The endosomal sorting complexes required for transport (ESCRT) pathway facilitates multiple fundamental membrane remodeling events. Previously, we determined X-ray crystal structures of ESCRT-III subunit Snf7, the yeast CHMP4 ortholog, in its active and polymeric state (Tang et al., 2015). However, how ESCRT-III activation is coordinated by the upstream ESCRT components at endosomes remains unclear. Here, we provide a molecular explanation for the functional divergence of structurally similar ESCRT-III subunits. We characterize novel mutations in ESCRT-III Snf7 that trigger activation, and identify a novel role of Bro1, the yeast ALIX ortholog, in Snf7 assembly. We show that upstream ESCRTs regulate Snf7 activation at both its N-terminal core domain and the C-terminus α6 helix through two parallel ubiquitin-dependent pathways: the ESCRT-I-ESCRT-II-Vps20 pathway and the ESCRT-0-Bro1 pathway. We therefore provide an enhanced understanding for the activation of the spatially unique ESCRT-III-mediated membrane remodeling.https://doi.org/10.7554/eLife.15507.001
The endosomal sorting complex required for transport (ESCRT) pathway mediates topologically unique membrane budding events. In multivesicular body (MVB) biogenesis, ESCRT-0, I and II sort ubiquitinated cargo by binding ubiquitin and endosomal lipids. ESCRT-III assembles into spiraling polymers for cargo sequestration, and together with the AAA-ATPase Vps4, remodels the membranes to generate cargo-laden intralumenal vesicles (ILVs).
ESCRT-III is a metastable and conformationally dynamic hetero-polymer of four 'core' subunits, Vps20, Snf7/Vps32, Vps24 and Vps2 (Babst et al., 2002). All subunits share a common domain organization of an N-terminal helical core domain and a flexible C-terminus, but provide distinct functions. ESCRT-II engages Vps20 to nucleate the polymerization of the most abundant ESCRT-III subunit, Snf7, which then recruits Vps24 and Vps2 (Teis et al., 2008). Finally, Vps2 engages Vps4 for ESCRT-III disassembly (Obita et al., 2007).
How is Snf7 activated to promote ESCRT-III assembly and cargo sequestration? Previous studies have shown that ESCRT-II and Vps20 modulate Snf7 protofilaments, emphasizing a role of the upstream ESCRTs in defining the assembly and architecture of the ESCRT-III complex (Henne et al., 2012; Teis et al., 2010). Recently, we have determined X-ray crystal structures of Snf7 protofilaments in the active conformation (Tang et al., 2015). Here, using genetics and biochemistry, we identify two parallel ubiquitin-dependent pathways that regulate Snf7 activation through both the Snf7 N-terminal core domain and the C-terminal α6 helix, providing an enhanced understanding of the activation of ESCRT-III-mediated membrane remodeling at endosomes.
Although Vps20 and Snf7 display a high degree of homology, they cannot complement each other. In order to identify regions of Vps20 essential for its function, we designed a series of Vps20-Snf7 chimeras and analyzed them by an established quantitative Mup1-pHluorin MVB sorting assay (Henne et al., 2012). Although a full-length Vps20 is required for function, retaining only the α1/2 hairpin of Vps20 while replacing the remainder of Vps20 with Snf7 (Vps201-105-Snf7107-240) is sufficient for sorting, albeit at ~70% efficiency (Figure 1A, Figure 1—figure supplements 1–2), suggesting that α1/2 is the minimal region unique to Vps20. This is consistent with the role of α1 of Vps20 in binding to the ESCRT-II subunit Vps25 (Im et al., 2009).
To investigate the role of Vps20 in nucleating Snf7 in vivo, we next applied an unbiased random mutagenic approach. We performed error-prone polymerase chain reaction on SNF7 and selected mutants that suppress the vps20Δ phenotype by growth on L-canavanine (Figure 1B). Two snf7 point mutations in conserved residues, snf7Q90L and snf7N100I, showed a partial rescue of the canavanine sensitivity of vps20Δ (Figures 1C-1D). Remarkably, in 'closed' Snf7, Gln90 of α2 is proximal to α4 (Tang et al., 2015), and Asn100 is an asparagine cap of the α2 helix (Figure 1E). We propose that these mutations destabilize closed Snf7 by displacing α4 from α2 and extending the α2/3 helix.
Since conformationally active Snf7 resides on membranes, we performed liposome sedimentation assays. As predicted, Q90L enhances Snf7 membrane association from 41% to 78% (Figure 1F). To further identify whether these substitutions trigger 'opening' in the core domain, we applied circular dichroism (CD) spectroscopy (Greenfield, 2006; Peter et al., 2004) on Snf7α1-α4, a truncated Snf7 construct with reduced membrane binding compared to the full-length proteins (Buchkovich et al., 2013). In the presence of liposomes, we observed a decrease of the negative absorption band at 208 nm and an increase at 222 nm in Q90L and N100I mutants, indicating an increase of α-helicity (Figure 1G). These data agree with the hypothesis that Snf7Q90L and Snf7N100I trigger structural rearrangements, where the α2/3 loop becomes α-helical and extends into one elongated α-helix (Figure 1—figure supplement 3) as observed in the open structures (McCullough et al., 2015; Tang et al., 2015). Notably, this structural rearrangement still occurs only upon membrane binding. Moreover, snf7Q90L and snf7N100I complement snf7Δ in vivo, and Snf7Q90L assembles into protofilaments in vitro (Figure 1—figure supplement 4), confirming a functional role of the mutants in activating Snf7.
Given that snf7Q90Land snf7N100I only modestly suppress vps20Δ, we hypothesized that a more stabilized 'open' Snf7 on endosomal membranes would improve the suppression. We combined the activation mutations with R52E (Henne et al., 2012) to further trigger 'opening', and swapped α0 of Snf7 with the N-terminal myristoylation motif of Vps20 to enhance its membrane-binding affinity (Buchkovich et al., 2013). This yielded myr-snf7R52E Q90L and myr-snf7R52E Q90L N100I, hereafter denoted as snf7** and snf7***, which sorted cargo with increased efficiencies, albeit not completely restoring wild-type levels (Figure 2A, Figure 2—figure supplements 1–2).
Consistent with these observations, the ESCRT-dependent cargo GFP-Cps1 partially localized to the vacuolar lumen in vps20Δ with snf7** or snf7*** (Figure 2B), indicating a substantial level of MVB sorting. Moreover, snf7** and snf7*** were also able to rescue the canavanine sensitivity of vps20Δ (Figure 2C). Thus, these snf7 suppressors exhibit the ability to sort cargo at MVB.
To visualize whether the snf7 suppressors could produce ILVs in vivo, we utilized a temperature sensitive allele of the vacuolar SNARE vam7 to accumulate MVBs and examined yeast with thin-section TEM (Buchkovich et al., 2013; Sato et al., 1998) (Figure 2D). We observed that while ILVs in wild-type cells have a diameter of ~32 nm, snf7** and snf7*** show a decrease in ILV number and an increase in ILV diameter to ~43 nm (Figures 2E–F, See Materials and methods). Since ESCRT-II and Vps20 set the architecture of ESCRT-III, we propose that the variation in ILV size is a result of aberrant ESCRT-III architecture, although we cannot completely rule out the possibility of changes in dynamics of ESCRT-III disassembly by Vps4 (Nickerson et al., 2010).
Intrigued by the vps20Δ suppression, we next wanted to test if these auto-activated Snf7 mutants could also bypass the loss of other ESCRT components (Figure 3A). Among them, the downstream ESCRT-III subunits Vps24 and Vps2 are known to modulate Snf7 architecture (Henne et al., 2012; Teis et al., 2008) and recruit the AAA-ATPase Vps4 via their C-terminal MIM motifs for ESCRT-III disassembly (Obita et al., 2007). We found that auto-activated Snf7 does not suppress vps24Δ, vps2Δ or vps4Δ (Figure 3B, Figure 3—figure supplement 1). This is consistent with the role of the suppressors in activating but not modulating or disassembling Snf7 filaments, reinforcing the division of labor among ESCRT-III subunits.
Previous studies showed that ESCRT-III assembly is regulated by ESCRT-II (Henne et al., 2012; Teis et al., 2010) (Figure 3A). ESCRT-II is a Y-shaped heterotetramer of Vps36, Vps22 and two Vps25 (arms). Vps36 GLUE domain binds ubiquitinated cargo and endosome-specific phosphatidylinositol 3-phosphate, PI3P; and each Vps25 'arm' binds one molecule of the ESCRT-III nucleator, Vps20. Since repurposing Snf7 to bind ESCRT-II does not improve the suppression (Figure 3—figure supplement 2), we next tested the functionality of the suppressors in ESCRT-II single and double deletion mutants. Strikingly, snf7** and snf7*** resulted in better suppression in ESCRT-II deletion compared to vps20Δ, with sorting efficiencies of ~60%–70% (Figure 3C, Figure 3—figure supplements 3–4).
We next tested ESCRT-I mutants. ESCRT-I is a heterotetramer of Vps23, Vps28, Vps37 and Mvb12. Vps23 UEV domain recognizes ubiquitinated cargo, Vps37 N-terminal helix binds to membranes, and Vps28 CTD engages Vps36 GLUE domain of ESCRT-II. We expressed the suppressors in ESCRT-I single and ESCRT-I/II double deletion mutants and we observed near wild-type sorting efficiencies (Figure 3D) with enlarged ILV sizes (Figure 3—figure supplements 5–6). Our data suggest that ESCRT-I and ESCRT-II set up the ESCRT-III architecture to program vesicle dimension.
Because ESCRT-I and ESCRT-II cluster ubiquitinated cargo prior to their packaging into ILVs, the observed suppression indicated that the auto-activated Snf7 might sort cargo in a ubiquitin-independent manner. We next tested whether auto-activated Snf7 could bypass the remaining ubiquitin-binding ESCRT components, ESCRT-0 (Vps27 and Hse1) and, the yeast ALIX ortholog, Bro1/Vps31. Interestingly, the engineered snf7 suppressors do not sort cargo in vps27Δ or bro1Δ (Figures 3E and G), or hse1Δ in combination with vps20Δ, vps25Δ (ESCRT-II) or vps23Δ (ESCRT-I) (Figure 3F). To test whether ubiquitin-binding of ESCRT-0 and Bro1 is critical, we expressed ubiquitin-binding mutants vps27S270D S313D (vps27UIM) and bro1I377R L386R (bro1UBD) (Bilodeau et al., 2002; Pashkova et al., 2013). They reduced the functionality of snf7*** in vps20Δ, vps25Δ or vps23Δ (Figure 4A, Figure 4—figure supplement 1). These data suggest that despite the ESCRT-I/II-independence, the suppression is still ubiquitin-dependent (Figure 3—figure supplement 7), perhaps through another subset of machinery of ESCRT-0 and Bro1. We thus propose that ESCRT-0/Bro1 are required to sort ubiquitinated cargo for ESCRT-III sequestration in parallel to ESCRT-I/II.
Bro1 has been shown to directly interact with Snf7, and X-ray crystal structures suggest that the C-terminal α6 helix of Snf7 binds to the Bro1 domain of Bro1 (Kim et al., 2005; McCullough et al., 2008; Wemmer et al., 2011). To test whether this interaction is required for snf7 suppression, we mutated residues at the Snf7-Bro1 interface. Notably, neither the Bro1-binding defective Snf7*** L231K L234K mutant (snf7*** BRO1), nor the Snf7-binding defective Bro1I144D L336D mutant (bro1SNF7), suppresses vps20Δ, vps25Δ or vps23Δ (Figure 4B, Figure 4—figure supplement 1). This strongly suggests that α6 of Snf7 is also auto-inhibitory, and that a physical binding between Snf7 α6 and Bro1 is a prerequisite for Snf7 activation.
We next tested if the Snf7-Bro1 interaction would release the α6 auto-inhibition. While the recombinant Snf7WT does not assemble due to auto-inhibition, coincubation with Bro1 resulted in Snf7 protofilament assembly (Figure 4C), indicating that Bro1 directly triggers Snf7 activation. In agreement with this, the α6 truncated Snf7 (Snf71-225) releases auto-inhibition and assembles into protofilaments (Figure 4D). Therefore, our data suggest that while Snf7 N100I, Q90L, and R52E release auto-inhibition in α3, α4, and α5, respectively, α6 of Snf7 is also auto-inhibitory and its activation is Bro1-dependent (Figure 4E).
The ancient and conserved ESCRT-III membrane-remodeling machinery plays a critical role in numerous fundamental cellular processes, including MVB biogenesis, viral budding and cytokinesis. Building on our previous study (Tang et al., 2015), we focused on the predominant ESCRT-III subunit, Snf7, to understand the molecular mechanisms governing ESCRT-III for its dynamic conversion from an auto-inhibited soluble monomer to a membrane-bending polymer. Remarkably, a recent cryo-EM study on ESCRT-III IST1/CHMP1B co-polymer suggested that CHMP1B (Did2/Vps46) undergoes a similar structural rearrangement for assembly (McCullough et al., 2015), implying that the core domain extension is a common theme of ESCRT-III activation.
Here, using a mutagenic approach, we identified novel Snf7 point mutations that release the auto-inhibition of α3 and α4 as observed in the conformationally open structures. Surprisingly, this leads to Snf7 activation that functionally bypasses the ESCRT-III nucleator Vps20, as well as the ESCRT-II and ESCRT-I complexes. This suggests that Snf7, along with its downstream ESCRT components, Vps24, Vps2 and Vps4, but not ESCRT-I/II, are among the minimal machinery required for membrane remodeling.
Our data suggest that ESCRT-III activation is mediated by two parallel pathways, ESCRT-I/II and ESCRT-0/Bro1 (Figure 4F). Bro1, directly triggers ESCRT-III assembly by binding to the C-terminal α6 of Snf7 (Figure 4C). Given that ESCRT-0 directly engages Bro1 (Lee et al., 2016) to recognize ubiquitinated cargo (Pashkova et al., 2013), we showed that Snf7 α6 binding to Bro1 relieves autoinhibiton of Snf7. This adds to the roles for Bro1, besides its recruitment of the Doa4 deubiquitinase in the MVB pathway (Luhtala and Odorizzi, 2004).
Consistent with our observation, a very recent study suggested that ALIX and ESCRT-I/II function as parallel CHMP4B (Snf7 ortholog in human) recruiters in cytokinetic abscission (Christ et al., 2016).
While biochemical data suggest that Snf7 can be activated by specific point mutations in the core domain or truncation at the C-terminus in vitro, our genetic evidence indicat that the conformational equilibrium of Snf7 is tightly regulated by two pathways in vivo to achieve ubiquitin-dependent cargo sorting at endosomes: 1) ESCRT-I/ESCRT-II/Vps20 activates the N-terminal core domain of Snf7; 2) ESCRT-0/Bro1 activates the C-terminal α6 of Snf7 (Figures 4E–F). Our results provide novel insights into a two-stage activation pathway for ESCRT-III-mediated membrane remodeling.
Fluorescence microscopy, western blotting and recombinant Snf7 purification for CD, TEM and liposome sedimentation analysis were performed as described (Buchkovich et al., 2013; Henne et al., 2012; Tang et al., 2015), and canavanine plating assay as described (Lin et al., 2008).
For Bro1 purification, Saccharomyces cerevisiae BRO1 was cloned into the pET23d vector (Novagen, Billerica, MA, USA) with an N-terminal His6-tag, induced by 1 mM IPTG at 18oC overnight from BL21 E. coli cells, and purified by TALON metal affinity resin (Clontech). Protein-bound TALON resins were washed in 500 mM NaCl, 20 mM HEPES pH 7.4, 20 mM imidazole, and eluted in 150 mM NaCl, 20 mM HEPES pH 7.4, 400 mM imidazole.
The quantitative Mup1-pHluorin ESCRT cargo-sorting flow cytometry assay was performed as described (Buchkovich et al., 2013; Henne et al., 2012; Tang et al., 2015). Briefly, mid-log yeast cell cultures grown with the addition of 20 μg/mL L-methionine for 2 hr were resuspended in 1x PBS buffer. Mean green fluorescence (FL1-A channel) of 100,000 events was recorded and gated on a BD Accuri C6 flow cytometer. For single ESCRT mutants, take Figure 1A for example: NBY42 (vps20Δ MUP1-PH) yeast cells were transformed with 1) pRS416 empty vector, 2) pRS416 VPS20, or 3) different mutants, respectively. Gated mean FL1-A values, F, of each sample are recorded and sorting scores are calculated as:
Sorting scores of 3 to 5 independent experiments are used to calculate standard deviation.
For double ESCRT mutants, take Figure 3D panel vps23Δvps25Δ for example. STY64 (vps23Δ vps25Δ MUP1-PH) yeast cells were co-transformed with 1) pRS415 empty vector and pRS416 empty vector, 2) pRS415 VPS25 and pRS416 VPS23, 3) pRS415 empty vector and pRS416 VPS20, 4) pRS415 empty vector and pRS416 SNF7, 5) pRS415 empty vector and pRS416 snf7**, or 6) pRS415 empty vector and pRS416 snf7***, respectively. MVB sorting scores are calculated as:
Sorting scores of 3 to 5 independent experiments are used to calculate standard deviation.
See Supplementary file 1 for a list of plasmids and yeast strains used in this study.
The DNA sequence of Saccharomyces cerevisiae SNF7 with 500bp of 5’UTR and 500bp of 3’UTR was amplified by Taq DNA polymerase with 20 µM MnCl2 and manipulated dNTP (N=A, T, G, or C) concentrations of 250 µM for three dNTPs and 25 µM for the other dNTP. Four individual 50 µL PCR reactions with different dNTP ratios were mixed, purified and transformed in vps20Δ yeast, along with a restriction enzyme digested vector of 3’UTR-pRS416-5’UTR. Yeast cells were plated and grown on YNB-uracil for 3 days at 26oC, and replica plated on YNB-uracil with 4.0 µg/mL of L-canavanine. Canavanine-resistant yeast colonies were selected, and gap-repaired pRS416 snf7 mutant were prepped, amplified and sequenced.
CD experiments were carried out using an Aviv Biomedical CD spectrometer Model 202–01. 10 μM Snf7core mutants were buffer exchanged by Superdex-200 gel filtration (GE Healthcare Life Sciences) to 10 mM sodium phosphate buffer pH 7.5. For solution samples, Snf7core was mixed with an equal volume of buffer. For liposome samples, Snf7core was mixed with an equal volume of 1.0 mg/mL liposomes of 800 nm diameter, with 60% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 30% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 10% phosphatidylinositol 3-phosphate (PI(3)P). The preparation of liposomes was performed as previously described (Henne et al., 2012).
The degrees of ellipticity were measured at 4oC and scanned from 260 nm to 200 nm. Molar ellipticity, θ, was then normalized using the following equation and plotted versus wavelength, where n=142 is the number of peptide bonds.
Visualization of ESCRT-III assembly using purified recombinant ESCRT components was performed as previously described (Henne et al., 2012).
Visualization of MVB in vam7tsf yeast cells was performed as previously described (Buchkovich et al., 2013). Briefly, 30 ODV of mid-log vam7tsf yeast cells were grown at 38oC for 3 hr, and then fixed with 2.5% (v/v) glutaraldehyde for 1 hr and spheroplasted with zymolyase and gluculase before embedding in 2% ultra-low temperature agarose. Cells were incubated in 1% osmium tetroxide/1% potassium ferrocyanide for 30 min, 1% thiocarbohydrazide for 5 min, and 1% osmium tetroxide/1% postassium ferrocyanide for 5 min. After dehydration through an ethanol series, samples were transitioned into 100% propylene oxide and embedded in Spurr’s resin. Note that osmotic gradients during fixation or dehydration might account for the MVB morphological defects and the larger mean ILV diameter compared to samples prepared by high-pressure freezing and automated freeze-substitution. However, all yeast cells used in these experiments were treated equally. All TEM was performed on a Morgnani 268 transmission electron microscope (FEI) with an AMT digital camera.
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William I WeisReviewing Editor; Stanford University, United States
In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.
Thank you for submitting your article "ESCRT-III activation mutants mediate MVB biogenesis independent of ESCRT-I and ESCRT-II" for consideration by eLife. Your article has been favorably evaluated by John Kuriyan (Senior editor) and three reviewers, one of whom, (William Weis) is a member of our Board of Reviewing Editors.
The following individual involved in review of your submission has agreed to reveal their identity: Alexey Merz (peer reviewer).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
The authors previously determined the active, polymerizing conformation of Snf7 as part of ESCRT-III function in MVB formation. This Research Advance exploits these structural data in combination with genetic screens to understand how the active conformation of Snf7 is regulated. They show that the two N-terminal α helices of Snf7 are essential for Vps20 (the most proximal upstream component) regulation, and find mutations that can bypass upstream ESCRT-I and II mutants. Intriguingly, although ESCRT-I and II cluster ubiquitylated cargo, the activated Snf7 mutants still require such cargo for MVB formation. Perhaps the most interesting finding is that Bro1, which is known to bind to Snf7 helix 6, does so to relieve autoinhibition of Snf7, demonstrating that Snf7 helix 6 also confers autoinhibition to Snf7 filament assembly in addition to the previously documented autoinhibition that prevents remodeling of helices 3,4 and 5. This two-stage mechanism of activation is intriguing and probably adds an important level of regulation to the system. Overall, this is a logical follow up to the previous eLife paper and adds considerable new data to understanding regulation of ESCRT-III.
The authors should address the following in a revised manuscript:
1) More background is needed for the non-specialist – it is a bit too 'insider'. The Introduction is very minimal, and going through the mutation data on ESCRT-I and -II suppression is difficult without more background on the roles of some of these factors. For example, apart from a cartoon no background is provided regarding the 'arms' of the ESCRT-II complex.
2) In the CD experiments shown in Figure 1G, a reference is needed to explain the red shift of the spectrum in the presence of liposomes. In general, more helical structure is associated with increased negative ellipticity at 208 and 222 nm, not a spectral shift. Also, comparing 1F and 1G, why is there no change with WT in 1G when there is 41% binding in 1F? Is this due to use of the truncated construct? This is not made clear.
3) Figure 2D; Figure 3—figure supplement 6: The variation in ILV size relies on samples prepared from vam7tsf vacuole-fusion-deficient spheroplasts fixed in conventional fixative. This accounts for the relatively poor preservation of the MVB morphology. Osmotic gradients during fixation or dehydration probably account for the morphological defects and the larger mean vesicle sizes versus MVBs in the same strains prepared by high-pressure freezing and automated freeze-substitution. The limitations of this approach should be mentioned particularly since there is a strong possibility that the composition (lipids, proteins, lipid:protein ratios) in the vesicles of suppressed cells vary, resulting in divergent robustness to the stresses of conventional fixation. Also, the legend for Figure 2 should specify whether n=150 refers to the number of ILVs, MVBs, or cells, and whether n=150 summed over the experiment or per-treatment. Finally, could some of the size differences be due to the somewhat smaller amount of Snf7 mutant protein relative to WT (Figure 2—figure supplement 2B)?
4) At the end of the subsection “Auto-activated Snf7 bypasses Vps20“: an alternative possibility is that the variation in ILV sizes is due to changes in disassembly of the ESCRT III polymers by Vps4. The possibility that the vesicle sizes reflect divergent ESCRT III dynamics (vs. static architecture) is underscored by previous work (Odorizzi et al., 2006; 2010) showing that mutants deficient for Did2, a Vps4 ATPase regulator, have vesicle morphology phenotypes highly similar to those described in the present paper.
5) The MVB sorting assays should be briefly described in Methods. In particular, the authors should reiterate how 0% and 100% are calibrated, and the figure legends need to specify the number of independent experiments summarized for each panel or experiment.
6) A concern in the experiments in Figure 3E is that expression of Hse1-DUB might mildly interfere with normal Hse1 function through a mechanism other than de-ubiquitination. Such a defect might not be evident in a WT background where MVB formation is robust, but it could potentially manifest in the suppressed background where MVB formation might be barely possible. The standard control in experiments using the Piper group's Hse1-DUB fusions is to use fusions containing a catalytically dead DUB domain. These controls need to be included in a supplemental panel. The related experiments in Figure 4A and B (Vps27 and Bro1 Ub-interaction mutants) are strong, however, so it is recommended that either the Hse1-DUB experiment be omitted, or that it should be presented with these needed controls.https://doi.org/10.7554/eLife.15507.025
The authors should address the following in a revised manuscript: 1) More background is needed for the non-specialist – it is a bit too 'insider'. The Introduction is very minimal, and going through the mutation data on ESCRT-I and -II suppression is difficult without more background on the roles of some of these factors. For example, apart from a cartoon no background is provided regarding the 'arms' of the ESCRT-II complex.
We agree with the reviewers that more introduction and background information is needed for general readers. We have now expanded the Introduction, and included more background information regarding the key functional and structural features of each ESCRT complex when going through the mutational data on ESCRT-I and ESCRT-II suppression in Figure 3 in the subsection “Auto-activated Snf7 bypasses ESCRT-I and ESCRT-II”.
2) In the CD experiments shown in Figure 1G, a reference is needed to explain the red shift of the spectrum in the presence of liposomes. In general, more helical structure is associated with increased negative ellipticity at 208 and 222 nm, not a spectral shift.
We have now included two references of CD spectra in the second paragraph of the subsection “Screening for Vps20-independent Snf7 activation mutants”. This seems to be a common observation. We also agree with αthe reviewers that more -helical structure gave decreased ratio of molecular ellipticity at negative bands of 208 and 222 nm. We havecorrected this in the aforementioned paragraph.
Also, comparing 1F and 1G, why is there no change with WT in 1G when there is 41% binding in 1F? Is this due to use of the truncated construct? This is not made clear.
Yes, the discrepancy is due to the use of a truncated construct. The liposome sedimentation experiment (Figure 1F) is performed using intact full-length Snf7 proteins. To observe the major structural rearrangement in the core domain by CD spec (Figure 1G), however, we utilized a construct of Snf7 that only contains α1-α4, Snf7α1-α4. We have previously reported in Buchkovich et al., Dev Cell2013, Snf7α1-α4 (a.k.a. ΔN-Snf7core) has a reduced membrane-binding affinity (at ~5%) due to 1) the deletion of the N-terminal amphipathic ANCHR motif that inserts into the membranes, and 2) the partially buried electrostatic membrane binding surface. Therefore, wild-type Snf7 α1-α4 in 1G has no ellipticity change with and without liposomes. However, activation mutations Q90L and N100I trigger Snf7 core domain conformational change and, thus, expose the electrostatic membrane-binding surface, which resulted in CD spectra change in the presence of liposome. We added a sentence in the second paragraph of the subsection “Screening for Vps20-independent Snf7 activation mutants” to clarify this difference.
3) Figure 2D; Figure 3—figure supplement 6: The variation in ILV size relies on samples prepared from vam7tsf vacuole-fusion-deficient spheroplasts fixed in conventional fixative. This accounts for the relatively poor preservation of the MVB morphology. Osmotic gradients during fixation or dehydration probably account for the morphological defects and the larger mean vesicle sizes versus MVBs in the same strains prepared by high-pressure freezing and automated freeze-substitution. The limitations of this approach should be mentioned particularly since there is a strong possibility that the composition (lipids, proteins, lipid:protein ratios) in the vesicles of suppressed cells vary, resulting in divergent robustness to the stresses of conventional fixation.
We appreciate this comment. However, our chemical fixation protocols have been wildly used in the field and were treated equally and applied to all experimental samples shown in these figures. We observed ILV formation in the suppressors’ condition, indicating that these suppressors have the ability to generate ILVs in the absence of selected ESCRT mutants. We have now added in the last paragraph of the subsection “Auto-activated Snf7 bypasses Vps20 “and in the subsection “Negative Stain Transmission Electron Microscopy” to comment on this potential technical limitation.
Also, the legend for Figure 2 should specify whether n=150 refers to the number of ILVs, MVBs, or cells, and whether n=150 summed over the experiment or per-treatment.
Finally, could some of the size differences be due to the somewhat smaller amount of Snf7 mutant protein relative to WT (Figure 2—figure supplement 2B)? Yeast cells used for ILV size quantification (Figures 2D and Figure 3—figure supplement 6) have two populations of Snf7 expressed: the snf7* suppressor (off the plasmid) and the wild-type SNF7 (off the chromosome). We thus believe that the functional wild-type Snf7 proteins available for ILV biogenesis should be the same between the mutant and the wild-type cells. But in Figure 2—figure supplement 2B, in order to identify the expression levels of different Snf7 mutants, these yeast cells used for Western blotting analysis have only one copy of mutant Snf7 (off the plasmid).
4) At the end of the subsection “Auto-activated Snf7 bypasses Vps20“: an alternative possibility is that the variation in ILV sizes is due to changes in disassembly of the ESCRT III polymers by Vps4. The possibility that the vesicle sizes reflect divergent ESCRT III dynamics (vs. static architecture) is underscored by previous work (Odorizzi et al., 2006; 2010) showing that mutants deficient for Did2, a Vps4 ATPase regulator, have vesicle morphology phenotypes highly similar to those described in the present paper.We agree with the reviewers that our observed aberrant ILV is similar to the Greg Odorizzi lab’s did2 mutants. We have shown in Figure 3B that both Vps2 and Vps4 are required for the suppressor’s pathway, indicating that the disassembly of the ESCRT-III polymer is still dependent upon the AAA-ATPase Vps4 machinery. Although the MIM motifs of ESCRT-III for the recruitment of Vps4 are all intact in our system, we cannot completely rule out the kinetics and dynamics of ESCRT-III have subtle differences between the suppressor mutant and the wild-type cells. We thus have added one sentence and a reference at the end of the subsection “Auto-activated Snf7 bypasses Vps20 “to comment on this concern.
5) The MVB sorting assays should be briefly described in Methods. In particular, the authors should reiterate how 0% and 100% are calibrated, and the figure legends need to specify the number of independent experiments summarized for each panel or experiment. We have now included this information in Methods (subsection “Flow Cytometry) and in all figure legends.
6) A concern in the experiments in Figure 3E is that expression of Hse1-DUB might mildly interfere with normal Hse1 function through a mechanism other than de-ubiquitination. Such a defect might not be evident in a WT background where MVB formation is robust, but it could potentially manifest in the suppressed background where MVB formation might be barely possible. The standard control in experiments using the Piper group's Hse1-DUB fusions is to use fusions containing a catalytically dead DUB domain. These controls need to be included in a supplemental panel. The related experiments in Figure 4A and B (Vps27 and Bro1 Ub-interaction mutants) are strong, however, so it is recommended that either the Hse1-DUB experiment be omitted, or that it should be presented with these needed controls.
We removed the Hse1-DUB experiments, because we agree with the reviewers that the related UIM and UBD mutant experiments in Figure 4A are sufficient. However, although we agree that Hse1-DUB might mildly interfere with normal Hse1 function that is not detectable under WT condition, yeast cells used for these Hse1-DUB experiments express their endogenous wild-type Hse1 from the chromosome, which would provide normal ESCRT-0 function. Interestingly, we have found that hse1 deletion, which has mild defects in MVB sorting in WT condition, abolishes the snf7* suppression. We thus have reorganized Figure 3E–G. We now included the observation of hse1 deletion to complete the global analysis of ESCRT mutations and modified the results in the subsection “Auto-activated Snf7 does not bypass Bro1 and ESCRT-0“.https://doi.org/10.7554/eLife.15507.026
- Shaogeng Tang
- Shaogeng Tang
- Nicholas J Buchkovich
- W Mike Henne
- Scott D Emr
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We gratefully thank Sarah T. Griffin, Yi-Chun Yeh, Leonid A. Timashev, Ming Li and Lu Zhu for technical expertise and sharing reagents. We thank Anthony C Gatts VI for TEM, Nattakan Sukomon and Brian R. Crane for CD spectroscopy. We thank Yuxin Mao for critical reading of the manuscript, and J. Christopher Fromme, Peter P. Borbat and William J. Brown for helpful discussion.
- William I Weis, Stanford University, United States
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