Dimerization of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein) II depends on specific residues within the transmembrane segment.
Synaptobrevin is an integral membrane protein of presynaptic vesicles and is essential for neurotransmitter release. Previously, a dimeric quaternary structure has been proposed by cross-linking experiments performed on brain fractions. Here, we demonstrate that heterologously expressed and solubilized synaptobrevin II forms a homodimer. The dimers were detected upon cross-linking with a homobifunctional lysine-reactive reagent or by oxidation of the single cysteine residue located within the transmembrane segment. Dimerization was also observed without prior cross-linking upon SDS/PAGE under mild conditions. Interestingly, dimerization required the presence of the transmembrane segment which therefore is inferred to be the principal site of subunit-subunit interaction. The residues comprizing this segment were individually mutated. Dimerization of some point mutants was significantly impaired, which proved the sequence specificity of interaction and identified residues contributing to the subunit-subunit interface. The distribution of these residues (Leu99, Ile102, Cys103, Leu107, Ile110, and Ile111) suggests that the transmembrane segment has an alpha-helical structure and that the helices pair in a right-handed fashion. The importance of the transmembrane segment for subunit-subunit interaction relates synaptobrevin to fusogenic membrane proteins of enveloped viruses where transmembrane segments have been implicated in both oligomerization and membrane fusion.
Laage
R
R
Universität Heidelberg, Neurobiologie Department, Germany.
Langosch
D
D
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D003545:Cysteine; D019281:Dimerization; D004926:Escherichia coli; D008239:Lysine; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D008961:Models, Structural; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D050683:R-SNARE Proteins; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013572:Synaptic Vesicles / Q000378:metabolism*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D008239:Lysine; D003545:Cysteine