Identification of phosphorylated residues in She2. (A) Binding of She2-myc on the Pro-Q phosphoprotein enrichment column. Detection of She2-myc by western blot from the whole lysate (I: input), flow-through (FT), the first wash (W1), third wash (W3), and eluate (E) from the Pro-Q column. Phosphorylated ribosomal protein S6 (phospho-S6) was used as a positive control (bottom). (B) Binding of She2 to the Pro-Q column depends on its phosphorylation state. Yeast extracts were treated with Fast phosphatase (+phosphatase) or incubated without phosphatase (−phosphatase) prior to binding to the Pro-Q column. Detection of She2-myc was performed as in A. (C) Denaturation of She2 with urea does not reduce its binding to the Pro-Q column. Yeast extracts were treated with 8M urea (+urea) or not (−urea) prior to binding to the Pro-Q column. Detection of She2-myc was performed as in A. (D) Sequence of She2 with phosphorylated amino acids identified by LC–MS/MS colored in red. Amino acids at the dimerization interface of She2 are underlined. Amino acids in the dimer-dimer interface of She2 are in bold. Amino acids in blue are phosphosites identified in previous studies. (E) Structure of the She2 tetramer. The locations of the phosphorylated residues T47, S91, S101, and T109 in the 3D structure are highlighted. She2 3D structure was generated with the PyMOL software using the 5M0J structure from the PDB database.
