Gin4 regulates Elm1 localization through direct binding and phosphorylation. (A) Top: Representative image of YEF10672 (gin4^∆KA1ELM1-GFP CDC3-mCherry) cells with brightfield (left) and maximum-intensity projection of Elm1-GFP in green (middle) and Cdc3-mCherry in magenta (right). Gray dashed line is the cell periphery. Scale bar = 5 μm. Bottom: Protein map of Gin4^ΔKA1 with relevant domains labeled. Numbers indicate amino acids of labeled domain boundaries. The KA1 domain of Gin4 (amino acids 1008–1142) has been removed. (B) Top: In vitro binding assay results for indicated GST-tagged proteins bound to glutathione resin and indicated 6xHis-SUMO–tagged protein separated by SDS-PAGE and Coomassie Blue stained. Yellow boxes indicate purified GST-tagged protein. Bottom: In vitro binding assay results for indicated GST-tagged proteins bound to glutathione resin and indicated 6xHis-SUMO–tagged protein separated by SDS-PAGE and immunoblotted with anti-6xHis antibody. This experiment was repeated three times with consistent interaction detected for the full-length Elm1, strong interaction for its C-terminal fragment, and weak or no interaction for its N-terminal fragment. (C) Montages of representative YEF10673 (Elm1-GFP in green and Gin4^KD-mScarlet in magenta) cells showing maximum-intensity projections from 12 min before to 60 min after bud emergence from time-lapse series taken with a 2-min interval. T = 0 is bud emergence, scale bars = 1 μm. (D) Quantification of Elm1-GFP signal from cells in C. Shown is integrated measured background subtracted intensity in Gin4^WT (YEF10802, green) and Gin4^KD (light green) from the sum projection of given number cells for each strain. The mean is plotted with error bars being the SD. a.u. = arbitrary units. (E) Quantification of Gin4-mScarlet signal from cells in C. Shown is the integrated measured background subtracted intensity in Gin4^WT (YEF10802, magenta) and Gin4^KD (tan) from the sum projection of the given number of cells for each strain. The mean is plotted with error bars being the SD. a.u. = arbitrary units. (F) Top: In vitro binding assay results for the indicated GST-tagged proteins bound to glutathione resin and indicated 6xHis-SUMO–tagged protein separated by SDS-PAGE and Coomassie Blue stained. Yellow boxes indicate purified GST-tagged protein. Bottom: In vitro binding assay results for the indicated GST-tagged proteins bound to glutathione resin and indicated 6xHis-SUMO–tagged protein separated by SDS-PAGE and immunoblotted (IB) with anti-6xHis antibody. This experiment was repeated two times with indistinguishable interaction differences detected between 6xHis-SUMO-Gin4^WT and 6xHis-SUMO-Gin4^KD to the C-terminal non-kinase domain of Elm1. Source data are available for this figure: SourceData F4.