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• W4312372118 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This landmark study 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 provides a convincing update of earlier, lower-resolution models. Interestingly, the SNARE-binding module is attached to the core of the complex. These results suggest possible mechanisms by which HOPS could catalyze SNARE-dependent fusion. https://doi.org/10.7554/eLife.80901.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Our cells break down the nutrients that they receive from the body to create the building blocks needed to keep us alive. This is done by compartments called lysosomes that are filled with a cocktail of proteins called enzymes, which speed up the breakdown process. Lysosomes are surrounded by a membrane, a barrier of fatty molecules that protects the rest of the cell from being digested. When new nutrients reach the cell, they travel to the lysosome packaged in vesicles, which have their own fatty membrane. To allow the nutrients to enter the lysosome without creating a leak, the membranes of the vesicles and the lysosome must fuse. The mechanism through which these membranes fuse is not fully clear. It is known that both fusing membranes must contain proteins called SNAREs, which wind around each other when they interact. However, this alone is not enough. Other proteins are also required to tether the membranes together before they fuse. To understand how these tethers play a role, Shvarev, Schoppe, König et al. studied the structure of the HOPS complex from yeast. This assembly of six proteins is vital for lysosomal fusion and, has a composition similar to the equivalent complex in humans. Using cryo-electron microscopy, a technique that relies on freezing purified proteins to image them with an electron microscope and reveal their structure, allowed Shvarev, Schoppe, König et al. to provide a model for how HOPS interacts with SNAREs and membranes. In addition to HOPS acting as a tether to bring the membranes together, it can also bind directly to SNAREs. This creates a bridge that allows the proteins to wrap around each other, driving the membranes to fuse. HOPS is a crucial component in the cellular machinery, and mutations in the complex can cause devastating neurological defects. The complex is also targeted by viruses – such as SARS-CoV-2 – that manipulate HOPS to reduce its activity. Shvarev, Schoppe, König et al.’s findings could help researchers to develop drugs to maintain or recover the activity of HOPS. However, this will require additional information about its structure and how the complex acts in the biological environment of the cell. Introduction Lysosomal fusion underlies a plethora of cellular processes. It is essential in the maintenance and upkeep of eukaryotic membranes and fundamental to secretion, endocytosis, and autophagy (Saftig and Puertollano, 2021). Macromolecules from different trafficking pathways end up in lysosomes where they are degraded (Klionsky et al., 2021; Saftig and Puertollano, 2021). This process relies on multiple fusion events within the endomembrane system. In general, fusion depends on SNAREs, which are present on opposite membranes and zipper into four-helix bundles with the help of Sec1/Munc1 (SM) proteins (Jahn and Fasshauer, 2012; Südhof and Rothman, 2009; Wickner and Rizo, 2017). Each assembled SNARE complex contains three Q-SNAREs (Qa, Qb, and Qc) and one R-SNARE, which are categorized according to the interaction of the glutamine and arginine residues in the central hydrophilic layer of the otherwise hydrophobic interfaces within the SNARE complex (Jahn and Fasshauer, 2012; Südhof and Rothman, 2009; Wickner and Rizo, 2017). Prior to fusion, specialized tethering complexes establish tight links between organelles and interact with SM proteins to promote fusion (Baker and Hughson, 2016; Kuhlee et al., 2015; Ungermann and Kümmel, 2019). Despite their central position in trafficking, the underlying mechanics of tethering complexes and how they catalyze membrane fusion remain unresolved. The heterohexameric HOPS complex mediates fusion of late endosomes, autophagosomes, and AP-3 vesicles with mammalian lysosomes or vacuoles in yeast (van der Beek et al., 2019; Spang, 2016; Wickner and Rizo, 2017), and is probably the best-studied tethering complex. Fusion assays using yeast vacuoles or reconstituted SNARE-bearing proteoliposomes showed that HOPS is essential for membrane fusion at physiological SNARE concentrations (D’Agostino et al., 2017; Mima et al., 2008; Zick and Wickner, 2016). HOPS is the target of viruses such as SARS-CoV2 (Miao et al., 2021), and its inactivation blocks Ebola infections (Carette et al., 2011). Furthermore, multiple HOPS mutations can cause severe diseases ranging from Parkinson’s to lysosomal disorders (van der Beek et al., 2019; Sanderson et al., 2021; van der Welle et al., 2021). Five out of six HOPS subunits (Vps11, Vps16, Vps18, Vps39, and Vps41) are predicted to share a similar architecture, comprising an N-terminal β-propeller and a C-terminal α-solenoid domain (Figure 1A). Vps11 and Vps18 form the core and carry conserved C-terminal RING finger domains (Rieder and Emr, 1997), which are essential for HOPS formation (Hunter et al., 2017), but also show E3 ligase activity on their own (Segala et al., 2019). At the opposite sites, Vps41 and Vps39 bind to membrane-anchored small GTPases (the Rab7-like Ypt7 in yeast) (Bröcker et al., 2012; Lürick et al., 2017; Ostrowicz et al., 2010), while Vps16 and the SM protein Vps33 establish the SNARE-binding module (Baker et al., 2015; Graham et al., 2013). Low-resolution negative-stain electron microscopy (EM) analyses revealed the overall arrangement of HOPS (Bröcker et al., 2012; Chou et al., 2016), yet were insufficient to localize the exact position of its subunits and suggested significant flexibility within the particle. Figure 1 with 4 supplements see all Download asset Open asset Composition and architecture of the yeast HOPS complex. (A) Domain architecture and size of HOPS subunits. Predicted domains and structural features are indicated. (B) Size exclusion chromatography (SEC) of the affinity-purified HOPS. Purification was done as described in Materials and methods. (C) Mass photometry analysis. Peak fractions from SEC were analyzed for size. (D) Purified HOPS. Proteins from affinity purification (eluate) and SEC (red dashes in (B)) were analyzed by SDS-PAGE. (E) Overall architecture of the HOPS complex. Composite map from local refinement maps (Figure S1-3) was colored by assigned subunits. One of the consensus maps used for local refinements was low-pass-filtered and is shown as a transparent envelope. (F) Atomic model of the HOPS complex. For the N-terminal fragments of Vps41 and Vps39, which were not resolved to high resolution by local refinements, AlphaFold models are used and manually fitted into the densities of consensus maps (Figure 1—figure supplements 1 and 2). The triangular shape of the complex is highlighted with the approximate distance between the β-propellers of Vps41 and Vps39. (G) Schematic representation of the HOPS complex indicates central features. Figure 1—source data 1 Gels and graphs for Figure 1b, c and d. https://cdn.elifesciences.org/articles/80901/elife-80901-fig1-data1-v2.zip Download elife-80901-fig1-data1-v2.zip The way HOPS fulfills its function remains speculative, and multiple mechanisms have been proposed, including a role as a bulky membrane stressor (D’Agostino et al., 2017) or, conversely, as a highly flexible membrane tether (Bröcker et al., 2012; Chou et al., 2016). In the absence of detailed structural data, it remains obscure how HOPS facilitates lysosomal fusion. Results Structure of the HOPS complex Previously, structural studies were hampered by the low stability and flexibility of the complex, which required fixation through mild crosslinking for sample preparation and confined structural studies to negative stain analyses (Bröcker et al., 2012; Chou et al., 2016). To enable high-resolution cryo-EM of non-crosslinked HOPS, we vastly improved and accelerated our purification protocols and removed any delays during the sample preparation procedure (Figure 1B–D). Single-particle analysis including extensive classifications followed by local refinements led to a composite structure with resolutions between 3.6 and 5 Å (Figure 1E and Figure 1—figure supplements 1–3). HOPS forms a largely extended, slender structure extending approximately 430 Å in height and 130 Å in width, resembling a triangular shape (Figure 1E–G, Video 1). In the center of the modular complex, Vps11 and Vps18 align antiparallel through their elongated α-solenoids, establishing a large interface area of 1972 Å2 (Figure 1E-G, Figure 2, Figure 2—figure supplement 4), which resulted in the highest resolution obtained within HOPS (Figure 1—figure supplement 3) and is comparable with protein interfaces in other complexes with similar structural elements as in HOPS (e.g. Kschonsak et al., 2022). Interestingly, AlphaFold predicts a long unstructured region within Vps11 (Q760 to D784), resulting in an upper and lower part of the subunit. However, this region is clearly resolved and organized in our density. In our model, the two core subunits create a central assembly hub for the four other subunits (Vps39, Vps41, Vps16, and Vps33) that fulfill specific functions and localize to the periphery of the complex. The N-termini of Vps11 and Vps18 are located distally from the core and each carries a β-propeller, which can be deleted without affecting the complex formation (Figure 3A and D, Figure 1—figure supplement 4). At the C-termini, both Vps11 and Vps18 have long α-helices which are followed by RING finger domains (Figures 1E–G–2A and B, Figure 2—figure supplement 1). Both features are key elements for the stability of the modular architecture and serve as anchor points for the additional subunits. In agreement, HOPS, carrying mutations in the RING finger domains of Vps11 (vps11-1) (Peterson and Emr, 2001), selectively lost Vps39, whereas only Vps41 was obtained from a similar Vps18 mutant. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Overall architecture of HOPS tethering complex: transition between ribbon and molecular surface representation. Figure 2 with 4 supplements see all Download asset Open asset Vps11 and Vps18 C-termini as central interaction hubs for all other subunits. Atomic model of HOPS with highlighted interaction sites between subunits. (A, B) Coiled-coil motifs followed by the RING finger domains (violet) are the key structural features of HOPS. (A) The Vps18 C-terminal hub. Vps18 and Vps41 interact via the coiled coil and the Vps18 RING finger domain (displayed as non-transparent cartoons). (B) The Vps11 C-terminal hub. Vps11 interacts via its RING finger domain and the coiled coil with Vps39 (displayed as non-transparent cartoons). (C) Connection of the SNARE binding module (Vps33 and Vps16) to the backbone of HOPS via interactions with the structured loop at the RING finger domain and the C-terminus of Vps18 (displayed as non-transparent cartoons). (D) Vps39 connects by its C-terminal helix the β-propeller of Vps18, which provides additional stability in this part of HOPS. Figure 3 with 1 supplement see all Download asset Open asset HOPS couples tethering and fusion activities. (A) Schematic representations of HOPS wild-type (as in Figure 1G) and mutants lacking N-terminal β-propeller domains (indicated by pink asterisks). (B) Tethering assay. Fluorescently labeled liposomes loaded with prenylated Ypt7-GTP or none were incubated with HOPS and mutant complexes. Tethering was determined as described in Materials and methods. Data shown from three biological replicates, bars indicate standard deviation. (C) Fusion assay. Fusion of proteoliposomes carrying vacuolar SNAREs were preincubated with Ypt7-GDI, GTP, and Mon1-Ccz1. For fusion, HOPS wild-type or mutant and the soluble Vam7 SNARE were added (Langemeyer et al., 2020; Langemeyer et al., 2018). Analysis was done as described (Zick and Wickner, 2016). See Materials and methods. Data shown from three biological replicates, bars indicate standard deviation. (D) Representative 2D class averages obtained from negative-stain analyses of wild-type HOPS and mutants. Pink asterisks indicate missing densities in the mutants. Figure 3—source data 1 Raw data for tethering (Figure 3B) and fusion assay (Figure 3C). https://cdn.elifesciences.org/articles/80901/elife-80901-fig3-data1-v2.zip Download elife-80901-fig3-data1-v2.zip Vps41 and Vps39 provide the Ypt7-interaction sites at their peripheral N-terminal regions. Their extended C-terminal helices, similar to those of Vps11 and Vps18, are tightly interlocked through coiled-coil motifs with the long C-terminal α-helices and RING finger domains from the respective core subunits (Vps41 with Vps18, and Vps39 with Vps11) (Figure 2A and B). Additional stability of Vps39 within the complex is provided by the interaction of the long α-helix at the C-terminus of Vps39 with the β-propeller of Vps18 (Figure 2D). In our density, peripheral portions of both Vps41 and Vps39 are least well resolved indicating their considerable flexibility. Multi-modular classification analyses revealed angular re-orientations of about 9° for Vps41 and 20° for Vps39 (Figure 2—figure supplement 3A-D) relative to the core, resulting in variable positions of the N-terminal β-propellers. At the top, Vps41 reaches out by approximately 100 Å in length and, similarly, Vps39 forms an elongated arch at the bottom (Figure 1E–G), positioning both Ypt7-interacting units at the farthest ends of the complex. The SNARE-binding element, composed of Vps16 and the SM protein Vps33, branches out to the lateral side of the complex approximately at the center of the structure (Figures 1E–G–2). Vps16 shares a large interface with the coiled-coil motif formed by Vps18 and Vps41 and the N-terminus of Vps18, which is stabilized through interactions between hydrophobic and charged residues (Figure 2C). Vps33 is in immediate contact with the structured loop of Vps18 (residues 824–831) that connects the elongated helix with the RING finger domain (Figure 2C). This, as well as the role of RING finger domains in the interlocking of other subunits, explains, why mutations at RING domains result in devastating human diseases and HOPS dysfunction (van der Beek et al., 2019; Edvardson et al., 2015; Robinson et al., 1991; van der Welle et al., 2021; Zhang et al., 2016) and cause failure of correct HOPS assembly (Figure 2—figure supplement 2). Overall, the SNARE binding module appears to be stably connected to the central core, while only the short C-terminal section of Vps16 α-solenoid (residues 739–798) displays high variability and is not resolved in our structure. HOPS couples tethering to fusion activity Tethering complexes bridge membranes by binding small GTPases, but also harbor or bind SM proteins (Ungermann and Kümmel, 2019). Reconstituted vacuole fusion is strictly HOPS and Ypt7-dependent at physiological SNARE concentrations (Langemeyer et al., 2018; Zick and Wickner, 2016), suggesting that HOPS is not just a tether, but part of the fusion machinery (Baker et al., 2015; Wickner and Rizo, 2017). However, so far, it was unknown how tethering and fusion activities of HOPS may be linked mechanistically. To address this, we first analyzed the N-terminal β-propellers of Vps41 and Vps39, the likely binding sites with Ypt7 (Lürick et al., 2017; Ostrowicz et al., 2010; Plemel et al., 2011). The intrinsic low affinity between HOPS and Ypt7 (Lürick et al., 2017) prevented reconstitution of the complex for structural studies, therefore, we instead relied on AlphaFold predictions. Additionally, we solved the structure of the β-propeller of Chaetomium thermophilum Vps39 by X-ray crystallography, which largely confirmed the predicted model (Figure 3—figure supplement 1A). Surprisingly, in the AlphaFold model, Ypt7 binding occurs at the α-solenoid of Vps39 where it does not directly interact with the β-propeller (Figure 3—figure supplement 1C), as originally expected. Furthermore, the binding site on Vps39 is placed approximately 5–6 nm above the membrane if Ypt7-anchored HOPS is in an upright position on supported lipid bilayers (Füllbrunn et al., 2021). Membrane-bound Ypt7 can still reach this site due to its 10 nm long hypervariable domain (not shown in the prediction). In the predicted complex of Vps41 (residues 1–919) with Ypt7 (residues 1–185), the GTPase binds directly to the Vps41 β-propeller, as anticipated. However, it interacts on the opposite side from the membrane-interacting amphipathic lipid-packing sensor (ALPS) motif (Cabrera et al., 2010; Figure 3—figure supplement 1B), suggesting that the hypervariable region of Ypt7 is required for binding, in analogy to Vps39. Curiously, in the predicted model, the ALPS motif faces away from the membrane which would hamper membrane binding. We noted, however, that this distal region of Vps41 (Figure 3—figure supplement 1B) displays substantial flexibility. The β-propeller of Vps41 might, therefore, be oriented differently in the structure than predicted by AlphaFold, which may bring the ALPS motif in contact with the bilayer if Vps41 is bound to Ypt7. Nevertheless, future experimental data will need to confirm the predicted AlphaFold model of Vps41 and Vps39 interaction with Ypt7. Vps39 binds Ypt7 far stronger than Vps41 (Auffarth et al., 2014; Lürick et al., 2017), and may be assisted by Vps18 to sandwich Ypt7, whereas Vps41 binds to Ypt7 apparently alone (Figure 3—figure supplement 1D). Such a dual interaction could explain both tighter binding and a preferred orientation of HOPS on membranes (Füllbrunn et al., 2021). To test the functional importance, we generated HOPS complexes lacking the β-propeller of Vps11, Vps18, or Vps41 (Figure 3A). All complexes were purified in equimolar stoichiometry (Figure 1—figure supplement 4A), and interacted with Ypt7, but not the Golgi Rab Ypt1 in GST pull-down assay, suggesting that at least one Rab-binding site is maintained in all truncated complexes (Bröcker et al., 2012; Lürick et al., 2017; Ostrowicz et al., 2010; Zick and Wickner, 2016; Zick and Wickner, 2012; Figure 1—figure supplement 4B). To determine the activity of HOPS mutants, we compared tethering and fusion. For tethering, we incubated liposomes bearing Ypt7-GTP with each complex and quantified clustering after centrifugation (Füllbrunn et al., 2021; Lürick et al., 2017). HOPS lacking the Vps41 β-propeller was inactive as shown (Lürick et al., 2017), whereas HOPS with truncated Vps11 or Vps18 was fully functional and as efficient as wild-type HOPS (Figure 3B). In contrast, when added to SNARE and Ypt7-GTP bearing liposomes, only wild-type HOPS, but none of the mutant complexes promoted fusion (Figure 3C). This was particularly puzzling for HOPS lacking either the Vps18 or Vps11 β-propeller as they had full tethering activity (Figure 3B). Therefore, we compared the structural features of HOPS mutants with the wild type using negative stain EM. Deletions of the β-propellers of Vps41, Vps11, or Vps18 indeed resulted in a loss of protein density at the expected positions, while preserving the densities of all other subunits (Figure 3D). Interestingly, HOPS complexes lacking β-propellers in Vps11 or Vps18 showed an alteration in the relative orientation of Vps39 within the complex in some 2D class averages. This observed structural variation might be a result of the increased flexibility of mutant HOPS due to the lack of structural support by β-propellers of Vps18 and Vps11. We conclude that the β-propellers of Vps18 and Vps11 contribute to the overall structure of the HOPS complex or may play a stabilizing role during the fusion process, which would explain, why they are essential for the full activity of HOPS. Discussion Our data suggest a working model of how HOPS catalyzes fusion at lysosomes and vacuoles (Figure 4). According to our structure, the three major ligand binding sites of HOPS are arranged in a triangular fashion. While the two Ypt7-binding sites show significant conformational variability, the SNARE-interacting unit is firmly connected to the stable backbone formed by Vps11 and Vps18. For tethering, HOPS Vps39 and Vps41 bind Ypt7 on target membranes. During this process, HOPS remains upright on membranes (Füllbrunn et al., 2021). Then, the SM protein Vps33 and possibly other sites on HOPS (Krämer and Ungermann, 2011; Baker et al., 2015; Lürick et al., 2017; Lürick et al., 2015; Song et al., 2020) bind SNAREs from the opposing membranes, and zipper them up toward their membrane anchor (Figure 4). Note, that this process can be blocked by Orf3a in the COVID-19 SARS-CoV-2 virus (Miao et al., 2021). In our model, the backbone of HOPS dampens the movement of the vesicles and acts as a lever arm holding on to SNAREs during zippering (D’Agostino et al., 2017). This three-point arrangement would cause membrane stress and could explain how HOPS catalyzes membrane fusion (Figure 4). The physiological function of HOPS can be bypassed if large complexes are redirected to SNAREs at the fusion site (D’Agostino et al., 2017; Song et al., 2017), which can even promote fusion of deficient SNARE complexes (Orr et al., 2022; Song et al., 2021). Zippered SNAREs may then dissociate from HOPS and allow access for α-SNAP and NSF to recycle SNAREs (Zhang and Hughson, 2021). Figure 4 with 1 supplement see all Download asset Open asset Working model for HOPS-mediated membrane tethering and fusion. The HOPS complex binds to Ypt7 on the vacuole and vesicles via Vps39 (dark green) and Vps41 (light green). SNARE proteins are recruited to HOPS by their N-terminal domains and the SNARE-binding module (dark and light brown). The stable central core of HOPS keeps the membranes in place, while Vps41 and Vps39 may function as dampers due to their limited flexibility. Consequentially, zippering of SNAREs, which is initiated by binding to Vps33 (dark brown), begins. As the N-terminal domains of SNAREs bind to HOPS, further SNARE zippering may occur with the HOPS backbone acting as a lever (not shown here). This may cause membrane stress and thus catalyzes fusion. HOPS may let go of Ypt7 and SNAREs thereafter. For details see text. How tethering complexes contribute to fusion poses a long-standing question in the field. The process necessitates the binding of two opposing membranes and exact coordination of the zippering procedure of membrane-bound SNAREs. Previous analyses suggested strong flexibility along the entire HOPS structure, which was interpreted as a hallmark of tethering complexes and an essential prerequisite to their function (Bröcker et al., 2012; Chou et al., 2016; Füllbrunn et al., 2021; Ha et al., 2016). Structural models of a largely open HOPS, based on negative stain EM. Chou et al., 2016, supported the importance of HOPS structural flexibility. Flexibility was also observed in our previous negative stain EM structure (Bröcker et al., 2012). Instead, our cryo-EM data, collected on a highly pure and homogeneous sample that was not modified by any crosslinker or other fixative agents like negative stain, show that HOPS flexibility is limited. Taking the obtained resolution as a measure of sample flexibility (Rawson et al., 2016), we conclude that the backbone and the SNARE-binding module are the least flexible parts of HOPS and appear to be stably associated with each other. In contrast, the membrane interacting units of Vps41 and Vps39 show some flexibility with 10–20° movements between particles, which substantially reduced their resolution (Figure 2—figure supplement 3). We relate this flexibility to their function within HOPS, where it may dampen the motion of HOPS between membranes and stabilizes the SNARE interaction, which are necessary for fusion (Laage and Ungermann, 2001; Krämer and Ungermann, 2011; Lürick et al., 2015; Baker et al., 2015; Song et al., 2020). Mammalian HOPS contains the same six subunits as yeast HOPS, which are generally highly conserved and will therefore function similarly (van der Beek et al., 2019; van der Kant et al., 2015). Our structure can hence be used to map disease prone mutations (Figure 4—figure supplement 1) and may thus explain the consequences on HOPS function. Due to low binding affinities, no structures of tethering complexes bound to Rabs have been solved. Here, we used AlphaFold to predict the interfaces of HOPS with its bound Rab7-like Ypt7 protein to understand the positioning of HOPS during tethering (Figure 3—figure supplement 1). We suspect that this low binding affinity helps tethering complexes to also let go of the Rab when fusion progresses (Figure 4). Even though long coiled-coil tethers such as EEA1 can promote SNARE-mediated membrane fusion (Murray et al., 2016), their cooperation with Rab GTPases in fusion is likely quite different from the mechanism proposed here (Ungermann and Kümmel, 2019). Some tethering complexes such as CORVET, CHEVI, or FERARI have attached SM proteins (van der Beek et al., 2019; Solinger et al., 2020), others such as COG, GARP, Exocyst, and Dsl cooperate with SM proteins (Ungermann and Kümmel, 2019), which may catalyze fusion similar to HOPS. For each of these complexes, in vivo models for their function exist, yet proteoliposome fusion assays in the presence of the required small GTPases are either not available or not yet completely developed (Balderhaar et al., 2013; Ha et al., 2016; Lee et al., 2022; Maib and Murray, 2022; Ren et al., 2009; Rossi et al., 2020; Solinger et al., 2020). We expect that similar approaches as established for HOPS will further support the key role of tethering complexes and reveal their cooperation with SM proteins in SNARE-mediated membrane fusion. The overarching principle suggested here for HOPS is not limited to lysosomal fusion but may extend to synaptic transmission, where the tether Munc13 and the SM protein Munc18 cooperatively catalyze the N- to C-terminal zippering of SNAREs during fusion (Lai et al., 2017; Rizo, 2022; Stepien et al., 2022; Stepien and Rizo, 2021). Both Munc18 and Vps33 interact similarly with the N-terminal part of the SNARE domains of the R- and Qa-SNARE and may promote assembly until the central zero-layer of the SNARE domain (Baker et al., 2015; Stepien et al., 2022). During HOPS-mediated fusion, SNARE zippering beyond the zero-layer could then proceed, while the Vps33 lets go of the forming four-helix bundles (Stepien et al., 2022). However, HOPS binds both the N-terminal extensions of the Qa-SNARE Vam3 and other SNAREs, possibly via different binding sites along the HOPS complex (Laage and Ungermann, 2001; Lürick et al., 2015; Song et al., 2020). This association may thus maintain the force on membranes to catalyze full fusion, even if the SM protein lets go of the assembling SNARE complex (Figure 4). In agreement, HOPS complexes with deficient Vps33 can catalyze fusion of proteoliposomes only if the SNARE density is high (Baker et al., 2015). In turn, vacuoles expressing a Qa-SNARE Vam3 variant lacking the N-terminal extension, which is needed to bind HOPS, show diminished fusion (Laage and Ungermann, 2001). This suggests that HOPS supports SNAREs by templating the association of R- and Qa-SNAREs and by binding the N-terminal regions of SNAREs. We believe that the deletion of the β-propeller in Vps11 or Vps18 result in a similarly deficient HOPS due to a less stable backbone (Figure 3). In either case, coupling between a stable backbone of HOPS and SNARE binding may be impaired and could result in less specific activity as fusion catalysts. Future experiments are required to determine the precise reason for their fusion deficiency. Overall, our insights provide a novel blueprint to understand HOPS function, dynamics, and regulation both in fusion and in other functions of its subunits (van der Beek et al., 2019; Elbaz-Alon et al., 2014; Hönscher et al., 2014; González Montoro et al., 2021; González Montoro et al., 2018; Wong et al., 2020), and imply a general role of tethering complexes as a catalytic part of the fusion machinery. Materials and methods Yeast strains Request a d" @default.
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• W4312372118 title "Editor's evaluation: Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery" @default.
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• W4312372118 workType "peer-review" @default.