Rules for the self-assembly of ESCRT-III on endosomes
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Evaluation Summary:
ESCRT-III is a conserved hetero-oligomeric membrane remodeling machine known to impact a number of cellular phenomena, yet mechanistic details of its function have remained enigmatic. This work identifies critical inter-subunit contact sites critical for ESCRT-III assembly and function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
We, the authors, have withdrawn this manuscript. During our work on the revision of this manuscript for eLife, it became clear that several key results of the manuscript were not reproducible by my lab. The rules for ESCRT-III assembly and the model that we have proposed in this manuscript are no longer supported by the data. Currently we are performing additional experiments to test some of our conclusions further. Therefore, we do not wish this work to be cited.
We sincerely apologize to the scientific community for any inconvenience resulting from this manuscript.
If you have any questions, please contact the corresponding author.
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Reviewer #3 (Public Review):
The article by Sprenger et al. uses the power of yeast genetics to generate mutants of the ESCRT-III subunits, and study their impact on the formation of a functional ESCRT-III complex. By using functional FP tags of subunits Snf7, Vps2 and Vps24, and of the CPS cargo, they essentially follow recruitment of subunits to the vacuolar membrane, formation of Class E compartments and sorting of CPS as readouts of the endosomal ESCRT-III function. They found that recruitment of Vps2, Vps24 and Snf7 is unaffected by deletions of other subunits (Did2, Ist1, Vps60), supporting the view that Vps2-Vps24 and Snf7 form an initial subcomplex.
To decipher molecular interactions between Vps2-Vps24 and Snf7 subunits, they use point mutants to replace well-chosen hydrophobic residues in two subunits by cysteines, and …
Reviewer #3 (Public Review):
The article by Sprenger et al. uses the power of yeast genetics to generate mutants of the ESCRT-III subunits, and study their impact on the formation of a functional ESCRT-III complex. By using functional FP tags of subunits Snf7, Vps2 and Vps24, and of the CPS cargo, they essentially follow recruitment of subunits to the vacuolar membrane, formation of Class E compartments and sorting of CPS as readouts of the endosomal ESCRT-III function. They found that recruitment of Vps2, Vps24 and Snf7 is unaffected by deletions of other subunits (Did2, Ist1, Vps60), supporting the view that Vps2-Vps24 and Snf7 form an initial subcomplex.
To decipher molecular interactions between Vps2-Vps24 and Snf7 subunits, they use point mutants to replace well-chosen hydrophobic residues in two subunits by cysteines, and cross-link them to probe the interactions of those residues in the functional case. They also change hydrophobic residue pairs into charged residue pairs to replace the hydrophobic interaction by an electrostatic interaction, and restore functionality (only when both mutants were used).
Overall, it is an elegant study, with very clear and well executed experiments, and which give strong support to a so far hypothetical architecture of the Vps2-Vps24-Snf7 as a double-strand filament, one of which is Snf7 only, and the other is an alternative repeat of Vps2 and Vps24.
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Reviewer #2 (Public Review):
ESCRT-III is a filament-forming machinery that is necessary for a variety of physiological and pathophysiological membrane remodelling events. These events are linked to an ability of an ESCRT-III filament to assemble and remodel cellular membranes. In recent years, it has become clear that whilst the ESCRT-III component Snf7 is likely the major component of ESCRT-III, individual filaments can form lateral interactions with alternate filaments, that remodelling the composition of ESCRT-III subunits within a filament likely allows its geometric changes and that it is unclear what role the Vps2/Vps24 subunits of ESCRT-III have alongside the major Snf7 filament. Building upon a previous publication in eLIFE, in which the authors used advanced microscopical approaches to quantitatively document the assembly …
Reviewer #2 (Public Review):
ESCRT-III is a filament-forming machinery that is necessary for a variety of physiological and pathophysiological membrane remodelling events. These events are linked to an ability of an ESCRT-III filament to assemble and remodel cellular membranes. In recent years, it has become clear that whilst the ESCRT-III component Snf7 is likely the major component of ESCRT-III, individual filaments can form lateral interactions with alternate filaments, that remodelling the composition of ESCRT-III subunits within a filament likely allows its geometric changes and that it is unclear what role the Vps2/Vps24 subunits of ESCRT-III have alongside the major Snf7 filament. Building upon a previous publication in eLIFE, in which the authors used advanced microscopical approaches to quantitatively document the assembly kinetics of ESCRT-III upon endosomes (demonstrating transient co-assembly of Snf7, Vps2 and Vps24), Sprenger et al have now used biochemical and microscopical approaches to understand individual interactions within the ESCRT-III holo-filament.
Protein-protein interactions are typically driven by two different modes that rely upon the physicochemical properties of the amino acids involved (namely electrostatics, or the shielding of hydrophobic residues by mutual interaction). Using published and modelled structural data, Sprenger et al., identify hydrophobic interactions governing longitudinal interaction of ESCRT-III monomers and electrostatic interactions that govern lateral interactions. They make elegant use of targeted mutagenesis to switch the interaction mode between individual monomers, and employ pairwise mutagenesis to rescue the disrupted interactions. They also employ chemical crosslinking to stabilise these transient interactions, and integrate this with an analysis of cargo sorting to the vacuole lumen, which is the archetypal function of ESCRT-III in yeast. In contrast to models proposing the Vps2/Vps24 unit as a 'cap' for a Snf7 filament, the authors propose that these subunits instead form a parallel filament that has important implications for our understanding of how Vps4 can access subunits within the ESCRT-III holo-filament.
The strengths of this manuscript are the integration of molecular and biochemical data with clear functional readouts of vacuolar sorting and the use of knock-in techniques bearing functionally tagged versions of the ESCRT-III proteins to analyse phenotypes. I think some improvement could be made to the description of the author's selection of residues for mutagenesis and to the degree of quantification of the data throughout the manuscript. I also wonder if there are different interpretations of the cross-linking experiments that could be integrated into their discussion.
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Reviewer #1 (Public Review):
In this work the authors address inter-subunit interactions leading to ESCRT-III function during MVB sorting in a yeast model system. ESCRTs mediate function in multiple biological processes, however the fundamental question of how ESCRT-III mediates membrane remodeling is not understood. As such this represents a topic of considerable interest despite significant technical limitations surrounding the issue. Random and rational mutagenic strategies, including compensatory mutations, are combined with protein-protein interaction studies and in vivo functional assays to identify residues within Vps24 and Vps2 mediating associations with each other and Snf7. Based on these analyses the authors put forth a series of "rules" governing ESCRT-III assembly and function. While beneficial to our conceptual …
Reviewer #1 (Public Review):
In this work the authors address inter-subunit interactions leading to ESCRT-III function during MVB sorting in a yeast model system. ESCRTs mediate function in multiple biological processes, however the fundamental question of how ESCRT-III mediates membrane remodeling is not understood. As such this represents a topic of considerable interest despite significant technical limitations surrounding the issue. Random and rational mutagenic strategies, including compensatory mutations, are combined with protein-protein interaction studies and in vivo functional assays to identify residues within Vps24 and Vps2 mediating associations with each other and Snf7. Based on these analyses the authors put forth a series of "rules" governing ESCRT-III assembly and function. While beneficial to our conceptual understanding of ESCRT-III these rules fall short in explicitly defining the structural basis of assembly and function including explaining requisite heterodimerization of Vps24-Vps2. This work represents a significant step forward in addressing this challenging question, the experimental design and implementation are convincing, however the limitations of this work could be conveyed more clearly.
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Evaluation Summary:
ESCRT-III is a conserved hetero-oligomeric membrane remodeling machine known to impact a number of cellular phenomena, yet mechanistic details of its function have remained enigmatic. This work identifies critical inter-subunit contact sites critical for ESCRT-III assembly and function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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