Deletion of tricalbin proteins causes vacuole fragmentation

(A, B) Cells (FKY2577 and FKY2927 in A; FKY2577, FKY2909, FKY3819, FKY2924, FKY3023, FKY3820 and FKY3008 in B) were grown overnight at 25 °C in YPD. Then vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of three independent experiments, each based on more than 100 cells. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t-test compared with WT.

(C) Cells (FKY2577 and FKY2927) were grown overnight at 25 °C in YPD. Cells were then incubated in sterile distilled water (SDW) for more than 45 minutes or YPD with 200 nM of rapamycin (Rap) for 2 hours. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of more than three independent experiments, each based on more than 100 cells. *p < 0.05, **p < 0.01 by Student’s t-test compared with none treated cells. (A-C) Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

(D) Cells (FKY2577 and FKY2927) transformed with pRS416-SCH9-5HA were cultured in YPD, treated with 200 nM rapamycin (control) or untreated. The extracts from cells expressing Sch9-5HA were reacted with 2-nitro-5-thiocyanobenzoic acid and analyzed by immunoblotting using anti-HA. Phosphorylated Sch9 relative to the total Sch9 was calculated and shown in comparison to untreated WT cells. The data represent mean ± SE of three independent experiments. *p < 0.05 by Student’s t-test.

E) Illustration shows that tricalbin proteins negatively regulate the vacuole fission in a TORC1-independent manner.

Effects of domain deletion and artificial tether on vacuole morphology

(A) Diagram of domain organization of Tcb3 protein. TM, transmembrane domain; SMP, synaptotagmin-like mitochondrial lipid-binding protein; C2, calcium-dependent lipid-binding domain; GBP, GFP-binding protein.

(B) Cells (FKY2577 (i), FKY2924 (ii), FKY3903 (iii), FKY3904 (iv), FKY3905 (v) and FKY4754 (vi)) were grown overnight at 25 °C in YPD. Then vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of three independent experiments, each based on more than 100 cells. *p < 0.05 and **p < 0.01 by Student’s t-test compared with Tcb1 Tcb2 Tcb3 (i). Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

(C) The modeled structures of the Tcb1p, Tcb2p and Tcb3p proteins. The ribbons and arrows indicate alpha-helices and beta-sheets, respectively.

(D) TM domain complex in Tcb1p (red), Tcb2p (green), and Tcb3p (blue). The rank “X” indicates the order in which the complexes are most likely to form.

(E) Illustrations show that TM domain of Tcb3 contributes to mediating protein interactions between the tricalbin family to maintain vacuolar morphology.

Accumulated PHS in tcb1Δ2Δ3Δ causes vacuole fragmentation.

(A) Cells (FKY2577 and FKY2927) were grown at 25°C, and labeled with [3H]DHS for 3 hours. Labelled lipids were applied to TLC plates using solvent system (Chloroform-methanol-4.2N ammonium hydroxide (9:7:2, v/v/v)). Incorporation of [3H]DHS into each lipid was quantified and the percentage of the total radioactivity (%) in WT cells was determined. Data represent mean ± SE of four independent experiments. ** P < 0.01 by Student’s t-test.

(B-D, F) Cells (FKY5687 and FKY5688 in B; FKY3340 and YKC121-59 in C; FKY36, FKY37, FKY33 and FKY38 in D; FKY2927 in F) were grown overnight at 25 °C in YPD. PHS was added at 160 µM (C) or 80 µM (D) for 2 hours. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of three independent experiments, each based on more than 100 cells. *p < 0.05, **p < 0.01 and ***p<0.001 by Student’s t-test compared with WT (B, D) or empty cells (F). Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

(F) Illustration showing intracellular utilization pathway of exogenous PHS.

NVJ is required for PHS-induced vacuole fragmentation

(A-C) Cells (FKY2929 in A; FKY3868 and FKY5560 in B; FKY6187, FKY6189, FKY6190, FKY6188 and FKY6409 in C) were grown overnight at 25 °C in YPD. PHS was added at 40 µM for 2 hours at 30 °C (A) and 25 °C (A, B). Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of three independent experiments, each based on more than 100 cells. *p < 0.05, **p < 0.01 and ***p<0.001 by Student’s t-test. Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

(D) Membrane contact sites regulate vacuole morphology via sphingolipid metabolism. See the main text for details.

NVJ and PHS accumulation mediate hyperosmotic shock-induced vacuole fission

(A) Cells (FKY6187 and FKY6140) were grown overnight at 25 °C in YPD, incubated with 80 µM of PHS or 0.2 M of NaCl for 2 hours. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. (B) Cells (FKY2577) were grown at 25°C then labeled with [3H]DHS and incubated with 0.2 M or 0.8 M of NaCl for 2 hours. Labelled lipids were applied to TLC plates using solvent system (Chloroform-methanol-4.2N ammonium hydroxide (9:7:2, v/v/v)). Incorporation of [3H]DHS into each lipid was quantified and the percentage of the total radioactivity (%) in WT cells was determined. Data represent mean ± SE of four independent experiments. (C) Cells (FKY2577) were grown overnight at 25 °C in SD, then incubated with 0.2 M of NaCl for 2 hours. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. (A-C) The data represent mean ± SE of three independent experiments. *p < 0.05, **p < 0.01 and ***p<0.001 by Student’s t-test. Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

(A) Cells (FKY2577, FKY2909, FKY3819, FKY2924, FKY2927, FKY3340 and YKC112-01) were adjusted to OD600 = 1.0 and fivefold serial dilutions were then spotted on YPD plates of indicated pH, then incubated at 25 °C.

(B) GFP-Cps1p fusion protein was transformed into WT (FKY2577), tcb3Δ (FKY2924), WT (FKY3340) and vps4Δ (YKC149-61) strains and analyzed its subcellular localization with respect to FM4-64-stained vacuoles using by fluorescent microscopy.

Coimmunoprecipitation assay between Tcb3-HA and Tcb1-GFP or Tcb2-GFP. Tcb1-GFP (FKP1127), Tcb2-GFP (FKP1131) or GFP (FKP851) plasmids were transformed into TCB3-HA pep4Δ strain (FKY3209). Cells were grown overnight at 25 °C in semi-SD (SD medium containing 0.2% yeast extract). Enriched ER fractions were processed for coimmunoprecipitation. Bound material (IP) was separated by SDS-PAGE and analyzed by immunoblotting using antibodies against HA and GFP. Total represents a fraction of the solubilized input material. Single star (*) indicates presumable degradation products. Double star (**) indicates presumable phosphorylation or other modification bands of Tcb1-GFP or Tcb2-GFP.

Cells (FKY3340, YKC145-21 and YKC149-61) were grown overnight at 25 °C in YPD. PHS was added at 80 µM for 2 hours. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of three groups. The data represent mean ± SE of three independent experiments, each based on more than 100 cells. *p < 0.05, **p < 0.01 and ***p<0.001 by Student’s t-test. Significant differences analysis between the pairwise combination of groups was performed using two-way ANOVA.

Yeast strains used in this study, Related to All Figures.

Plasmids used in this study, Related to All Figures.