Figure 1.

Tup1 binds PI(3,5)P₂ lipid with a high specificity. (A, top panel) GST-Tup1 and GST-Atg18 bound PI(3,5)P₂ with a high specificity, while GST-PH_PLCδ specifically bound PI(4,5)P₂. GST protein alone did not bind any PI lipid. (Bottom panel) Equivalent amount of purified, bacterially expressed GST fusion proteins (50 μg) were used for protein–lipid overlay assay. (B) Vph1-mCherry marks the limiting membrane of vacuoles (red). A pool of GFP-Tup1 localized at the vacuolar membrane (arrowhead), in addition to the nuclear Tup1 (arrow), was observed in some wild-type (WT) cells (see Supplemental Fig. S2B). In contrast, the vacuolar localization of Tup1 was not observed in fab1Δ cells.