Figure 4.

Cti6 binds PI(3,5)P₂ lipid, and the Cys residues in Cti6's PHD domain are important for nuclear localization of Cti6 in Gal medium. (A) Cti6 contains a PHD domain and a CTI domain. The PHD domain contains several Cys residues (red) that are potentially involved in zinc binding and important for PIP binding. Two Cti6 mutants were generated, such as Cti6^C75S,C77S (Cys⁷⁵–Cys⁷⁷ → Ser⁷⁵–Ser⁷⁷) and Cti6^C92S,C95S (Cys⁹²–Cys⁹⁵ → Ser⁹²–Ser⁹⁵). (B, top panel) Full-length Cti6 bound PI(3,5)P₂ with high specificity, while the region (amino acids 51∼150) encompassing the PHD domain of Cti6 bound PI(3)P, PI(3,5)P₂, and PI(4)P. (Bottom panel) Bacterially expressed GST fusion proteins were purified, and the same amounts of proteins were used for the protein–lipid overlay assay. (C) Wild-type Cti6 localized primarily in the nucleus in wild-type (WT) cells in both Glu and Gal media. The Cti6 mutants Cti6^C75S,C77S and Cti6^C92S,C95S localized in the nucleus in wild-type cells in Glu medium, but a significant portion of the mutant Cti6 proteins accumulated in the cytoplasm upon Gal shift.