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. 2024 Nov 15;15:9910. doi: 10.1038/s41467-024-54141-0

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Exponentially growing ypk1-as ypk2Δ cells (GA-5893) were treated with 1% DMSO (mock), 75 μg/ml Zeocin (Ypk1^on+Zeo), 0.5 μM 1NM-PP1 (Ypk1^off), or both for 80 min, and genomic DNA was subjected to CHEF gel analysis as in Fig. 1b. b Treatments as in a, as well as wild-type yeast in 750 μg/ml Zeocin (high Zeo), were for 80 min, performed in triplicate. Extracted proteins were analyzed by label-free mass spectrometry (see Methods⁵⁸). Phosphopeptides were triaged for change >2-fold vs DMSO control. Venn diagrams show phopshopeptides up (reddish) or down (green tones) in Ypk1^off + Zeo vs. Ypk1^off or Ypk1^on + Zeo. Ypk1^off + Zeo-specific targets are darker in the Venn diagrams. From this subgroup we subtracted phosphopeptides altered by Zeocin alone (80 up; 27 down), leaving 201 up- and 188 downregulated phosphopeptides, in 302 proteins. c GO-term analysis of the Ypk+Zeocin-specific phosphoproteome was carried out, and significance was determined vs a total number of factors in yeast in that category using the hypergeometric distribution. Cytoskeletal organization was most significantly enriched. d A cluster of interacting regulators of yeast endocytosis and actin cytoskeleton (image based on⁶⁴), of which all except two (blue) were recovered as Ypk+Zeocin-sensitive phosphoacceptors. Red-labeled proteins gain phosphorylation, green lose it. e Each bar represents a specific phosphotarget site involved in actin filament regulation and endo- and exocytosis, plotted as log2 change of increased (reddish) or decreased (green) phosphorylation by indicated treatments vs DMSO. Individual values are from biological triplicates; tops of bars are median values. Relevant target sites are: Akl1: S541, S496, S504; Prk1, S553, S556, S560; Las17, T380; Pan1, S1003; S1253 or T1256; S991. T993, T995; Sla1, S449; Abp1, T206; T211; Ent1, T160, T163; T395, T388; Ent2, T468, T470, T479; Ark1. S478; Sla2, T468. T470, T479; Bni1, S325, S327, some defining overlapping phosphopeptides. Arrows indicate proteins studied further.