title: Investigation of the juxtamembrane region of neuronal-Synaptobrevin in synaptic transmission at the Drosophila neuromuscular junction.
abstract: In this study, the juxtamembrane region of the Drosophila SNARE protein neuronal-Synaptobrevin (n-Syb) was tested for its role in synaptic transmission. A transgenic approach was used to express n-Syb mutant genes. The transgenes carried engineered point mutations that alter the amino acid sequence of the conserved tryptophan residues in the juxtamembrane sequence. Such transgenes were expressed in an n-syb hypomorphic background, which produces little endogenous protein. On their own, hypomorphic flies displayed severe motor inhibition, limited life span, reduced evoked junctional potentials (EJPs), decreased synchronicity in EJP time to peak, and potentiation of EJPs with 10-Hz stimulation. All of these deficits were restored to wild-type levels with the expression of wild-type transgenic n-syb, regulated by the endogenous promoter (n-syb(WT)). We created transgenic mutants with one additional tryptophan (n-syb(WW)) or one less tryptophan (n-syb(AA)) than the wild-type sequence. While n-syb(WW) resembled n-syb(WT) in all variables listed, n-syb(AA) exhibited decreased EJP amplitude, synchronicity, and quantal content. To determine whether the n-syb juxtamembrane region is important for transduction of force arising from SNARE complex assembly during membrane fusion, we introduced short 6-amino acid (n-syb(L6)) or long 24-amino acid (n-syb(L24)) flexible linkers into the n-syb transgene. We observed a reduced EJP amplitude in n-syb(L6) but not n-syb(L24), while both linker mutants showed a decreased quantal content and an effect on the readily releasable and recycling vesicle pools. In conclusion, mutation of the juxtamembrane region of n-syb deleteriously affected synaptic transmission at the Drosophila neuromuscular junction.
authors.affiliation: Department of Biology, University of Toronto Mississauga, Mississauga, Canada; and Department of Cell and Systems Biology, University of Toronto, Toronto, Canada cdemill@uoguelph.ca.
authors.forename: Colin M
authors.initials: CM
authors.lastname: DeMill
authors.affiliation: Department of Biology, University of Toronto Mississauga, Mississauga, Canada; and.
authors.forename: Xinping
authors.initials: X
authors.lastname: Qiu
authors.affiliation: Department of Biology, University of Toronto Mississauga, Mississauga, Canada; and.
authors.forename: Marta
authors.initials: M
authors.lastname: Kisiel
authors.affiliation: Department of Biology, University of Toronto Mississauga, Mississauga, Canada; and.
authors.forename: Alanna
authors.initials: A
authors.lastname: Bolotta
authors.affiliation: Department of Biology, University of Toronto Mississauga, Mississauga, Canada; and Department of Cell and Systems Biology, University of Toronto, Toronto, Canada.
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Stewart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000378:metabolism* / Q000502:physiology; D009474:Neurons / Q000378:metabolism / Q000502:physiology; D050683:R-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000378:metabolism
chemical_list: D050683:R-SNARE Proteins; D050600:SNARE Proteins
keywords: SNARE protein; juxtamembrane region; membrane fusion; neuronal-Synaptobrevin; synaptic transmission