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Vol. 19, Issue 9, 3769-3781, September 2008

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The SNAP-25 Linker as an Adaptation Toward Fast Exocytosis

Gábor Nagy^*,,, Ira Milosevic^*,,, Ralf Mohrmann^*, Katrin Wiederhold^||, Alexander M. Walter^*, and Jakob B. Sørensen^*

*Molecular Mechanism of Exocytosis, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; and ^||Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

Submitted December 6, 2007; Revised May 23, 2008; Accepted June 18, 2008
Monitoring Editor: Thomas F. J. Martin

InCytes from MBC

The assembly of four soluble N-ethylmaleimide-sensitive factor attachment protein receptor domains into a complex is essential for membrane fusion. In most cases, the four SNARE-domains are encoded by separate membrane-targeted proteins. However, in the exocytotic pathway, two SNARE-domains are present in one protein, connected by a flexible linker. The significance of this arrangement is unknown. We characterized the role of the linker in SNAP-25, a neuronal SNARE, by using overexpression techniques in synaptosomal-associated protein of 25 kDa (SNAP-25) null mouse chromaffin cells and fast electrophysiological techniques. We confirm that the palmitoylated linker-cysteines are important for membrane association. A SNAP-25 mutant without cysteines supported exocytosis, but the fusion rate was slowed down and the fusion pore duration prolonged. Using chimeric proteins between SNAP-25 and its ubiquitous homologue SNAP-23, we show that the cysteine-containing part of the linkers is interchangeable. However, a stretch of 10 hydrophobic and charged amino acids in the C-terminal half of the SNAP-25 linker is required for fast exocytosis and in its absence the calcium dependence of exocytosis is shifted toward higher concentrations. The SNAP-25 linker therefore might have evolved as an adaptation toward calcium triggering and a high rate of execution of the fusion process, those features that distinguish exocytosis from other membrane fusion pathways.

These authors contributed equally to this work.

Present addresses: National Centre for Stereotactic Radiosurgery, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom;

Department of Cell Biology, Yale University, School of Medicine, New Haven, 06510 CT.

Address correspondence to: Jakob B. Sørensen (jsoeren{at}gwdg.de)

Abbreviations used: LDCV, large dense-core vesicle; RRP, readily-releasable pool; SNAP-25, synaptosome-associated protein of 25 kDa; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SRP, slowly releasable pool; WT, wild type.

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