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. Author manuscript; available in PMC: 2020 Oct 21.
Published in final edited form as: Dev Cell. 2019 Sep 26;51(2):173–191.e5. doi: 10.1016/j.devcel.2019.08.017

Figure 4. Bem1 interaction with Slt2 facilitates translocation of septin rings at CRMs during ER stress.

Figure 4.

(A) Bem1-GFP in unstressed WT cells was localized to the bud tip, bud neck, and CRMs. All scale bars, 2 μm.

(B) Bem1-GFP localization was altered in Tm-treated WT cells. CRMs were visualized by staining with WGA-594. Zoomed-in views of Bem1-GFP at CRMs are shown.

(C) Split-YFP PCA between Slt2 and Bem1 in untreated WT cells (no Tm).

(D) Split-YFP PCA between Slt2 and Bem1 in cells treated with 1μg/ml Tm. Close-up views of Slt2 interacting with Bem1 at CRMs are also shown.

(E) co-IP of experiment with Bem1-GFP and Slt2-Myc (lane 6). Spa2 is a previously identified Slt2 binding protein (lane 5).

(F) The reconstituted YFP of the Slt2-Bem1 interaction (strain shown in C–D) was pulled down using anti-GFP beads. Cells were treated or untreated with Tm. Western blots for phosphorylated Slt2 (P-Slt2) and total Slt2 are shown.

(G) Split-YFP PCA between Shs1 and Bem1 in untreated (no Tm) and Tm-treated WT cells. Close-up views show Shs1 interacting with Bem1 localized at the bud neck in untreated cells and at CRMs in Tm-treated cells. Zoomed-in views showing PCA signals at the bud neck for unstressed and at CRMs for ER-stressed cells are shown.

(H) Shs1-GFP or Cdc10-GFP in unstressed cells was localized to the bud neck of bem1Δ cells, whereas Shs1-GFP or Cdc10-GFP was mislocalized away from the bud neck but was localized outside of CRMs in Tm-treated bem1Δ cells. Zoomed-in views show Shs1-GFP at the bud neck in unstressed cells and at a location distinct from CRMs in ER-stressed cells.

(I) Quantification of Shs1-GFP in bem1Δ cells shows that significant levels of Shs1-GFP become mislocalized upon ER stress induction. *, p < 0.01; **, p < 0.001; ns, not significant.

(J) Cartoon diagram summarizing the findings in this figure. Under normal conditions, Slt2 interacts with Bem1 at the bud tip while Shs1 is at the bud neck. None of these polarity components is seen at CRMs. When ER stress is triggered, Bem1 recruits Shs1 and phosphorylated Slt2 to CRMs.