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Structure of a membrane tethering complex incorporating multiple SNAREs

Nature Structural & Molecular Biology volume 31, pages 246–254 (2024)Cite this article

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Abstract

Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here we present a cryo-electron microscopy structure of the simplest known MTC, the 255-kDa Dsl1 complex of Saccharomyces cerevisiae, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

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Fig. 1: The Dsl1 complex bound to Sec20 and Use1.
Fig. 2: Structure of Dsl1:Qb:Qc.
Fig. 3: Use1:Sec39 binds Sec20:Tip20 via inter-SNARE interactions.
Fig. 4: SNARE incorporation into Dsl1:Qb:Qc is essential in yeast.
Fig. 5: Comparison of Dsl1:Qb:Qc and exocyst.
Fig. 6: Model for retrograde vesicle capture by Dsl1:Qb:Qc.

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Data availability

Structural coordinates for Dsl1:Qb:Qc were deposited in the PDB with the accession code 8EKI. The cryo-EM density maps were deposited in the Electron Microscopy Data Bank with accession numbers EMD-28204 (composite map), EMD-29447, EMD-28760, EMD-28768, EMD-28762 (local refinement maps) and EMD-28774 (pre-local-refinement consensus map). The updated K. lactis Use1:Sec39:Dsl1 X-ray structure was deposited in the PDB with the accession code 8FTU. Source data are provided with this paper.

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Acknowledgements

We thank X. Fan, P. Shao, V. Vandavasi and members of the Hughson laboratory past and present for helpful advice and discussion. We are grateful to the Princeton University Biophysics and Macromolecular Crystallography core facilities for technical assistance. We acknowledge the use of Princeton’s Imaging and Analysis Center (IAC), which is partially supported by the Princeton Center for Complex Materials (PCCM), a National Science Foundation (NSF) Materials Research Science and Engineering Center (MRSEC, DMR-2011750). This work was supported by National Institutes of Health grants R01GM071574 (F.M.H.), T32GM007388 (K.A.D., A.E.S., J.D.S. and S.M.T.) and F31GM12676 (S.M.T.).

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Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Kevin A. DAmico, Abigail E. Stanton, Jaden D. Shirkey, Sophie M. Travis, Philip D. Jeffrey & Frederick M. Hughson

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Contributions

K.A.D. performed the structural experiments and the in vivo functional analysis. K.A.D. and A.E.S. performed the biochemical experiments. K.A.D., S.M.T., P.D.J. and F.M.H. analyzed the structural data. K.A.D. and J.D.S. computed the AF predictions. K.A.D. and F.M.H. designed the research and wrote the paper with input from A.E.S., J.D.S., S.M.T. and P.D.J.

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Correspondence to Frederick M. Hughson.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Representative cryo-EM images.

a, Representative cryo-EM micrograph of Dsl1:Qb:Qc prepared as described in Methods. Individual Dsl1:Qb:Qc particles have been marked with white circles. 5,857 micrographs were collected. b, Representative 2D class averages of the Dsl1:Qb:Qc complex used in the construction of the cryo-EM density. Classes were generated using cryoSPARC.

Extended Data Fig. 2 Workflow of cryo-EM data processing pre-local refinement.

a, Flowchart describing the training of the template picker used in the initial EM map determination of the Dsl1:Qb:Qc complex. b, Flowchart describing the method for generating an initial 3D model of Dsl1:Qb:Qc to use for density-guided template picking. c, Flowchart describing the method for generating a pre-local-refinement EM map of Dsl1:Qb:Qc using a template picker trained on the 8.0 Ã… map generated in (b). The EM map is inverted relative to the final map.

Extended Data Fig. 3 Local refinement of the Dsl1:Qb:Qc complex.

a, Output from Non-Uniform Refinement that was used for mask generation and local refinement. b, Angular distribution of the consensus map. c, GS-FSC curve of the consensus map. d, Flowchart describing the masking process for local refinement of the EM map. Four separate masks were applied, covering approximately half of the complex in four different orientations. At the outset of this process, the EM map was inverted by non-uniform refinement. Local refinement was performed on both the inverted and corrected EM map, and the higher-resolution output was used in the final composite map. e, GS-FSC curve of the composite EM map of the Dsl1:Qb:Qc complex. The curve was generated by combining the half maps from each of the four local refinement jobs and processing with the Validation (FSC) tool in cryoSPARC.

Extended Data Fig. 4 Local resolution of the Dsl1:Qb:Qc complex cryo-EM map.

a, Local refinement map generated from Mask 1 colored by local resolution from two different viewing angles. The map is superimposed on an outline of the complete Dsl1:Qb:Qc complex at a lower contour level for reference. b, Local refinement map generated from Mask 2 colored by local resolution. c, Local refinement map generated from Mask 3 colored by local resolution. d, Local refinement map generated from Mask 4 colored by local resolution.

Extended Data Fig. 5 Crystallographic and Alphafold (AF) contributions to the Dsl1:Qb:Qc complex model.

a, Model of the Dsl1:Qb:Qc complex surrounded by an outline of the EM map. Each of the crystal structures (denoted with PDB codes) and AF predictions used in the modeling process are also shown with their relative location specified. b, Dsl1ΔN (356-754) was generated by AF to supplement the available crystallographic data. Dsl1ΔN (excluding the lasso 378-488) is overlaid onto the outline of the cryo-EM density of the Dsl1:Qb:Qc complex, to demonstrate fit. c, Sec39 (1-100), generated by AF, is overlaid onto the outline of the cryo-EM density of the Dsl1:Qb:Qc complex, to demonstrate fit. d, The N-terminal domains of Dsl1 and Tip20 interact directly. The AF-predicted interface of Dsl1 and Tip20 is overlaid onto the outline of the cryo-EM density of the Dsl1:Qb:Qc complex, to demonstrate fit. e, AF was used to model K. lactis Use1 bound to Sec39, and the resulting prediction was fit into our previously reported electron density map (6WC4). In the resulting model (8FTU), the position of Use1 is the same, but the orientation is flipped, agreeing well with the orientation we observe for S. cerevisiae Use1 bound to Sec39.

Extended Data Fig. 6 Statistics on AF contributions to the Dsl1:Qb:Qc complex model.

a, Table listing AF predictions utilized in the model building process of the Dsl1:Qb:Qc complex. Left: protein name and residues included in the AF job. Center: depiction of the Rank_0 model generated by AF, colored by pLDDT values. Dsl1 (378-488) and Sec20 (38-65), though depicted, were not used in the modeling process as there was no corresponding density for these regions. Right: statistics on the AF jobs. pLDDT was calculated by averaging the score of each residue in a given job used in the final model. pTM (for monomeric jobs) and pTM+ipTM (for multimeric jobs) were extracted from AF directly. CC fit was generated in ChimeraX at the local resolution of the fitted portion of the map. b, Predicted aligned error (PAE) graph generated by AF for Dsl1 (344-754). Green indicates a lower distance error for a given residue pair. c, PAE graph generated by AF for Sec39 (1-112). d, PAE graph generated by AF for Dsl1 (1-131), Tip20 (1-66). e, PAE graph generated by AF for Sec20 (1-184), Use1 (2-86).

Extended Data Fig. 7 Mutations in Sec20 and Use1 that abolish SNARE-SNARE binding do not affect protein migration on size-exclusion chromatography.

a, His7-Use1 (1-212):Sec39 migrates with a similar profile to His7-Use1 (1-212) (L34A, F46A, F58A):Sec39 on size-exclusion chromatography. b, Sec20-His7 (1-275):Tip20 migrates with a similar profile to Sec20-His7 (1-275) (D129R, L132R, D136R):Tip20. Data presented in this figure are identical to data presented in Fig. 3.

Extended Data Fig. 8 Mutations designed to disrupt tether:SNARE interactions abolish binding in vitro.

a, His7-Sec39 pulls down Use1 (1-212). b, His7-Sec39 does not pull down mutant Use1 (1-212) (F9A, V13A). c, His7-Tip20 pulls down Sec20 (1-275). d, His7-Tip20 does not pull down Sec20 (1-275) (C79R, V82R, Y86A). Each experiment was performed twice.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

3DVA of the Dsl1:Qb:Qc complex.

Source data

Source Data Fig. 1

Unprocessed SDS–PAGE gel.

Source Data Fig. 3

Unprocessed SDS–PAGE gels.

Source Data Extended Data Fig. 8

Unprocessed SDS–PAGE gels.

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DAmico, K.A., Stanton, A.E., Shirkey, J.D. et al. Structure of a membrane tethering complex incorporating multiple SNAREs. Nat Struct Mol Biol 31, 246–254 (2024). https://doi.org/10.1038/s41594-023-01164-8

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