From an extract of Drosophila melanogaster head homogenates, a membrane fraction can be isolated that has the same sedimentation properties as vertebrate synaptic vesicles and contains Drosophila synaptotagmin. The fraction disappears from homogenates of temperature-sensitive (ts) mutant shibiretsl (shitsl) flies paralyzed by exposure to nonpermissive temperatures, and reappears on return to permissive temperatures. Since reversible, temperature-dependent depletion of synaptic vesicles is known to occur in shibiretsl flies, we conclude that the fraction we have identified contains synaptic vesicles. We have examined the fate of synaptic vesicle membrane proteins in shibire flies at nonpermissive temperatures and found that all of these vesicle antigens are transferred to rapidly sedimenting membranes and codistribute with a plasma membrane marker by both glycerol velocity and metrizamide density sedimentation and by confocal microscopy. Three criteria were used to establish that other neuronspecific antigens-neuronal synaptobrevin and cysteinestring proteins-are legitimate components of synaptic vesicles: cosedimentation with Drosophila synaptotagmin, immunoadsorption, and disappearance of these antigens from the vesicle fractions in paralyzed shibire flies.Synaptic vesicles contain high concentrations of neurotransmitter, which is released when exocytosis is triggered by calcium influx into the nerve terminal. To balance this loss of vesicles due to exocytosis, synaptic activity induces a rapid rate of endocytosis. Stimulation of endocytosis correlates with the dephosphorylation of a cytoplasmic nerve-terminal GTPase, dynamin, probably by the calcium-activated phosphatase, calcineurin (1, 2). Dynamin is known to play a major role in synaptic vesicle recycling from characterizations of the shibire mutation in Drosophila in which dynamin is defective. When flies bearing temperature-sensitive (ts) alleles of the shibire locus (shi) are exposed to high temperatures, they are paralyzed within a minute, and they recover rapidly when returned to permissive temperatures. Electron microscopy of the nervous system shows that paralysis correlates with depletion of synaptic vesicles from nerve terminals (3).Because synaptic vesicles can be isolated, we can learn about the state of their proteins prior to docking and fusion. To identify the changes that synaptic vesicle proteins undergo as they fuse with the membrane and recycle back to form new vesicles, it would be advantageous to be able to freeze the terminal at different parts of the exo-endocytotic cycle. The temperature-sensitive alleles of shibire potentially provide a tool to halt the cycle after fusion but before endocytosis.To take advantage of the only known ts mutation of vesicle recycling, the shibire mutation, we set out to develop biochemical assays for the synaptic vesicle content of the adult Drosophila melanogaster central nervous system. We identified synaptic vesicles from Drosophila using antibodies that recogThe publication costs of this artic...
Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane‐bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane‐bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane‐associated ChAT sedimented in fractions containing plasma membrane marker. Membrane‐bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 m hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester‐linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane‐bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.
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