Protein relocalization in response to SDS treatment.

(A) Schematic representation of screening methodology and images of Pkc1-GFP with or without 0.02 % SDS treatment are shown. Scale bar, 5 μm. (B) Overlap of screening hits of C- and N-terminal libraries. P value for the significance of the overlap is indicated. Fisher’s exact test was performed. (C) GO analysis of biological processes for the screening hits. Successfully observed proteins were used as the background protein sets. (D) Screening hits for six relocalization classes and the images of representative proteins in each class were shown. Numbers in parentheses indicate the number of proteins in the class. Scale bar, 2 μm.

Laser damage assays identified 80 repair protein candidates.

(A) Schematic representation of screening methodology. Cells were imaged for 25 min in 30-second intervals after 405 nm laser damage. (B) 90 proteins change localization after laser damage. The classification of the repair protein candidates based on their localization changes, representative proteins, and representative biological processes in each category is shown. Scale bar, 2 μm. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. (C) Classification of repair protein candidates based on subcellular localization and domain. TMD+ represents the proteins that have transmembrane domains. The existence of transmembrane domains was predicted by TMHMM (60) (D) GO analysis of molecular functions for the repair protein candidates. The proteins whose localization changes in response to SDS treatment were used as background protein sets. (E) GO analysis of biological processes for the repair protein candidates. The proteins whose localization changes in response to SDS treatment were used as background protein sets.

The temporal order of the Pkc1 accumulation, polarized exocytosis, and CME at the damage site.

(A) The representative images and normalized fluorescence intensity of Exo70-mNG (Green) and Pkc1-mSc-I (Purple) at the damage site after laser damage. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. n = 10 cells. (B) The representative images and normalized fluorescence intensity of Pkc1-sfGFP (Green) and Dnf1-mSc-I (Purple) at the damage site after laser damage. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. n = 8 cells. (C) Representative images, kymograph at the damage site, and fluorescence intensity at the damage site of Exo70-mNG (Green) and Ede1-mSc-I (purple). n = 10 cells. Yellow arrows show the damage site. White arrows show the coaccumulation of Exo70-mNG and Ede1-mSc-I at the damage site. (D) Representative images, kymograph at the damage site, and fluorescence intensity at the damage site of Dnf1-mNG (Green) and Ede1-mSc-I (purple). n = 9 cells. Yellow arrows show the damage site. White arrows show the coaccumulation of Dnf1-mNG and Ede1-mSc-I. Lines and shaded regions are the mean and the standard error of the mean.

CME proteins are required for polarized exocytosis at the damage site.

(A) The quantification results of fluorescence intensity of Myo2-sfGFP at the damage site and at the bud tip. n = 13 for WT, n = 12 for end3Δ, sla1Δ, rvs167Δ, and vrp1Δ. (B) The quantification results of the fluorescence intensity of Exo70-mNG at the damage site. n = 18 for WT, n = 12 for end3Δ, sla1Δ, and rvs167Δ, n = 14 for vrp1Δ. Lines and shaded regions are the mean and standard error of the mean. Maximum fluorescence intensity at the damage site or fluorescence intensity changes at the bud tip were compared between WT and mutants using Dunnett’s multiple comparison test.

CME at the bud tip directs repair proteins with TMDs to the damage site.

(A) Representative images and the normalized fluorescence intensity at the bud tip of Dnf1-mNG and Ede1-mSc-I. Yellow arrows show the damage site. White arrows show the recruitment of fluorescence signals. (B) Kymograph of Dnf1-mNG and Ede1-mSc-I at the bud tip in WT and rvs167Δ. n = 10 cells. (C)-(F) Max fluorescence intensity at the damage site and fluorescence intensity changes at the bud tip in WT and rvs167Δ. n = 10 cells for Dnf1-mNG. n = 11 cells for Slg1-sfGFP. n = 12 cells for Sho1-GFP. n = 10 cells for mNG-Snc1. Welch’s t-test was performed.

mNG-Snc1 is recovered from the damage site to the bud tip after PM repair.

(A) Schematic of transient expression induction of mNG-Snc1 by Gal1 promoter and representative images of mNG-Snc1 and Ede1-mSc-I. After transcription activation of the Gal1 promoter by adding 3% galactose, we stop the expression by transferring the cells to glucose media. The cells were incubated for at least 3 hours before the laser damage assay. Yellow arrows show the damage site. White arrows showed the recruitment of fluorescence signals. (B) Quantification of mNG-Snc1 (Green) and Ede1-mSc-I (purple) at the damage site. (C) Quantification of mNG-Snc1 at the bud tip (Green) and at the damage site (tomato). (D) The changes in the normalized mNG-Snc1 signal at the bud tip. n = 8 cells.

Model of spatiotemporal cellular responses to PM damage in budding yeast.

We showed the hypothetical model of spatiotemporal PM damage responses in budding yeast. First, the degradation of Sec3 and Bni1 resolved the polarity competition between the bud tip and the damage site (26). Within 10 min after laser damage, CME directs repair proteins with TMDs to the damage site from the bud tip. At the damage site, polarized exocytosis and CME simultaneously occur, with exocytosis predominating approximately within 20 min and with CME predominating approximately 20 min after laser damage. CME targets repair proteins with TMDs from the bud tip to the damage site. The endocytosed PM proteins are retargeted to the bud tip again after the PM repair is finished. The retargeted PM proteins may be involved in the resumption of cell growth after PM repair. The numbers represent the temporal order of events.