Phosphorylation at S519 in Elm1 is not necessary for Elm1 localization to the bud neck. (A) Montages of representative cells of Elm1^WT, Elm1^S519A, and Elm1^S519D tagged with GFP in green and Cdc3-mCherry shown in magenta. The images show maximum-intensity projections of the indicated fluorescent protein from 20 min before to 40 min after bud emergence with selected frames from time-lapse series taken with a 2-min interval. Strains used are as follows: YEF11688 (GFP-ELM1^WTCDC3-mCherry), YEF11689 (GFP-elm1^S519ACDC3-mCherry), and YEF11690 (GFP-elm1^S519DCDC3-mCherry). The dashed line indicates the cell periphery. Scale bars = 2 µm. (B) Quantification of the cells in A. Top panel is the background subtracted integrated intensity measured of GFP-Elm1 from the sum projection of a given number of cells for each strain. The mean is plotted with error bars being SD. a.u. = arbitrary units. Bottom panel is the background subtracted integrated intensity measured of Cdc3-mCherry (open circles) and GFP-Elm1 (crosses) in the given number of cells per strain at 20 min after bud emergence. Each plotted point is a single cell’s bud neck measured intensity. ns = not significant (P > 0.05) by unpaired Student’s t test. (C) Montages of representative cells of Elm1^WT, Elm1^S519A, and Elm1^S519D tagged with GFP in green and Cdc3-mCherry shown in magenta. The images show maximum-intensity projections of the indicated fluorescent protein from 40 min before to 8 min after septin HDR transition with selected frames from a time-lapse series taken with a 2-min interval. Strains used are the same as listed in A. The dashed line indicates the cell periphery. Scale bars = 2 µm. (D) Quantification of the cells in C. Top panel is the background subtracted integrated intensity measured of GFP-Elm1 from the sum projection of a given number of cells for each strain. The mean is plotted with error bars being SD. a.u. = arbitrary units. Bottom panel is the background subtracted integrated intensity measured of Cdc3-mCherry (open circles) and GFP-Elm1 (crosses) in the given number of cells per strain at 20 min after bud emergence. Each plotted point is a single cell’s bud neck measured intensity. ns = not significant (P > 0.05), * = P < 0.05, and ** = P < 0.01 by unpaired Student’s t test. (E) Western blot analysis of Elm1 phospho-site mutant protein expression compared with Elm1^WT. Strains used are as follows: YEF11665 (GFP-ELM1^WT), YEF11666 (GFP-elm1^S519A), YEF11667 (GFP-elm1^S519D), YEF11668 (GFP-elm1^7A) YEF11669 (GFP-elm1^S519D), and YEF2232 (no GFP control). Top: immunoblotted with antibody anti-GFP; bottom: immunoblotted with antibody anti-Cdc28 as the loading control. This experiment was repeated three times. Source data are available for this figure: SourceData FS3.