The SNAP-25 linker as an adaptation toward fast exocytosis.
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.
Nagy
Gábor
G
Molecular Mechanism of Exocytosis, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Milosevic
Ira
I
Mohrmann
Ralf
R
Wiederhold
Katrin
K
Walter
Alexander M
AM
Sørensen
Jakob B
JB
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D005089:Exocytosis; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050766:Qb-SNARE Proteins / Q000502:physiology; D050767:Qc-SNARE Proteins / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000502:physiology*; D013574:Synaptosomes / Q000378:metabolism
D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; C100493:SNAP23 protein, human; D050825:Synaptosomal-Associated Protein 25