Gin4 directly phosphorylates Elm1 to regulate its bud neck localization. (A) In vitro kinase assay results for GST-Elm1^KD incubated with 6xHis-SUMO-Gin4 separated by SDS-PAGE and Coomassie Blue stained. Yellow boxes indicate regions excised for mass spectrometry analysis. (B) Protein schematic of Elm1 with indicated domain boundaries labeled with amino acid positions. Green vertical lines indicate the position of a phosphorylated residue discovered via mass spectrometry. Those sites from left to right are S324, S422, S424, T463, S519, S604, and S605. (C) Representative images of cells for GFP-Elm1^WT (YEF11679), GFP-Elm1^S519A (YEF11680), GFP-Elm1^S519D (YEF11681), GFP-Elm1^7A (YEF11682), and GFP-Elm1^7D (YEF11683) in green and mRuby2-Tub1 in magenta. The images show maximum projections. Scale bars = 2 μm. (D) Representative images of the indicated gin4∆ cells with GFP-tagged Elm1 mutants in green and mRuby2-Tub1 in magenta. Strains used are as follows from left to right: YEF11708 (gin4∆ GFP-ELM1^WT), YEF11709 (gin4∆ GFP-elm1^S519A), YEF11710 (gin4∆ GFP-elm1^S519D), YEF11711 (gin4∆ GFP-elm1^7A), and YEF11712 (gin4∆ GFP-elm1^7D). The images show maximum projections. The dotted line indicates the cell periphery. Scale bars = 2 µm. (E) In vitro binding assay results for the indicated GST-tagged proteins bound to glutathione resin and their ability to pull down His-SUMO-Gin4. Top: Immunoblotted (IB) with antibody against 6x-His; bottom: Coomassie Blue stained. This experiment was repeated three times and a representative immunoblot is shown. (F) Quantification of GST-Elm1 mutants binding ability to 6xHis-SUMO-Gin4. The formula used to calculate values = (membrane band intensity of Gin4/gel band intensity of Elm1)/WT calculated value. Source data are available for this figure: SourceData F5.