(A) Growth of strains lacking genes encoding endosomal sorting nexins and retromer deletions in the presence of CFW. Serial dilutions of control (chs6Δapl2Δ), mih1-1 (chs6Δapl2Δmih1-1), snx3Δ (chs6Δapl2Δsnx3Δ), snx4Δ (chs6Δapl2Δsnx4Δ), vps5Δ (chs6Δapl2Δvps5Δ), vps17Δ (chs6Δapl2Δvps17Δ), mvp1Δ (chs6Δapl2Δmvp1Δ), vps26Δ (chs6Δapl2Δvps26Δ), vps29Δ (chs6Δapl2Δvps29Δ) and vps35Δ (chs6Δapl2Δvps35Δ) cells were spotted onto rich medium with or without 50 μg/ml CFW for three days at 30°C. (B) Localization of Chs3-GFP in control (chs6Δapl2Δ), mih1-1 (chs6Δapl2Δmih1-1) and vps29Δ (chs6Δapl2Δvps29Δ) (as a representative retromer deletion). A representative medial optical plane of a z-series is shown. Scale bar, 5 µm. (C) Endogenously expressed Vps35-GFP, Vps29-GFP, Vps26-GFP, Vps17-GFP or Vps5-GFP fusion proteins were immunopurified from chs6Δapl2Δmih1Δ cells and samples of the collected material were analyzed by immunoblotting with a pan phosphoryl amino acid antiserum (top) or anti-GFP (bottom). An amount of cell lysate equivalent to 0.5% of the starting material for the purifications was run in the ‘load’ lanes. The positions of molecular weight (kDa) protein standards are indicated on the left side of the gels. (D) Vps26 phosphorylation status. Endogenously expressed Vps26-GFP fusion protein was immunopurified from chs6Δapl2Δ (control), chs6Δapl2Δmih1-1 (mih1-1) and chs6Δapl2Δmih1Δ (mih1Δ) cells and subjected to immunoblot assay with a pan phosphoryl amino acid antiserum (top) or anti-GFP (bottom). The molecular weight (kDa) of protein standards is indicated on the left side of the gels. (E) Purified Mih1 proteins. The indicated Mih1 proteins were purified from yeast cell lysates. A coomassie blue stained gel of aliquots of the eluted fractions used for activity assays is shown. (F) In vitro assay of Vps26 dephosphorylation by Mih1. Purified Mih1 proteins or lambda protein phosphatase were incubated with immunopurified Vps26-GFP. Equivalent portions of each reaction were examined by immunoblot with a pan phosphoryl amino acid antiserum (top) or anti-GFP (bottom). ‘Mock’ indicates a reaction that received no purified phosphatase.