Ub binding by Mvb12 contributes to GFP-Cps1 sorting. (A) Mvb12 mutants lacking Ub binding correctly assemble with ESCRT-I. HA epitope–tagged wild-type Mvb12 and two mutants, mvb12^ΔUb1 and mvb12^ΔUb2, were expressed in mvb12Δ cells (pPL23713, pPL3709, and pPL3711 in JPY21). Lysates were prepared and immunoprecipitated with anti-HA antibodies and immunoblotted with α-Vps23, α-Vps28, or α-HA antibodies. IP, immunoprecipitation. (B) Sorting of Sna3-GFP and Ste3-GFP in mvb12Δ cells cotransformed with plasmids containing HA-tagged mvb12ΔUb alleles under the endogenous promoter. (C) Strains carrying the mvb12^ΔUb alleles alone or in combination with vps23^ΔUb and vps36^ΔUb were assessed for MVB sorting using GFP-Cps1, Sna3-GFP, and Ste3-GFP. The MVB12 alleles were expressed as HA epitope–tagged proteins from low copy plasmids (pPL23713, pPL3709, and pPL3711). Wild-type (WT), mvb12Δ-null, and vps23^ΔUb2 vps36^ΔUb mvb12Δ–null cells were also analyzed. The GFP fluorescence images and corresponding DIC images are shown. Bars, 5 µm.