Interactions between Vps4 and its substrate and regulators. (A) Vps4 interaction network based on previous studies (Scott et al., 2005b; Azmi et al., 2008; Kieffer et al., 2008; Bajorek et al., 2009a; Xiao et al., 2009). (B) Model for the recruitment and assembly of Vps4. The numbers indicating New Interactions or Lost Interactions refer to the numbers in A. (C) Alignments of the putative MIM1 and MIM2 motifs of yeast and mammalian ESCRT-III subunits (yeast Ist1 does not contain an obvious MIM2 consensus sequence). Mutations used in this study are marked in red.

MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIM1 mutant (L64D), or MIM2 mutant (I18D) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomal localization was determined (Loc.). For better visualization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brightness range (black is the brightest signal). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (B) Quantification of endosome-localized MIT-GFP relative to wild type (0.0) and vps4Δ (1.0). The data shown represent the results of at least 15 individually analyzed cells. Numbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing vps4^E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9–14) and the HA-tag (bottom panels, lanes 1–8 and 15–16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (D) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-terminal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4^E233Q was added in the presence of ATP or ADP to immobilized proteins; bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.

Phenotypic analysis of mutations affecting Vps4 interactions. (A) Fluorescence microscopy analysis of yeast strains expressing GFP-CPS. The efficiency of GFP-CPS sorting into the lumen of the vacuole is indicated (+, +/−, −). Vps4 interactions affected by the different mutations are listed. (B) Subcellular fractionation of yeast strains into soluble (S) and membrane-associated pellet fractions (P). The samples were analyzed by Western blot using antibodies specific for Vps24 and Snf7. Vps4 interactions affected by the different mutations are listed. (A and B) Int., numbers are based on the interactions in Figure 1A, and numbers in parentheses indicate partially disrupted interactions.

Localization of mutant Vps4 proteins. (A) Localization of MIT-deleted Vps4 (GFP- Vps4^ΔMIT,E233Q and GFP-Vps4^{ΔMIT,E233Q,S377A}) in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.). Vps4 interactions affected by the different mutations are listed. Int., numbers are based on the interactions in Figure 1A. (B) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by subcellular fractionation and Western blot analysis (S, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized membrane fractions. The resulting bound and unbound samples were analyzed by Western blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full-length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate partially disrupted interactions.

Ist1 and Did2 form a stable complex. GST-tagged full-length Did2 (GST-Did2) or C-terminal half of Did2 [GST-CT(Did2)] was immobilized on GSH-Sepharose, and purified Ist1, Vta1, or Vps4^E233Q protein was added. The resulting bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.