The ubiquitin hydrolase Doa4 directly binds Snf7 to inhibit recruitment of ESCRT-III remodeling factors in S. cerevisiae

Abstract

The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.

Keywords: ESCRT-III; Endosome; Membrane; Scission; Ubiquitin.

Fig. 1.

Snf7 binds the N-terminus of Doa4. (A) Schematic diagram of Doa4 and Snf7. (B) Affinity isolation of GST-Doa4 fusion proteins incubated with the indicated ESCRT-III subunits. The asterisk indicates GST-Doa4^1-80, which was detected because the anti-Snf7 antiserum was raised against GST-tagged Snf7. (C) Affinity isolation of GST-Doa4^1-348 incubated with Snf7, Snf7^ΔMIM2 (L199D, V202D), Snf7^ΔMIM1 (L231A, L234A) or Snf7^Δα6 (Δ226-240). (D) Live-cell BiFC fluorescence microscopy images showing the interaction of Doa4-VN or Doa4^1-348-VN (light gray bar) with Snf7-VC. Vacuolar membranes are stained with FM4-64. Dashed lines indicate cell outlines. Scale bar: 4 μm. (E) Quantification of the mean fluorescence per cell based on ≥3 independent experiments. Error bars represent the ±s.e.m.; *P≤0.05, ***P≤0.001. n.s., not significant.

Fig. 2.

Overexpression of the Doa4 N-terminus causes Snf7 polymer accumulation. (A,D) Western blots analyzing the distribution of Snf7 in fractions resolved by rate-zonal density gradient centrifugation. Indicated at the top is the migration of the protein standards aldolase (158 kDa), catalase (232 kDa) and ferritin (440 kDa). (B,C,E) Line-graphs representing the mean percentage of Snf7 in each gradient fraction examined in triplicate independent experiments. ±s.d. values of each fraction are indicated by the shaded margin surrounding each line. Statistical significance was calculated using two-tailed Student's t-test for each individual fraction. Differences in the levels of Snf7 protein in cells with overexpressed Doa4 (+DOA4 oe) or empty vector (+EV) were significant (P≤0.05) for fractions 7 and 8 in B, and fractions 2, 8 and 9 in E. All other differences in Snf7 protein levels were not statistically significant.

Fig. 3.

ILV membrane scission is inhibited by overexpression of the Doa4 N-terminus. (A-D) Cross-sectional tomographic slices and 3D models obtained using electron tomography of a 250-nm thick section of a yeast cell (strain as indicated). In each model, endosome-limiting membranes are traced in yellow, detached ILVs are traced in red and ILV budding profiles are traced in green. Scale bars: 100 nm. (E) Quantification of ILV budding profiles per endosome using the tomographic models of the indicated strains. The number of endosomes analyzed under each condition are as follows: wild-type+empty vector (+EV), n=12; wild-type+overexpressed DOA4 (+DOA4 oe), n=20; snf7^ΔMIM1+DOA4, n=15; wild-type+DOA4^1-348, n=19. Error bars represent ±s.d., *P≤0.05. (F) Distribution of the percentage of endosomes observed with the indicated number of ILV budding profiles. (G) Sum of ratios of ILVs:budding profiles for all endosomes under each condition. A lower ratio of ILVs:budding profiles indicates a delay at the membrane scission step of ILV biogenesis. n.s., not significant.

Fig. 4.

ILV protein sorting is inhibited by overexpression of the Doa4 N-terminus. (A) Live-cell fluorescence microscopy of GFP-Cps1 localization (green). Vacuolar membranes are stained with FM4-64 (purple). Cell outlines are traced by dashed lines. Error bar: 4 µM. (B) FLuc-Cps1 mis-sorting was quantified using the LUCID system. Higher FLuc-Cps1 levels represent increased mis-sorting of ILV cargo. Error bars represent ±s.e.m. calculated from 4 independent experiments (*P≤0.05).

Fig. 5.

Doa4 inhibits Snf7 polymer association with Vps2:24 in vitro. (A) Experimental setup and kymographs from time-lapse fluorescence microscopy of representative Snf7-AlexaFluor-488 patches grown on supported lipid membranes with (+) or without (−) Doa4^1-348. (B) Kymographs of representative Snf7 patch disassembly. Snf7-AlexaFluor-488 was polymerized together with Vps2 and Vps24. At t=0, these were washed out, followed by the addition of ATP and Vps4. (C,D) Quantification of mean Snf7 fluorescence during polymerization (C) and depolymerization (D) of Snf7 in the presence (+) or absence (−) of Doa4^1-348, of at least 58 patches and 3 independent experiments from A and B, respectively. (E) Kymographs of Vps2 associating with representative Snf7 patches polymerized on supported lipid membranes. At t=0, Snf7 was washed out and Vps2-Atto-565 along with Vps24 was added. (F) Kymographs of Vps2 dissociation from representative Snf7 patches polymerized with Vps2-Atto-565 and Vps24 on supported lipid membranes. At t=0, these were washed out, followed by the addition of ATP and Vps4. (G,H) Quantification of mean Vps2 fluorescence during association (G) and dissociation of Vps2 (F) in the presence (+) or absence (−) of Doa4^1-348, of at least 56 patches and 3 independent experiments each from E and F, respectively. (I,J) Experimental setup for Vps2 associating with representative ESCRT-III patches. Snf7 was polymerized on supported lipid membranes. At t=0, Vps2-Atto-565 together with Vps24 was added. Doa4^1-348 was added either at t=0 (I) or during the initial Snf7 polymerization and washed out at t=0 (J). (K,L) Quantification of mean Vps2 fluorescence during Vps2 association in the presence (+) or absence (−) of Doa4^1-348 of at least 91 patches and 4 independent experiments each, from set-up described for I (K) and set-up described for J (L). Error bars in all graphs represent ±s.d.

Fig. 6.

Overexpression of Doa4 inhibits association of Snf7 with Vps2:24 and Vps4 in vivo. (A) Live-cell BiFC fluorescence microscopy showing interaction between Snf7-VC and Vps2-VN (Vps2 – Snf7 BiFC) at basal levels and in response to Doa4 overexpression (DOA4 oe). Vacuolar membranes are stained with FM4-64 (purple). Cell outlines are traced with dashed lines. Scale bar: 4 μM. (B) Quantification of mean BiFC fluorescence per cell from 4 independent experiments during an experimental set-up described in A. Error bars represent ±s.e.m.; a.u., arbitrary unit. (C) Schematic of the experimental setup for BiFC mating experiments. (D) Representative images of BiFC fluorescence derived from interaction of Vps2-VN with Snf7-VC in zygotes at 240 min after mixing haploid strains. Venus fluorescence is shown merged with the corresponding bright-field image. Scale bar: 4 μM. (E-H) Line graphs of BiFC fluorescence intensity per zygote showing interaction between Snf7-VC and Vps2-VN (E), and between Snf7-VC and Vps4-VN (Vps4–Snf7 BiFC) (F) after mixing haploid strains expressing the VN or VC fusions at t=0. The same analysis was performed in strains expressing the Snf7^ΔMIM1-VC mutant (F-H). Data were normalized to BiFC fluorescence intensity at the final time-point under ‘empty vector’ condition. As a negative control (NC), BiFC was measured in cells expressing Snf7-VC and His2-VN, the latter of which being a cytosolic protein not involved in ESCRT-III function. Data are from at least 3 independent experiments and error bars represent ±s.e.m. (*P≤0.05, **P≤0.01).