Abstract
The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.
Highlights
SNARE1 proteins play an essential role in all membrane fusion steps of the secretory and endocytic pathway [1, 2]
Since in the closed conformation the SNARE motif of syntaxin cannot bind to other SNAREs, the N-terminal domain can down-regulate the capability of syntaxin to form SNARE complexes
We have shown previously that the four SNARE motifs form a SNARE complex with remarkable similarities to the neuronal SNARE complex [8, 9] with endobrevin corresponding to synaptobrevin 2, syntaxin 7 corresponding to syntaxin 1, and vti1b and syntaxin 8 corresponding to the N- and C-terminal SNARE motifs of SNAP-25, respectively
Summary
SNARE1 proteins play an essential role in all membrane fusion steps of the secretory and endocytic pathway [1, 2]. Since assembly and disassembly of SNARE motifs is essential for fusion, major efforts have been made to understand these reactions in detail From these studies, a few principles have emerged that appear to be valid for all SNAREs. First, SNARE complexes are represented by elongated four-helix bundles whose structure is remarkably conserved despite considerable sequence heterogeneity [7, 8]. In the middle of the bundle is a highly conserved layer of four interacting polar side chains (three glutamines and one arginine), each contributed by one of the SNARE motifs. In the best studied examples, the neuronal syntaxin 1 and its yeast plasma membrane homologue Sso1p, the N-terminal domain consists of a three-helix bundle [17, 18] that interacts intramolecularly with the SNARE motif, resulting in an equilibrium between a “closed” and an “open” conformation (19 –21). Removal of the N-terminal domain of Sso1p has been shown to accelerate SNARE complex assembly [22], and removal of this domain in syntaxin 1 accelerates SNARE-medi-
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