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  • The EMBO Journal (2000) 19, 6000 - 6010
  • doi:10.1093/emboj/19.22.6000

Exocytosis requires asymmetry in the central layer of the SNARE complex

Rainer Ossig1,2, Hans Dieter Schmitt3, Bert de Groot4, Dietmar Riedel1, Sirkka Keränen5, Hans Ronne6, Helmut Grubmüller4 and Reinhard Jahn1

  1. Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
  2. Present address: Department of Physiology, University of Münster, Robert-Koch-Strasse 27a, D-48149 Münster, Germany
  3. Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
  4. Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
  5. VTT, Biotechnology, Tietotie 2, Espoo, PO Box 1500, FIN-02044 VTT, Finland
  6. Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, S-75007 Uppsala, Sweden

Correspondence to:

Reinhard Jahn, E-mail: rjahn@gwdg.de

Received 15 June 2000; Accepted 20 September 2000; Revised 18 September 2000

Assembly of SNAREs (soluble N-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q–R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror Rright arrowQ substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

  • Keywords:

    • exocytosis,
    • membrane fusion,
    • SNARE,
    • yeast