Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. The structure will be of interest to a wide range of cell biologists and structural biologists who study membrane traffic. However, while the structural data are elegant, the functional interpretations need further support.
(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 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Structural Biology and Molecular Biophysics (eLife)
Abstract
Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery.
Article activity feed
-
-
Evaluation Summary:
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. The structure will be of interest to a wide range of cell biologists and structural biologists who study membrane traffic. However, while the structural data are elegant, the functional interpretations need further support.
(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 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. HOPS has been characterized extensively through biochemical studies, which indicate that HOPS cooperates with SNAREs to facilitate membrane fusion. The authors conclude that HOPS is not a highly flexible structure as has been proposed, but instead has a stiff backbone to which the SNARE-binding Vps33 subunit is tightly anchored. Because the ends of HOPS bind to opposing membranes, the implication is that HOPS acts as a lever and membrane stressor, thereby amplifying the effects of SNARE assembly and catalyzing fusion.
The structural biology analysis was based on an improved purification protocol and appears to be well done. An …
Reviewer #1 (Public Review):
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. HOPS has been characterized extensively through biochemical studies, which indicate that HOPS cooperates with SNAREs to facilitate membrane fusion. The authors conclude that HOPS is not a highly flexible structure as has been proposed, but instead has a stiff backbone to which the SNARE-binding Vps33 subunit is tightly anchored. Because the ends of HOPS bind to opposing membranes, the implication is that HOPS acts as a lever and membrane stressor, thereby amplifying the effects of SNARE assembly and catalyzing fusion.
The structural biology analysis was based on an improved purification protocol and appears to be well done. An atomic-level structure is always valuable, and this contribution will undoubtedly guide further research involving HOPS. Initial steps in this direction are presented in the form of functional studies of structure-guided mutants.
Structures are most useful when they help to define mechanisms, and the authors argue that the HOPS structure explains how HOPS catalyzes membrane fusion. The key conclusion is that the antiparallel association of the Vps11 and Vps18 subunits create a rigid core for the complex, leaving flexible ends that bind the Ypt7 GTPase to anchor the two membranes. This model is inconsistent with earlier suggestions that HOPS bends to bring the two membranes together. Instead, the inferred rigidity of the HOPS core, combined with the central location of the SNARE-binding module, suggests that HOPS acts as a lever that exerts a force on the membranes to promote SNARE-driven membrane fusion.
This interpretation is interesting and potentially exciting, but I question why the authors are certain that the Vps11-Vps18 core is truly rigid. Proteins can undergo all sorts of rearrangements. Is there evidence that Vps11 and Vps18 interact strongly and in a unique configuration? Portions of a protein that have a consistent structure in vitro might nevertheless rearrange during functional interactions in vivo. If there is any flexibility of the Vps11-Vps18 core, this property combined with the evident flexibility of the Ypt7-binding portions and the low affinity of Vps41 for Ypt7 would make HOPS anything but a rigid membrane stressor. If the authors wish to make a strong point about the functional implications of the HOPS structure, these points need to be addressed.
-
Reviewer #2 (Public Review):
HOPS is central to the trafficking of endocytosed pathogens to the lysosome, and thus vital to human disease pathways. It has received extensive functional biochemical study, but its three-dimensional structure has proven elusive. In a technical tour-de-force, Shvarev and colleagues now report the extended, elegant structure of HOPS, exhibiting an overall triangular shape with Rab-GTPase binding sites at 2 vertices of the triangle and an SM-family subunit at the third (SM proteins catalyze SNARE complex assembly). This structure is fundamental for the molecular understanding of membrane fusion.
This study is a very important contribution to the membrane fusion field. HOPS has received extensive functional study, yet there has been no high-resolution structure. The structure presented here will be a basis for …
Reviewer #2 (Public Review):
HOPS is central to the trafficking of endocytosed pathogens to the lysosome, and thus vital to human disease pathways. It has received extensive functional biochemical study, but its three-dimensional structure has proven elusive. In a technical tour-de-force, Shvarev and colleagues now report the extended, elegant structure of HOPS, exhibiting an overall triangular shape with Rab-GTPase binding sites at 2 vertices of the triangle and an SM-family subunit at the third (SM proteins catalyze SNARE complex assembly). This structure is fundamental for the molecular understanding of membrane fusion.
This study is a very important contribution to the membrane fusion field. HOPS has received extensive functional study, yet there has been no high-resolution structure. The structure presented here will be a basis for substantial further work in many labs.
-
Reviewer #3 (Public Review):
This is an exciting new cryoEM structure of the HOPS tethering complex, which is necessary for membrane fusion at the vacuole/lysosome in eukaryotic cells. Finally, we can visualize, at moderate resolution, the positioning of HOPS subunits with respect to each other, and predict how HOPS and its various binding partners, such as Rab GTPases and SNAREs, can interact and control fusion. A conceptual advance put forward by this structure seems to be a rigid central core of HOPS that may contribute to helping drive the efficiency of the SNARE-mediated fusion mechanism.
As exciting as this new structure is, however, the study seems to fall a bit short of its promise to explain "why tethering complexes are an essential part of the membrane fusion machinery, or how HOPS "catalyzes fusion." As such, the title is …
Reviewer #3 (Public Review):
This is an exciting new cryoEM structure of the HOPS tethering complex, which is necessary for membrane fusion at the vacuole/lysosome in eukaryotic cells. Finally, we can visualize, at moderate resolution, the positioning of HOPS subunits with respect to each other, and predict how HOPS and its various binding partners, such as Rab GTPases and SNAREs, can interact and control fusion. A conceptual advance put forward by this structure seems to be a rigid central core of HOPS that may contribute to helping drive the efficiency of the SNARE-mediated fusion mechanism.
As exciting as this new structure is, however, the study seems to fall a bit short of its promise to explain "why tethering complexes are an essential part of the membrane fusion machinery, or how HOPS "catalyzes fusion." As such, the title is also misleading with regard to HOPS being the "lysosomal membrane fusion machinery."
Overall, the manuscript could benefit greatly, especially for a non-HOPS specialist reader, in providing more introduction and context to the complex and tethering/fusion mechanisms in general. Additionally, the examination of the structure, in light of decades of biochemistry and cell biology studies of HOPS (and homologous proteins that regulate fusion), seems superficial and suggests that deeper analyses may reveal additional insights and lead to a more detailed and impactful model for HOPS function. Moreover, are the insights gained here applicable to other tethering complexes, why or why not?
-
Author Response
Reviewer 1
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. HOPS has been characterized extensively through biochemical studies, which indicate that HOPS cooperates with SNAREs to facilitate membrane fusion. The authors conclude that HOPS is not a highly flexible structure as has been proposed, but instead has a stiff backbone to which the SNARE-binding Vps33 subunit is tightly anchored. Because the ends of HOPS bind to opposing membranes, the implication is that HOPS acts as a lever and membrane stressor, thereby amplifying the effects of SNARE assembly and catalyzing fusion.
The structural biology analysis was based on an improved purification protocol and appears to be well done. An …
Author Response
Reviewer 1
This manuscript reports the cryo-EM structure of HOPS, a heterohexameric tether that participates in the fusion of late endosomes, autophagosomes, and AP-3 vesicles with lysosomes. HOPS has been characterized extensively through biochemical studies, which indicate that HOPS cooperates with SNAREs to facilitate membrane fusion. The authors conclude that HOPS is not a highly flexible structure as has been proposed, but instead has a stiff backbone to which the SNARE-binding Vps33 subunit is tightly anchored. Because the ends of HOPS bind to opposing membranes, the implication is that HOPS acts as a lever and membrane stressor, thereby amplifying the effects of SNARE assembly and catalyzing fusion.
The structural biology analysis was based on an improved purification protocol and appears to be well done. An atomic-level structure is always valuable, and this contribution will undoubtedly guide further research involving HOPS. Initial steps in this direction are presented in the form of functional studies of structure-guided mutants.
Structures are most useful when they help to define mechanisms, and the authors argue that the HOPS structure explains how HOPS catalyzes membrane fusion. The key conclusion is that the antiparallel association of the Vps11 and Vps18 subunits create a rigid core for the complex, leaving flexible ends that bind the Ypt7 GTPase to anchor the two membranes. This model is inconsistent with earlier suggestions that HOPS bends to bring the two membranes together. Instead, the inferred rigidity of the HOPS core, combined with the central location of the SNARE-binding module, suggests that HOPS acts as a lever that exerts a force on the membranes to promote SNARE-driven membrane fusion.
This interpretation is interesting and potentially exciting, but I question why the authors are certain that the Vps11-Vps18 core is truly rigid. Proteins can undergo all sorts of rearrangements. Is there evidence that Vps11 and Vps18 interact strongly and in a unique configuration? Portions of a protein that have a consistent structure in vitro might nevertheless rearrange during functional interactions in vivo. If there is any flexibility of the Vps11-Vps18 core, this property combined with the evident flexibility of the Ypt7binding portions and the low affinity of Vps41 for Ypt7 would make HOPS anything but a rigid membrane stressor. If the authors wish to make a strong point about the functional implications of the HOPS structure, these points need to be addressed.
Based on our data we conclude that the Vps11-Vps18 core represents a rigid structure. Our extensive 2D and 3D classifications, as well as the 3D variability analysis of cryo-EM data indicate no flexibility in this region of the complex (in contrast to the Vps41- and Vps39-termini of the particle), as illustrated in Figure 1 and Figure 2 - Supplemental Figure 3. Additionally, the highest resolution achieved in this region within the whole structure suggests the least flexibility of this region in comparison to other parts of the complex.
To get a better idea, we mapped the interface between Vps11 and Vps18. The interface area between Vps11 and Vps18 is 1972 A2 according to the PDBePISA tool, which is large enough to form a strong interaction and is comparable with protein interfaces in other complexes with similar structural elements as in HOPS (e.g. Yang et al. Nat. Comm. 2021, Kschonsak et al. Nature 2022). To demonstrate this, we added an additional Supplement Figure to Fig. 2 (Fig. 2-S4) addressing the interaction area between Vps11 and Vps18 and revised the manuscript text in line 111 with words “…large interface area of 1972 Å2 which provides a…”.
Reviewer 3
This is an exciting new cryoEM structure of the HOPS tethering complex, which is necessary for membrane fusion at the vacuole/lysosome in eukaryotic cells. Finally, we can visualize, at moderate resolution, the positioning of HOPS subunits with respect to each other, and predict how HOPS and its various binding partners, such as Rab GTPases and SNAREs, can interact and control fusion. A conceptual advance put forward by this structure seems to be a rigid central core of HOPS that may contribute to helping drive the efficiency of the SNARE-mediated fusion mechanism.
As exciting as this new structure is, however, the study seems to fall a bit short of its promise to explain "why tethering complexes are an essential part of the membrane fusion machinery, or how HOPS "catalyzes fusion." As such, the title is also misleading with regard to HOPS being the "lysosomal membrane fusion machinery."
Overall, the manuscript could benefit greatly, especially for a non-HOPS specialist reader, in providing more introduction and context to the complex and tethering/fusion mechanisms in general. Additionally, the examination of the structure, in light of decades of biochemistry and cell biology studies of HOPS (and homologous proteins that regulate fusion), seems superficial and suggests that deeper analyses may reveal additional insights and lead to a more detailed and impactful model for HOPS function. Moreover, are the insights gained here applicable to other tethering complexes, why or why not?
We thank the Reviewer for her/his kind and helpful comments and have addressed the concerns below and in the revised manuscript.
-
-
-
-
-
