Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex.
In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical ... analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.
Mesh Terms:
Amino Acid Sequence, Cloning, Molecular, Epitopes, Evolution, Molecular, Exocytosis, Fungal Proteins, Membrane Fusion, Membrane Proteins, Microscopy, Electron, Models, Molecular, Molecular Sequence Data, Nerve Tissue Proteins, Protein Conformation, Protein Structure, Secondary, Qc-SNARE Proteins, R-SNARE Proteins, SNARE Proteins, Saccharomyces cerevisiae Proteins, Suppression, Genetic, Synaptosomal-Associated Protein 25, Vesicular Transport Proteins
Amino Acid Sequence, Cloning, Molecular, Epitopes, Evolution, Molecular, Exocytosis, Fungal Proteins, Membrane Fusion, Membrane Proteins, Microscopy, Electron, Models, Molecular, Molecular Sequence Data, Nerve Tissue Proteins, Protein Conformation, Protein Structure, Secondary, Qc-SNARE Proteins, R-SNARE Proteins, SNARE Proteins, Saccharomyces cerevisiae Proteins, Suppression, Genetic, Synaptosomal-Associated Protein 25, Vesicular Transport Proteins
EMBO J.
Date: Nov. 02, 1998
PubMed ID: 9799229
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