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A tethering complex drives the terminal stage of SNARE-dependent membrane fusion

Abstract

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore1,2,3. This last step is highly energy-demanding4,5. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

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Figure 1: Effect of soluble Vam7 on lipid and content mixing.
Figure 2: Fusion pore opening driven by ligands increasing SNARE complex size in vitro.
Figure 3: Effect of SNARE complex enlargement on vacuole fusion in vivo.
Figure 4: Molecular dynamics simulations of the influence of steric constraints at the (hemi)fusion site.

References

  1. Gao, Y. et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337, 1340–1343 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  2. Zhang, X. et al. Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex. Proc. Natl Acad. Sci. USA 113, E8031–E8040 (2016)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Reese, C., Heise, F. & Mayer, A. Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion. Nature 436, 410–414 (2005)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Chernomordik, L. V. & Kozlov, M. M. Protein–lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem. 72, 175–207 (2003)

    Article  CAS  PubMed  Google Scholar 

  5. Cohen, F. S. & Melikyan, G. B. The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J. Membr. Biol. 199, 1–14 (2004)

    Article  CAS  PubMed  Google Scholar 

  6. Shin, J., Lou, X., Kweon, D.-H. & Shin, Y.-K. Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states. Biochem. J. 459, 95–102 (2014)

    Article  CAS  PubMed  Google Scholar 

  7. Rizo, J. & Südhof, T. C. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices—guilty as charged? Annu. Rev. Cell Dev. Biol. 28, 279–308 (2012)

    Article  CAS  PubMed  Google Scholar 

  8. Hernandez, J. M., Kreutzberger, A. J. B., Kiessling, V., Tamm, L. K. & Jahn, R. Variable cooperativity in SNARE-mediated membrane fusion. Proc. Natl Acad. Sci. USA 111, 12037–12042 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Baker, R. W. et al. A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349, 1111–1114 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Orr, A., Wickner, W., Rusin, S. F., Kettenbach, A. N. & Zick, M. Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion. Mol. Biol. Cell 26, 305–315 (2015)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Kuhlee, A., Raunser, S. & Ungermann, C. Functional homologies in vesicle tethering. FEBS Lett. 589, 2487–2497 (2015)

    Article  CAS  PubMed  Google Scholar 

  12. Zick, M. & Wickner, W. The tethering complex HOPS catalyzes assembly of the soluble SNARE Vam7 into fusogenic trans-SNARE complexes. Mol. Biol. Cell 24, 3746–3753 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Price, A., Seals, D., Wickner, W. & Ungermann, C. The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein. J. Cell Biol. 148, 1231–1238 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Xu, H., Jun, Y., Thompson, J., Yates, J. & Wickner, W. HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion. EMBO J. 29, 1948–1960 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Thorngren, N., Collins, K. M., Fratti, R. A., Wickner, W. & Merz, A. J. A soluble SNARE drives rapid docking, bypassing ATP and Sec17/18p for vacuole fusion. EMBO J. 23, 2765–2776 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Pieren, M., Schmidt, A. & Mayer, A. The SM protein Vps33 and the t-SNARE Habc domain promote fusion pore opening. Nat. Struct. Mol. Biol. 17, 710–717 (2010)

    Article  CAS  PubMed  Google Scholar 

  17. Schwartz, M. L. & Merz, A. J. Capture and release of partially zipped trans-SNARE complexes on intact organelles. J. Cell Biol. 185, 535–549 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bröcker, C. et al. Molecular architecture of the multisubunit homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Proc. Natl Acad. Sci. USA 109, 1991–1996 (2012)

    Article  ADS  PubMed  CAS  PubMed Central  Google Scholar 

  19. Orr, A., Song, H., Rusin, S. F., Kettenbach, A. N. & Wickner, W. HOPS catalyzes the interdependent assembly of each vacuolar SNARE into a SNARE complex. Mol. Biol. Cell 28, 975–983 (2017)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yu, H. et al. Reconstituting intracellular vesicle fusion reactions: the essential role of macromolecular crowding. J. Am. Chem. Soc. 137, 12873–12883 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Haruki, H., Nishikawa, J. & Laemmli, U. K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008)

    Article  CAS  PubMed  Google Scholar 

  22. Michaillat, L., Baars, T. L. & Mayer, A. Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1. Mol. Biol. Cell 23, 881–895 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cabrera, M. et al. Vps41 phosphorylation and the Rab Ypt7 control the targeting of the HOPS complex to endosome–vacuole fusion sites. Mol. Biol. Cell 20, 1937–1948 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. LaGrassa, T. J. & Ungermann, C. The vacuolar kinase Yck3 maintains organelle fragmentation by regulating the HOPS tethering complex. J. Cell Biol. 168, 401–414 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Carr, C. M. & Rizo, J. At the junction of SNARE and SM protein function. Curr. Opin. Cell Biol. 22, 488–495 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Fisher, R. J., Pevsner, J. & Burgoyne, R. D. Control of fusion pore dynamics during exocytosis by Munc18. Science 291, 875–878 (2001)

    Article  ADS  CAS  PubMed  Google Scholar 

  27. Morgera, F. et al. Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol. Biol. Cell 23, 337–346 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hashizume, K., Cheng, Y.-S., Hutton, J. L., Chiu, C.-H. & Carr, C. M. Yeast Sec1p functions before and after vesicle docking. Mol. Biol. Cell 20, 4673–4685 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Grote, E., Carr, C. M. & Novick, P. J. Ordering the final events in yeast exocytosis. J. Cell Biol. 151, 439–452 (2000)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zick, M. & Wickner, W. T. A distinct tethering step is vital for vacuole membrane fusion. Elife 3, e03251 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Zick, M., Orr, A., Schwartz, M. L., Merz, A. J. & Wickner, W. T. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18. Proc. Natl Acad. Sci. USA 112, E2290–E2297 (2015)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Risselada, H. J., Bubnis, G. & Grubmüller, H. Expansion of the fusion stalk and its implication for biological membrane fusion. Proc. Natl Acad. Sci. USA 111, 11043–11048 (2014)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004)

    Article  CAS  PubMed  Google Scholar 

  34. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998)

    Article  CAS  PubMed  Google Scholar 

  35. Kuznetsova, I. M., Turoverov, K. K. & Uversky, V. N. What macromolecular crowding can do to a protein. Int. J. Mol. Sci. 15, 23090–23140 (2014)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Mage, M. G. in Immunochemical Techniques Vol. 70 (eds Vunakis, H. V. & Langone, J. ) 142–150 (Elsevier, 1980)

    Article  CAS  Google Scholar 

  37. Ostrowicz, C. W. et al. Defined subunit arrangement and Rab interactions are required for functionality of the HOPS tethering complex. Traffic 11, 1334–1346 (2010)

    Article  CAS  PubMed  Google Scholar 

  38. Hess, B., Kutzner, C., van der Spoel, D. & Lindahl, E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J. Chem. Theory Comput. 4, 435–447 (2008)

    Article  CAS  PubMed  Google Scholar 

  39. Monticelli, L. et al. The MARTINI coarse-grained force field: extension to proteins. J. Chem. Theory Comput. 4, 819–834 (2008)

    Article  CAS  PubMed  Google Scholar 

  40. Marrink, S. J., Risselada, H. J., Yefimov, S., Tieleman, D. P. & de Vries, A. H. The MARTINI force field: coarse grained model for biomolecular simulations. J. Phys. Chem. B 111, 7812–7824 (2007)

    Article  CAS  PubMed  Google Scholar 

  41. Risselada, H. J., Kutzner, C. & Grubmüller, H. Caught in the act: visualization of SNARE-mediated fusion events in molecular detail. ChemBioChem 12, 1049–1055 (2011)

    Article  CAS  PubMed  Google Scholar 

  42. Hub, J. S., De Groot, B. L. & van der Spoel, D. g_wham—a free weighted histogram analysis implementation including robust error and autocorrelation estimates. J. Chem. Theory Comput. 6, 3713–3720 (2010)

    Article  CAS  Google Scholar 

  43. Mayer, A. & Wickner, W. Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF). J. Cell Biol. 136, 307–317 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang, L., Merz, A. J., Collins, K. M. & Wickner, W. Hierarchy of protein assembly at the vertex ring domain for yeast vacuole docking and fusion. J. Cell Biol. 160, 365–374 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Karunakaran, S., Sasser, T., Rajalekshmi, S. & Fratti, R. A. SNAREs, HOPS and regulatory lipids control the dynamics of vacuolar actin during homotypic fusion in S. cerevisiae. J. Cell Sci. 125, 1683–1692 (2012)

    Article  CAS  PubMed  Google Scholar 

  46. Merz, A. J. & Wickner, W. T. Resolution of organelle docking and fusion kinetics in a cell-free assay. Proc. Natl. Acad. Sci. USA. 101, 11548–11553 (2004)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  47. Lürick, A. et al. Multivalent Rab interactions determine tether-mediated membrane fusion. Mol. Biol. Cell 28, 322–332 (2016)

    Article  PubMed  CAS  Google Scholar 

  48. Jackson, M. B. Minimum membrane bending energies of fusion pores. J. Membr. Biol. 231, 101–115 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ryham, R. J., Ward, M. A. & Cohen, F. S. Teardrop shapes minimize bending energy of fusion pores connecting planar bilayers. Phys. Rev. E 88, 062701 (2013)

    Article  ADS  CAS  Google Scholar 

  50. D’Agostino, M., Risselada, H. J. & Mayer, A. Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion-to-fusion transition. EMBO Rep. 17, 1590–1608 (2016)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Pieren, M., Desfougères, Y., Michaillat, L., Schmidt, A. & Mayer, A. Vacuolar SNARE protein transmembrane domains serve as nonspecific membrane anchors with unequal roles in lipid mixing. J. Biol. Chem. 290, 12821–12832 (2015)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank V. Comte and J. Gao for the purification of proteins and antibodies. This work was supported by grants from the DFG (SFB 944, to C.U.), State of Lower Saxony (life@nano, to H.J.R) and SNF and ERC to A.M. HLRN is acknowledged for CPU time.

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Contributions

H.J.R. conceived and interpreted simulation experiments and values derived from them. M.D.A. conceived, performed and interpreted all other experiments. A.M. conceived the study and interpreted the results. A.L. and C.U. provided purified CORVET, HOPS and HOPS subcomplexes. All authors jointly wrote the paper.

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Correspondence to Andreas Mayer.

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The authors declare no competing financial interests.

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Extended data figures and tables

Extended Data Figure 1 Optimization and production of reaction components.

a, ypt7Δ vacuoles lack HOPS. Ypt7 and HOPS content of total cell extracts and purified vacuoles from wild-type and ypt7Δ mutants in BJ3505 and DKY6281 cells, representing the background strains used for the content mixing assay. b, Purified HOPS, HOPS subcomplexes and CORVET. The complexes used for the in vitro experiments were analysed by SDS–PAGE and Coomassie staining. The gel represents the preparations, which followed published routine procedure, used for the experiments in Fig. 1 and Extended Data Figs 3 and 4. c, Production of Fab fragments from polyclonal antibodies to Vam3 and Nyv1. Schematic view of papain cleavage sites for Fab fragment generation (left). Affinity-purified antibodies and Fab fragments extracted after papain digestion were analysed by non-reducing SDS–PAGE and Coomassie staining. The gel shows the preparation used in Fig. 2. d, Expression of FKBP and FRB fusion proteins. Total cell extracts were prepared from 0.1 OD600 nm units of logarithmic cultures of yeast strains expressing Vam7–2×FKBP12 and/or Pfk1–FRB–GFP. Proteins were analysed by SDS–PAGE and western blotting against Vam7, Vam3 and GFP. e, As in d, but for cells expressing Vam7–LL–2×FKBP12, containing the 35-amino-acid linker. For a and ce, similar results were obtained from two independent preparations.

Extended Data Figure 2 Kinetics and efficiency of in vitro vacuole fusion, measured by content mixing.

Vacuoles were prepared from two different strains, which contain either the soluble 45-kDa maturase Pep4 (contained in DKY6281) or the pro-alkaline phosphatase (p-ALP) (contained in BJ3505). Formation of a sufficiently large fusion pore allows Pep4 to transfer into the p-ALP containing fusion partner, leading to proteolytic cleavage of its pro-sequence and activation of the enzyme (m-ALP). This activity is used as a measurement of fusion. Note that proteolytic maturation of p-ALP is fast and not limiting for the development of the content mixing signal46. Standard fusion reactions were started, and aliquots were withdrawn at the indicated time points and set on ice. a, At the end of the 60 min period, m-ALP activity was determined for all samples. Data are means ± s.d. from 3 biologically independent experiments. b, Aliquots from one of the experiments in (a) were trichloroacetic acid (TCA)-precipitated and analysed by SDS–PAGE and western blotting against ALP and Vam3. Signals were detected on a LICOR infrared scanner (left) and quantified (right) as the ratio of m-ALP to (m-ALP + p-ALP). Vam3 has been included as a loading control. Note that after one round of fusion, only a maximum of 50% of p-ALP can be matured, because half of the fusion events in the suspension will occur between like vacuoles (that is, Pep4/Pep4 or p-ALP/p-ALP) and will not produce a signal.

Extended Data Figure 3 HOPS and CORVET complexes stimulate fusion to similar degrees.

Two-stage fusion reactions with ypt7Δ vacuoles were run as in Fig. 1g, in the absence of ATP. rVam7 had been added in the first phase of the incubation (1°), and either 0.4 μM HOPS subcomplexes or 0.4 μM CORVET was added during the second incubation (2°). Half of the samples also received 6% Ficoll-400, an agent mimicking molecular crowding35, during the second incubation. At the end of the 75-min incubation period, content mixing was assayed. Fusion activity of a standard wild-type reaction performed in the presence of ATP served as a100% reference. Data are mean ± s.d. from 3 biologically independent experiments.

Extended Data Figure 4 ypt7Δ vacuoles require both Vam7 and HOPS for content mixing.

Two-stage fusion reactions were run as in Fig. 1g in the presence or absence of ATP. rVam7 was added in the first phase of the incubation, HOPS was added only in the only in the second incubation. At the end of the 75-min incubation period, content mixing was assayed. Data are mean ± s.d. from 3 biologically independent experiments.

Extended Data Figure 5 A molecular crowding agent cannot stimulate fusion in the absence of bulky SNARE ligands.

Two-stage fusion reactions with ypt7Δ vacuoles were run as in Fig. 2b, in the absence or presence of ATP. rVam7 was added in the first phase of the incubation where indicated, antibodies and various concentrations of the crowding agent Ficoll-400 were added only during the second incubation. At the end of the 75-min incubation period, content mixing was assayed. Fusion activity of a wild-type reaction performed in the presence of ATP served as a 100% reference. Data are mean ± s.d. from 3 biologically independent experiments.

Extended Data Figure 6 Effect of rapamycin-induced dimerization on in vivo vacuole fusion using the small fusion protein FRB–GFP.

a, Schematic view of rapamycin-induced FKBP12/FRB-tagged protein dimerization between Vam7–2×FKBP12 and FRB–GFP. b, Logarithmically growing cells, expressing tagged Vam7–2×FKBP12, were stained with the vacuole tracer FM4-64 and analysed by spinning disc microscopy before and 10 min after the addition of 10 μM rapamycin. Scale bar, 5 μm. Similar results were obtained in 3 biologically independent repetitions.

Extended Data Figure 7 Fusion can be prematurely triggered by protein recruitment after osmotically induced vacuole fragmentation.

a, Logarithmically growing cells, carrying Vam7–2×FKBP12 and Pfk1–FRB–GFP as indicated, were stained with the vacuole tracer FM4-64. Vacuole fission was induced by adding 0.5 M NaCl. Cells were analysed by spinning disc microscopy before and 10 and 60 min after salt addition. The cells were grouped into three categories according to the number of vacuoles visible per cell. 100 cells were analysed per sample. b, As in a, but 10 μM rapamycin was added before the salt shock. c, As in b, but with cells expressing non-tagged Vam7. Data are mean ± s.d. from 3 biologically independent experiments. Scale bars, 5 μm.

Extended Data Figure 8 Effect of HOPS on the free energy barrier of fusion pore formation.

This plot complements Fig. 4. a, The free energy barrier of fusion pore opening is derived for a simulated system consisting of three SNARE complexes (right) and a POPC membrane that contains 40% POPE (coloured orange). To this aim, we pull two hydrophilic probes (coloured purple) towards the centre of the stalk and estimate the work (ΔG) as a function of probe–probe distance (the stalk thickness)32. The arrows in the free energy profile indicate the nucleation barrier for the fusion pore. Beyond this stage, subsequent pore opening proceeds in the absence of additional work (the plateau region). Tethering complexes such as HOPS are attracted to the membrane by Rab-GTPases or through direct lipid interaction10,47. An attractive HOPS surface (green line) conserves the lowered nucleation barrier, even when the surface attractions fully compensate the membrane bending energy (no net bending work; Extended Data Fig. 9). Error bars are calculated via Bayesian resampling of 50 overlapping WHAM histograms42 Each parental WHAM histogram consists of more than 30,000 data points (autocorrelation up to approximately 1,500 data points). b, Pore formation in the absence of HOPS. A defect is frequently formed in the vicinity of the SNARE TMDs (black arrow), illustrating the presence of a high stress (the defect probably decreases the bending stress). Fusion pore formation is associated with a sudden reduction of the sharp curvature near the circumference of the stalk (dashed lines). Fusion pores tend to adopt a teardrop shape48,49. c, Fusion pore formation in the presence of HOPS. The pre-existing teardrop membrane shape imposed by HOPS likely provides a geometrical and therefore an energetic advantage for pore formation. d, Set up in which we artificially enforced formation of a leakage pore/defect in the direct vicinity of the stalk (the rationale behind this has been previously explained32). The induced defect (between 3.2 and 1.8 nm the probe pierced through the membrane) instantaneously recovers. This suggests that the stress that HOPS imposes on the fusion site does not prime fusion to become leaky.

Extended Data Figure 9 Detailed analysis of HOPS-mediated membrane bending in the presence of an inter-membrane restraint.

a, Simulation snapshot illustrating the geometry of the system. Shown is the central plane of the membrane (the lipid tail ends), the stalk, and HOPS. The SNARE complex present in the simulation setup is not illustrated. This setup serves as a motivation for the elastic continuum model. b, Bending work required to place HOPS at the (hemi-)fusion site and peristaltic force experienced by HOPS. Simulations were run to measure the work required to place HOPS-like spheres of 10–14 nm diameter at the site of hemifusion or at a fusion pore (FP). HOPS could be detached from the SNARE complex by a long spacer (link.). The influence of a SNARE complex with an unstructured, non-helical juxta-membrane region (unstr.) and of a HOPS mimic that was attractive to the membrane surface (attr.) was also analysed. The bottom panel shows averages obtained from the simulations. fd is the (peristaltic) force that pushes HOPS away from the inter-membrane restraint (for example, a stalk, fusion pore, or trans-SNARE complex). Note that surface attractions or Rab-GTPase interactions of HOPS (modelling the tethering of membranes)10,47 can yield a negative value of the average work required to bend the membrane (bending occurs spontaneously). Fusion pore formation reduces the required bending work—it moves HOPS away from the restraint because of additional SNARE association up into the TMD region. The errors in the averages are derived from block averaging over more than 10,000 data points until the error becomes independent of block size (autocorrelation up to approximately 300 data points). c, Elastic continuum model. The coordinate system is based on the snapshot of the molecular dynamics simulation shown in a. Because of symmetry along the xy plane and xz plane, we only model one-quarter of the original system. The cartoon illustrates the shape of minimal free energy for a membrane (modelled by a single sheet), subjected to two constraints: (1) A local constraint on the position (height) of the membrane illustrated by the black arrow at z = 2 nm. This mimics the inter-membrane constraint (stalk, fusion pore or partly-assembled SNARE complex). (2) The presence of a hemisphere, this mimics HOPS. The colour code illustrates the height of the membrane (the z axis) relative to the two constraints. d, Prediction of bending energies by the elastic continuum model. The bending energy is shown as a function of the size and distance of HOPS to the inter-membrane restraint. Top, bending energy decreases steeply when HOPS moves away from the restraint. The predicted values are about a factor of two lower than the ‘bending work’ predicted by the simulations (see Methods). Middle, the corresponding peristaltic force (fd) on HOPS (the derivative of bending energy). At short distances, fd becomes substantial (tens of pN). Note that making the surface of HOPS moderately attractive to the membrane affects fd only weakly, that is, it does not result in an attraction towards the stalk. Bottom, the relative reduction of membrane area as a result of HOPS-induced membrane bending. This property reflects the tension that HOPS induces by curving the membrane near the contact zone. In contrast to bending energy and force, tension only weakly depends on the distance (d) to the restraint.

Extended Data Figure 10 Effect of HOPS on the force exerted by a single SNARE complex.

a, One way of rationalizing the acceleration of fusion pore formation by a SNARE complex is to consider it as a mechanical device that exerts force on the luminal leaflets through its TMDs, thereby compressing the stalk. This can happen through a peristaltic force that pulls the SNARE complex away from the stalk, or through the elastic bending of the SNAREs. This latter mode of force transmission requires the juxta-membrane regions, which connect the coiled-coil domains of the SNAREs to their TMDs, to be structured and rigid. The compressing force that the SNARE complex exerts on the stalk can be rationalized from the apparent work (free energy) that one needs to perform in order to force the luminal C termini of Vam3 and Nyv1 in closer proximity. We estimated how HOPS binding affects the force that the C termini of the SNAREs Vam3 and Nyv1 exert on the stalk. b, The work required to slightly indent the stalk in the presence of repulsive or attractive HOPS-spheres of different diameter has been determined. It is shown relative to the situation without the sphere. Error bars are calculated via Bayesian resampling of 15 overlapping WHAM histograms42. Each parental WHAM histogram is comprised of more than 30,000 data points (autocorrelation up to approximately 1,200 data points). The lines shown result from fitting a power expansion (up to the fourth power) through the average of each data point. Error bars are calculated via Bayesian resampling of 15 overlapping WHAM histograms44. c, The corresponding forces on the SNARE TMDs were derived from this work. Apparent gains in the force exerted by the SNARE C termini (left) are shown as a function of their distance in the hemifusion structure. HOPS binding can double or triple the magnitude of the apparent force (10–20 pN) that a SNARE complex exerts on a stalk50. The gain dissipates, however, as zipping of the SNARE TMDs progresses and their C termini approach each other. d, Snapshots of three special scenarios. Top, The HOPS sphere is placed at a distal location with respect to the stalk (for example, via attachment with a flexible linker), which abolishes the force gain. Middle, A sphere that favourably attracts (and bends) the membrane, this conserves the force gain. Bottom, unstructured, flexible SNARE juxta-membrane regions partially disrupt the mechanical coupling between the coiled-coil domains and the TMDs; they decrease the apparent gain in SNARE pull force induced by HOPS. Structured (α-helical) SNARE juxta-membrane regions result in a high initial force gain which gradually reduces. In contrast, unstructured, flexible juxta-membrane regions, which impair vacuole fusion51, result in a near-constant force gain of only about 8 pN. Both cases converge to similar force values when the C termini of Vam3 and Nyv1 come in closer proximity. Because the SNARE complex is unable to exert bending force on the membrane, when the connection between its transmembrane anchors and the SNARE domains is completely flexible, we relate the remaining gain to an effective softening of the stalk because of the induced membrane curvature and to the peristaltic force generated by the interaction of the HOPS sphere with the SNAREs.

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D’Agostino, M., Risselada, H., Lürick, A. et al. A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638 (2017). https://doi.org/10.1038/nature24469

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