Exocytosis requires asymmetry in the central layer of the SNARE complex

Rainer Ossig, Hans Schmitt, Bert de Groot, Dietmar Riedel, Sirkka Keränen, Hans Ronne, Helmut Grubmuller, Reinhard Jahn (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

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 α‐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 R→Q 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.

Original languageEnglish
Pages (from-to)6000 - 6010
Number of pages11
JournalEMBO Journal
Volume19
Issue number22
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Exocytosis
Membrane Fusion
Proteins
Fusion reactions
Fungal Proteins
Eukaryotic Cells
Membranes
Molecular Dynamics Simulation
Glutamine
Leucine
Arginine
Cell growth
Yeast
Free energy
Molecular dynamics
Mirrors
Amino Acids
Substitution reactions
Mutation
Growth

Cite this

Ossig, R., Schmitt, H., de Groot, B., Riedel, D., Keränen, S., Ronne, H., ... Jahn, R. (2000). Exocytosis requires asymmetry in the central layer of the SNARE complex. EMBO Journal, 19(22), 6000 - 6010. https://doi.org/10.1093/emboj/19.22.6000
Ossig, Rainer ; Schmitt, Hans ; de Groot, Bert ; Riedel, Dietmar ; Keränen, Sirkka ; Ronne, Hans ; Grubmuller, Helmut ; Jahn, Reinhard. / Exocytosis requires asymmetry in the central layer of the SNARE complex. In: EMBO Journal. 2000 ; Vol. 19, No. 22. pp. 6000 - 6010.
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Ossig, R, Schmitt, H, de Groot, B, Riedel, D, Keränen, S, Ronne, H, Grubmuller, H & Jahn, R 2000, 'Exocytosis requires asymmetry in the central layer of the SNARE complex', EMBO Journal, vol. 19, no. 22, pp. 6000 - 6010. https://doi.org/10.1093/emboj/19.22.6000

Exocytosis requires asymmetry in the central layer of the SNARE complex. / Ossig, Rainer; Schmitt, Hans; de Groot, Bert; Riedel, Dietmar; Keränen, Sirkka; Ronne, Hans; Grubmuller, Helmut; Jahn, Reinhard (Corresponding Author).

In: EMBO Journal, Vol. 19, No. 22, 2000, p. 6000 - 6010.

Research output: Contribution to journalArticleScientificpeer-review

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AB - 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 α‐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 R→Q 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.

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Ossig R, Schmitt H, de Groot B, Riedel D, Keränen S, Ronne H et al. Exocytosis requires asymmetry in the central layer of the SNARE complex. EMBO Journal. 2000;19(22):6000 - 6010. https://doi.org/10.1093/emboj/19.22.6000