocalizes with a plasmid-expressed endosomal marker mRFP-FYVEEEA1 (see arrows) but not with the Golgi marker Sec7-dsRed. (B) Subcellular fractionation was performed on lysates from a wild-type strain and a strain in which endogenous Ist1 was tagged with FLAG. Ist1 was visualized by immunoblotting with an anti-FLAG antibody, and marker proteins Pep12, ALP, and PGK were detected by immunoblotting with antibodies specific for these proteins. The asterisk denotes a background band. (C) Localization of chromosomally expressed Ist1-GFP in wild-type, vps27Δ, and vps4Δ strains labeled with FM4-64. Arrows indicate class E compartments..