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Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Nature Structural Biology volume 5, pages 793–802 (1998)Cite this article

Abstract

The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in α-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of ∼3 M–1 s–1. An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.

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Figure 1: An N-terminal domain of Sso1p (Sso2p) inhibits binary and ternary SNARE complex formation.
Figure 2: Binary and ternary SNARE complex formation induces the folding of unstructured SNARE domains.
Figure 3: Binary and ternary SNARE complex stoichiometries.
Figure 4: The high thermal stability of binary and ternary SNARE complexes is independent of the Sso1p N-terminal domain.
Figure 5: Binary Sso1p–Sec9CT complex assembly approaches equilibrium slowly.
Figure 6: Formation of Sso1p–Sec9CT binary complex is inhibited by the N-terminal domain of Sso1p.
Figure 7: Formation of the binary complex is rate-limiting for ternary complex assembly.
Figure 8: Schematic model for SNARE assembly.

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Acknowledgements

We thank P. Brennwald, J. Gerst, S. Keränen and J. Walker for plasmids, S. Kyin and D. Little for DNA sequencing and mass spectrometry, and G. Waters, P. Brennwald, J. Carey, P. Hanson, G. McLendon, and A. Nagi for discussion and critical comments on the manuscript. The analytical ultracentrifuge was purchased with funds from a grant to R.F. from the Zimmer Corporation. This work was funded in part by an American Heart Association Fellowship (M.M.) and Searle Scholar and Beckman Young Investigator awards (F.M.H.).

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  1. Rebecca B. Miller

    Present address: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York, 10021, USA

Authors and Affiliations

  1. Department of Chemistry, Princeton University, Princeton, 08544, New Jersey

    Karin L. Nicholson

  2. Department of Molecular Biology, Princeton University, Princeton, 08544, New Jersey

    Mary Munson, Rebecca B. Miller & Frederick M. Hughson

  3. Department of Cell, Molecular, and Developmental Biology, Haverford College, Haverford, 19041, Pennsylvania, USA

    Thomas J. Filip & Robert Fairman

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Correspondence to Frederick M. Hughson.

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Nicholson, K., Munson, M., Miller, R. et al. Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p. Nat Struct Mol Biol 5, 793–802 (1998). https://doi.org/10.1038/1834

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