Figure 7.

Ist1 regulates the recruitment of Vps4 to MVBs. (A) Western blot analysis of cell extracts from yeast strains grown in galactose-containing medium expressing IST1-HA either from its own promoter (C, control) or the GAL1 promoter (G). (B) Subcellular fractionation of yeast strains expressing Vps4-HA into soluble (S) and membrane-bound pellet (P) fractions (WT, MBY3 pMB283; vps24Δ, MBY12 pMB283; did2Δ, EEY26-1 pMB283; and ist1Δ, MCY3 pMB283). The fractions were analyzed by Western blot using anti- HA antibodies. (C) Localization of Vps4(E233Q)-HA (expressed from pMB149) and Snf7 in cells overexpressing IST1 from a GAL1 promoter (Gal IST1: pMB286) was determined by immunofluorescence microscopy. The strains were grown in presence of galactose (WT, W303; did2Δ, MCY31). Strains containing the empty expression vector were used as controls (Gal con).