Mutational analysis of synaptobrevin transmembrane domain oligomerization.
Synaptobrevin 2 is thought to facilitate fusion of synaptic vesicles with the presynaptic membrane through formation of a soluble NSF attachment protein receptor complex (SNARE) with syntaxin 1a and a synaptosomal associated protein of 25 kDa (SNAP-25). Previous reports have described a homodimer of synaptobrevin that is dependent on the transmembrane domain. However, these reports disagree about the magnitude of dimerization, which makes it difficult to assess the biological relevance of this interaction. We used SDS-PAGE and the TOXCAT genetic assay to reexamine the homodimerization of the synaptobrevin transmembrane domain in detergents and the Escherichia coli inner membrane, respectively. To gauge the magnitude of synaptobrevin homodimerization, we used the well-characterized glycophorin A homodimer as a positive standard. In contrast to previous studies, we found synaptobrevin homodimerization in E. coli is very weak when compared to glycophorin A. Recombinant synaptobrevin forms a small amount of dimer and higher order oligomers in detergents that are highly dependent on solublization conditions. We estimate a dissociation constant of 10 mM for synaptobrevin dimerization in detergent. Thus, the dimerization of synaptobrevin in membranes is very weak, questioning any possible functional role for this association in vivo.
Bowen
Mark E
ME
The Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305-5489, USA.
Engelman
Donald M
DM
Brunger
Axel T
AT
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D003902:Detergents; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors / Q000737:chemistry; D006021:Glycophorins / Q000737:chemistry / Q000235:genetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013570:Synaptic Membranes / Q000737:chemistry / Q000235:genetics
D003902:Detergents; D006021:Glycophorins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D001216:Asparagine; D003545:Cysteine