Membrane contact sites regulate vacuolar fission via sphingolipid metabolism
- Graduate School of Integrated Sciences for Life, Hiroshima University, Japan
- School of Applied Biological Science, Hiroshima University, Japan
Figures
Figure 1 with 1 supplement
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 …
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Figure 1—source data 1
Excel file of numerical data represented as a graph in Figure 1A.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data1-v1.xlsx
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Figure 1—source data 2
Excel file of numerical data represented as a graph in Figure 1B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data2-v1.xlsx
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Figure 1—source data 3
Excel file of numerical data represented as two graphs in Figure 1C.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data3-v1.xlsx
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Figure 1—source data 4
Excel file of numerical data represented as a graph in Figure 1D.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data4-v1.xlsx
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Figure 1—source data 5
Original file for the Western blot analysis in Figure 1D.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data5-v1.zip
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Figure 1—source data 6
JPEG containing Figure 1D and original scans of the relevant Western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig1-data6-v1.zip
Figure 1—figure supplement 1
Vacuolar acidification and Cps1p delivery are not affected by tricalbin deletion.
(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 …
Figure 2 with 1 supplement
Effects of domain deletion 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, …
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Figure 2—source data 1
Excel file of numerical data represented as a graph in Figure 2B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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-synthetic dextrose (SD) (SD medium …
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Figure 2—figure supplement 1—source data 1
Original file for the Western blot analysis in Figure 2—figure supplement 1 (anti-HA).
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original file for the Western blot analysis in Figure 2—figure supplement 1 (anti-GFP).
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
JPEG containing Figure 2—figure supplement 1 and original scans of the relevant Western blot analysis (anti-HA and anti-GFP) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig2-figsupp1-data3-v1.zip
Figure 3
Accumulated phytosphingosine (PHS) in tcb1Δ2Δ3Δ causes vacuole fragmentation.
(A) Cells (FKY2577 and FKY2927) were grown at 25°C, and labeled with [3H]DHS for 3 hr. Labeled lipids were applied to thin-layer chromatography (TLC) plates using solvent system …
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Figure 3—source data 1
Original file for the thin-layer chromatography (TLC) analysis in Figure 3A.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data1-v1.zip
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Figure 3—source data 2
JPEG containing Figure 3A and original scans of the relevant thin-layer chromatography (TLC) analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data2-v1.zip
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Figure 3—source data 3
Excel file of numerical data represented as a graph in Figure 3A.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data3-v1.xlsx
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Figure 3—source data 4
Excel file of numerical data represented as a graph in Figure 3B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data4-v1.xlsx
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Figure 3—source data 5
Excel file of numerical data represented as two graphs in Figure 3C.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data5-v1.xlsx
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Figure 3—source data 6
Excel file of numerical data represented as two graphs in Figure 3D.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data6-v1.xlsx
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Figure 3—source data 7
Excel file of numerical data represented as a graph in Figure 3F.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig3-data7-v1.xlsx
Figure 4 with 1 supplement
Nucleus–vacuole junction (NVJ) is required for phytosphingosine (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 hr at 30°C (A) and 25°C (A…
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Figure 4—source data 1
Excel file of numerical data represented as two graphs in Figure 4A.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig4-data1-v1.xlsx
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Figure 4—source data 2
Excel file of numerical data represented as two graphs in Figure 4B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig4-data2-v1.xlsx
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Figure 4—source data 3
Excel file of numerical data represented as two graphs in Figure 4C.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
Cells (FKY3340, YKC145-21, and YKC149-61) were grown overnight at 25°C in YPD.
Phytosphingosine (PHS) was added at 80 µM for 2 hr. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. The number of vacuoles per cell was counted and categorized into one of …
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Figure 4—figure supplement 1—source data 1
Excel file of numerical data represented as two graphs in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig4-figsupp1-data1-v1.xlsx
Figure 5
Nucleus–vacuole junction (NVJ) and phytosphingosine (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 hr. Vacuoles were stained with FM4-64 and imaged by fluorescence microscopy. …
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Figure 5—source data 1
Excel file of numerical data represented as three graphs in Figure 5A.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig5-data1-v1.xlsx
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Figure 5—source data 2
Original file for the thin-layer chromatography (TLC) analysis in Figure 5B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig5-data2-v1.zip
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Figure 5—source data 3
JPEG containing Figure 5B and original scans of the relevant thin-layer chromatography (TLC) analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig5-data3-v1.zip
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Figure 5—source data 4
Excel file of numerical data represented as two graphs in Figure 5B.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig5-data4-v1.xlsx
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Figure 5—source data 5
Excel file of numerical data represented as two graphs in Figure 5C.
- https://cdn.elifesciences.org/articles/89938/elife-89938-fig5-data5-v1.xlsx
Additional files
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Supplementary file 1
Yeast strains used in this study.
Related to all figures.
- https://cdn.elifesciences.org/articles/89938/elife-89938-supp1-v1.xlsx
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Supplementary file 2
Plasmids used in this study.
Related to all figures.
- https://cdn.elifesciences.org/articles/89938/elife-89938-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89938/elife-89938-mdarchecklist1-v1.pdf
