ESCRTs function directly on the lysosome membrane to downregulate ubiquitinated lysosomal membrane proteins
- Cornell University, United States
- University of Michigan, United States
Figures
Figure 1 with 1 supplement
Genetic selection for mutations that block the Ypq1 sorting pathway.
(A) Fluorescent microscopy analysis of cells expressing Ypq1-SBP-GFP and vacuole membrane marker Vph1-mCherry in wild-type and (B) ssh4Δ cells grown to mid-log phase at 30°C. (C) Cartoon depicting genetic selection: wild-type cells constitutively degrade Ypq1-SBP-GFP-His3 via the vacuole membrane protein degradation pathway. This effectively removes His3 from the cytoplasm, making WT cells His-. Degradation mutants, however, prevent Ypq1 sorting, this leaves His3 exposed to the cytoplasm, making them His+. (D) Cell growth assay comparing wild-type and ssh4Δ mutant cells grown in the presence, or absence of histidine. Scale bars represent 2 μm.
Figure 1—figure supplement 1
Ypq1-SBP-pHluorin visual screen.
(A) Cartoon model of the Ypq1-SBP-pHluorin screen. (B) Fluorescent microscopy and flow cytometry analysis of Ypq1-SBP-pHluorin in wild-type and ssh4Δ mutant cells. (C) Plot showing the fold change in fluorescence intensity to WT cells of the BY4741 non-essential gene knock-out collection. X-axis plots mutants in order of fluorescence intensity, and Y-axis represents the fluorescence intensity fold change. Fold change=[fluorescence(mutant) – fluorescence(background)]/ [fluorescence(WT) – fluorescence(background)]. Forty-six mutants exhibit fivefold or greater fluorescence intensity were shown in the dotted black box. (D) Plot showing mutants with fivefold or greater fluorescence intensity to WT cells. The scale bar represents 2 μm.
Figure 2 with 4 supplements
Ypq1 ubiquitination defect is bypassed by RapiDeg system.
(A) Fluorescent microscopy analysis of cells expressing Ssh4-mNeonGreen and Vph1-mCherry in wild-type, and vps27Δ(ESCRT-0) cells. (B) Ypq1-GFP was immunoprecipitated using GFP-trap resin from doa4Δ (WT), and doa4Δvps27Δ cells expressing Myc-ubiquitin. Cells were collected before and 2 hr after lysine withdrawal at 26°C. Both input and immunoprecipitated protein samples were analyzed using Western blot and probed with GFP and Myc antibodies. (C) A cartoon depicting the RapiDeg system. One (or two) FKBP peptide is fused to the C-terminus of Ypq1-GFP. A chain of three ubiquitins is fused to the C-terminus of the FRB peptide. After adding rapamycin, FRB-3xUb is recruited to Ypq1-FKBP. (D) Fluorescent microscopy analysis of the RapiDeg assay. Images show cells co-expressing Ypq1-FKBP and FRB-3xUb before (top) and after rapamycin treatment (1 μg/ml) (bottom) at 30°C. Line scan of both Vph1-mCherry and Ypq1-FKBP was performed using ImageJ (right). (E) Western blot analysis of the RapiDeg assay. Whole cell lysates from 0, 15, 30, and 45 min after rapamycin treatment (1 μg/ml) at 30°C. Blots were probed with anti-GFP and anti-G6PDH antibodies. (F) Western blot analysis the RapiDeg assay in WT and ssh4Δ cells after rapamycin treatment. Blots were probed with anti-GFP and anti-G6PDH antibodies. Scale bars represent 2 μm.
Figure 2—figure supplement 1
Ssh4 localization and Ypq1 ubiquitination assay in endosome-vacuole fusion mutants.
(A) Ssh4-mNeonGreen localization and FM4-64 (vacuole membrane) straining in vam3ts and vps18ts mutants. Cells were grown at 26°C and shifted to -Lysine media at 37°C for 2 hr. Arrows indicate representative Ssh4-mNeonGreen localized at pre-vacuolar compartments. (B) Ypq1-GFP was immunoprecipitated using GFP-trap resin from doa4Δ (WT), doa4Δvps18ts, and doa4Δvam3ts cells expressing Myc-ubiquitin. Cells were grown at 26°C and shifted to 37°C (non-permissive temperature) 15 min prior to lysine starvation. Cells were harvested after 2 hr lysine starvation at 37°C. Both input and immunoprecipitated protein samples were analyzed using Western blot and probed with GFP and Myc antibodies. The scale bar represents 2 μm.
Figure 2—figure supplement 2
FRB-3xUb is required for rapamycin-induced Ypq1 degradation.
(A) Fluorescent microscopy analysis of the RapiDeg assay. Images show cells with or without FRB-3xUb after rapamycin treatment (1 μg/ml) at 30°C. (B) Western blot analysis of the RapiDeg assay with or without FRB-3xUb. Whole cell lysates from both conditions were taken 0, 15, 30 and 45 min after rapamycin treatment (1 μg/ml) at 30°C. Blots were probed with anti-GFP and anti-G6PDH antibodies. The scale bar represents 2 μm.
Figure 2—figure supplement 3
The RapiDeg assay is highly specific.
(A) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Ypq1-mCherry. Images show cells that are untreated (top), and rapamycin treated (bottom). (B) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Ypq1-mCherry. Images show cells grown in SC-Ura (Top), and cells starved of lysine for 6 hr (Bottom). (C) Fluorescent microscopy analysis of ssh4Δ cells expressing Ypq1-FKBP and Ypq1-mCherry. Images show cells which have been starved of lysine for 6 hr. Scale bars represent 2 μm.
Figure 2—figure supplement 4
The RapiDeg assay can be used to degrade membrane proteins from the PM, Golgi, vacuole, and endosome.
(A) Fluorescent microscopy analysis of cells expressing the PM transporter Can1-FKBP and vacuole transmembrane protein Vph1-mCherry in wild-type cells. Images show cells before and after rapamycin treatment at 30°C. (B) Western blot analysis of Can1-FKBP degradation in wild-type cells grown at 30°C after rapamycin treatment. The blot was probed with GFP and G6PDH antibodies. (C) Fluorescent microscopy analysis of cells expressing the Golgi transmembrane protein Kex2-FKBP and vacuole transmembrane protein Vph1-mCherry. Images show cells before and after rapamycin treatment. (D) Western blot analysis of Kex2-FKBP degradation in wild-type cells grown at 30°C after rapamycin treatment. The blot was probed with GFP and G6PDH antibodies. (E) Fluorescent microscopy analysis of cells expressing the vacuole membrane proteins Vph1-FKBP and mCherry-ALP(pho8). Images show cells before and after rapamycin treatment. (F) Western blot analysis of Vph1-FKBP degradation in wild-type cells grown at 30°C after rapamycin treatment. The blot was probed with GFP and G6PDH antibodies. (G) Fluorescent microscopy analysis of cells expressing the endosome transmembrane protein Nhx1-FKBP and vacuole transmembrane protein Vph1-mCherry. Images show cells before and after rapamycin treatment. (H) Western blot analysis of Nhx1-FKBP degradation in wild-type cells grown at 30°C after rapamycin treatment. The blot was probed with GFP and G6PDH antibodies. Scale bars represent 2 μm.
Figure 3 with 1 supplement
Ypq1 sorting does not require membrane fusion.
(A) Fluorescent microscopy analysis of Ypq1-FKBP and Vph1-mCherry in vps18ts cells at non-permissive temperature (37°C). The vps18ts cells were grown at 26°C then pretreated at 37°C for 15 min. Images show before (0 min) and after (30 min) rapamycin treatment (RapiDeg). (B) Western blot analysis of Ypq1-FKBP degradation. Wild-type and vps18ts cells were shifted to 37°C (15 min pretreatment) and collected at four time points (0, 15, 30, and 45 min) after rapamycin treatment. Blots were probed with GFP and G6PDH antibodies. (C) Fluorescent microscopy analysis of Ypq1-FKBP and Vph1-mCherry in vam7ts cells at 37°C. The vam7ts cells were grown at 26°C then pretreated at 37°C for 15 min. Images show before and after rapamycin treatment (RapiDeg). (D) Western blot analysis of Ypq1-FKBP degradation. Wild-type and vam7ts cells were shifted to 37°C (15 min pretreatment) and collected before and after rapamycin treatment. Blots were probed with GFP and G6PDH antibodies. (E) Fluorescent microscopy analysis of Ypq1-FKBP and Vph1-mCherry in sec18ts cells shifted to 34°C (non-permissive temperature). The sec18ts cells were grown at 26°C then pretreated at 34°C for 15 min. Images show before and after rapamycin treatment. (F) Western blot analysis of Ypq1-FKBP degradation. Wild-type and sec18ts cells were shifted to 34°C(15 min pretreatment) and collected before and after rapamycin treatment. Blots were probed with GFP and G6PDH antibodies. Scale bars represent 2 μm.
Figure 3—figure supplement 1
Mup1-GFP sorting is blocked in the sec18ts, vps18ts or vam7ts mutants at non-permissive temperature.
(A) Fluorescent microscopy analysis of cells expressing Mup1-GFP and Vph1-mCherry in the wild-type, sec18ts, vps18ts and vam7ts cells. Images show cells grown to mid-log phase at 26°C, and shifted to higher temperature before and 60 min after methionine treatment (20 μg/ml) (34°C for sec18ts, 37°C for all others). (B) Western blot analysis of Mup1-GFP degradation in wild-type and vps18ts cells. Cells were grown to mid-log phase at 26°C then shifted to 37°C 15 min prior to adding methionine. The blot was probed with GFP antibody and the blot against G6PDH antibody is used as a loading control. (C) Western blot analysis of Mup1-GFP degradation in wild-type and sec18ts cells. Cells were grown to mid-log phase at 26°C then shifted to 34°C 15 min prior to adding methionine. The blot was probed with GFP antibody and the blot against G6PDH antibody is used as a loading control. (D) Western blot analysis of Mup1-GFP degradation in wild-type and vam7ts cells. Cells were grown to mid-log phase at 26°C then shifted to 37°C 15 min prior to adding methionine. The blot was probed with GFP antibody and the blot against G6PDH antibody is used as a loading control. Scale bars represent 2 μm.
Figure 4 with 1 supplement
In ESCRT mutants, Ypq1 remains on the vacuole membrane after ubiquitination.
(A) Fluorescent microscopy analysis of Ypq1-FKBP and Vph1-mCherry localization in vps4ts cells at 37°C (non-permissive temperature). The vps4ts cells were grown at 26°C then pretreated at 37°C for 15 min. Images show before and after rapamycin treatment. (B) Western blot analysis of Ypq1-FKBP degradation. Wild-type and vps4ts cells were shifted to 37°C (15 min pretreatment) and collected before and after rapamycin treatment. The blot was probed with GFP and G6PDH antibodies. (C) Fluorescent microscopy analysis of GFP-FYVEVps27 and Vph1-mCherry in wild-type and vps34Δ cells. Scale bars represent 2 μm.
Figure 4—figure supplement 1
Ypq1-FKBP sorts to the vacuole lumen in the vps4ts mutant at permissive temperature.
(A) Fluorescent microscopy analysis of vps4ts cells expressing Ypq1-FKBP and Vph1-mCherry grown at 26°C. Images show cells before and after rapamycin treatment. (B) Western blot analysis of Ypq1-FKBP degradation in wild-type and vps4ts cells treated with rapamycin at 26°C. The scale bar represents 2 μm.
Figure 5
ATP-depletion traps ESCRT-dependent Ypq1 sorting intermediates.
(A) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Vph1-mCherry. Images show untreated cells (top), Rapamycin (1 μg/ml, pretreated for 1 min) and NaN3/NaF (10 mM each, final concentration) treated cells (middle), and cells treated with only NaN3/NaF. All cells were grown at 30°C (bottom). (B) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Vph1-mCherry. Images shows cells treated with Rapamycin and NaN3/NaF for 30 min (top), and cells from the same culture that have been washed with water and re-suspended in fresh YPD medium (30 min after wash) (bottom). (C) Western blot analysis of Ypq1-FKBP degradation. Wild-type cells were treated with rapamycin, with or without NaN3/NaF. After washing with deionized water and resuspended in fresh YPD medium, the NaN3/NaF-treated cells were collected at 15 min and 30 min. Cells were grown at 30°C. The blot was probed with GFP and G6PDH antibodies. (D) Fluorescent microscopy analysis of Ypq1-FKBP in wild-type and vps27Δ cells. Images show cells before and 15 min after rapamycin and NaN3/NaF treatment. Scale bars represent 2 μm.
Figure 6 with 3 supplements
ESCRT-0 and -I are recruited to the vacuole membrane after Ypq1 ubiquitination.
(A) Fluorescent microscopy analysis of Ypq1-FKBP and Hse1-mCherry in wild-type cells. Images show localization of Ypq1-FKBP and Hse1-mCherry before and after rapamycin and NaN3/NaF treatment for 15 min. (B) Fluorescent microscopy analysis of Ypq1-FKBP and Vps23-mCherry in wild-type cells. Images show localization of Ypq1-FKBP and Vps23-mCherry before and after rapamycin and NaN3/NaF treatment. (C) Fluorescent microscopy analysis of Ypq1-FKBP and FM4-64 (endosome dye) in wild-type cells after rapamycin and NaN3/NaF treatment at 30°C. Cells were stained with FM4-64 for 5 min prior to rapamycin and NaN3/NaF treatment. Arrows indicate representative co-localization between Ypq1-FKBP and Hse1-mCherry (or Vps23-mCherry). Scale bars represent 2 μm.
Figure 6—figure supplement 1
ESCRT-II protein Vps36 is recruited to the vacuole membrane after Ypq1 ubiquitination.
Fluorescent microscopy analysis of Ypq1-FKBP and Vps36-mCherry in wild-type cells. Images show localization of Ypq1-FKBP and Vps36-mCherry before and after rapamycin and NaN3/NaF treatment for 15 min. Arrows show co-localization between Ypq1-FKBP and Vps36-mCherry. The scale bar represents 2 μm.
Figure 6—figure supplement 2
Ypq1-FKBP and Vps23-mCherry do not co-localize after NaN3/NaF or rapamycin treatment alone.
Fluorescent microscopy analysis of Ypq1-FKBP and Vps23-mCherry in wild-type cells. Images show localization of Ypq1-FKBP and Vps23-mCherry before and after either rapamycin or NaN3/NaF treatment alone for 15 min. The scale bar represents 2 μm.
Figure 6—figure supplement 3
Expression of Hse1DUB, releases ESCRTs from endosomes, allowing for ESCRTs recruitment onto the vacuole membrane in the vps4ts mutant.
(A) Fluorescent microscopy analysis of vps4ts cells expressing Ypq1-FKBP and Vps23-mCherry. Images show cells at 26°C (permissive temperature) (top), cells shifted to 37°C for 15 min (middle), and cells co-expressing Hse1DUB shifted to 37°C for 15 min (bottom). (B) Fluorescent microscopy analysis of vps4ts cells expressing Ypq1-FKBP and Vps23-mCherry shifted to 37°C. Images show cells treated with rapamycin (top), and cells treated with rapamycin co-expressing Hse1DUB (bottom). Arrows indicate co-localization between Ypq1-FKBP and Vps23-mCherry. The scale bar represents 2 μm.
Figure 7
Visualization of Ypq1-FKBP ILVs.
(A–B) EM images of the vacuole in pep12Δpep4Δvma4Δatg9Δ cells (pep12Δ) with or without Hse1DUB (No Rapamycin treatment). (C) EM image of the vacuole in cells expressing Hse1DUB after rapamycin treatment (2 hr) at 26°C. Arrows indicate representative vesicles formed following rapamycin-induced Ypq1 sorting. (D) EM images of the vacuole in pep4Δvma4Δatg9Δ cells (PEP12) without Hse1DUB (No Rapamycin treatment). ‘V’ in the figures (7A~D) is abbreviated for vacuole. (E) Average number of ILVs (per cell per section) calculated by counting ILVs within vacuoles of cells (pep12Δ) without Hse1DUB, with Hse1DUB, and with both Hse1DUB and rapamycin treatment. The mean value of ILVs formed in vacuole lumen was quantified and error bars are standard deviation. Two-tail t-test was performed between pep12Δ(Hse1DUB-/Rap-) and pep12Δ(Hse1DUB+/Rap-), and between pep12Δ(Hse1DUB+/Rap-) and pep12Δ(Hse1DUB+/Rap+). α = 0.025 (Bonferroni correction), p-value<0.001 in each test (***, highly significant), N = 40 cells for each condition. (F) Average diameter of ILVs in cells (PEP12) without Hse1DUB or rapamycin treatment, and in cells (pep12Δ) without Hse1DUB or rapamycin treatment, as well as cells (pep12Δ) with both Hse1DUB and rapamycin treatment. The mean value of ILV size formed in the vacuole lumen was quantified and error bars are standard deviation. ANOVA (single factor) test was performed among PEP12(Hse1DUB-/Rap-), pep12Δ(Hse1DUB-/Rap-), and pep12Δ(Hse1DUB+/Rap+). α = 0.025 (Bonferroni correction), p-value>0.05 (n.s., non-significant), N = 64 vesicles for each condition. (G) Cartoon model depicting Ypq1 sorting from the vacuole membrane into the vacuole lumen. After lysine withdrawal, Ypq1 is ubiquitinated by the Ssh4/Rsp5 E3-ligase complex. Ubiquitinated Ypq1 and PtdIns(3)P recruit ESCRT-0 to the vacuole membrane. ESCRT-0 sorts Ypq1 and recruits downstream ESCRTs to the vacuole membrane. The ESCRTs internalize Ypq1 directly into the vacuole lumen for degradation.
Additional files
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Supplementary file 1
Compiled fluorescence fold change data form each mutant in the fluorescence based screen.
- https://doi.org/10.7554/eLife.26403.019
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Supplementary file 2
The mutants defective for Ypq1 sorting found by ‘spontaneous mutagenesis’ and ‘fluorescence screen’
- https://doi.org/10.7554/eLife.26403.020
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Supplementary file 3
Supplementary experimental materials.
- https://doi.org/10.7554/eLife.26403.021
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ESCRTs function directly on the lysosome membrane to downregulate ubiquitinated lysosomal membrane proteins
eLife 6:e26403.
https://doi.org/10.7554/eLife.26403
