Mixed and Non-cognate SNARE Complexes
CHARACTERIZATION OF ASSEMBLY AND BIOPHYSICAL PROPERTIES*
- From the Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
Abstract
Assembly of solubleN-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPaseN-ethylmaleimide-sensitive fusion protein and its cofactor α-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.
Footnotes
-
↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 523.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF132812.
-
↵‡ To whom correspondence should be addressed: Dept. of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany. Tel.: 49-551-201-1634; Fax: 49-551-201-1639; E-mail: rjahn{at}gwdg.de.
- Abbreviations:
- SNARE
-
SNAP receptor
- NSF
-
N-ethylmaleimide-sensitive fusion protein
- SNAP
-
soluble NSF attachment protein
- SNAP-25
-
synaptosomal-associated protein of 25 kDa
- BoNT
-
botulinum neurotoxin
- GST
-
glutathioneS-transferase
- CD
-
circular dichroism
- MALLS
-
multi-angle laser light scattering
- PMSF
-
phenylmethylsulfonyl fluoride
- PAGE
-
polyacrylamide gel electrophoresis
- PCR
-
polymerase chain reaction
- Tricine
-
N-tris(hydroxymethyl) methylglycine
-
- Received February 5, 1999.
- Revision received March 9, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
