Redistribution of synaptic vesicles and their proteins in temperature-sensitive shibire(ts1) mutant Drosophila.

Abstract

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...

Association of Drosophila cysteine string proteins with membranes

“…Thus, all Dcsp's was found to be membrane-bound. Unlike our findings with the protein Rop (a sec-1 homologue in yeast), none of the Dcsp's was recovered at the top of the gradient as soluble protein [3,11]. When subcellular fractionation was performed in Torpedo electric organ a considerable fraction of the Dcsp was recovered as soluble material [5,6].…”

“…At restrictive temperatures the recycling of synaptic vesicles in shibire tsl mutant becomes blocked at an early stage of endocytotic retrieval of vesicles from the plasma membrane. We have shown previously that all synaptic vesicles are depleted from the nerve terminals during exposure of shibire flies to elevated temperatures [3]. The synaptic vesicle antigens, synaptotagmin, synaptobrevin and Dcsp's were dramatically absent from synaptic vesicle fractions in shibire flies at restrictive temperatures.…”

“…Our previous study showed that Drosophila csp's are present on synaptic vesicles as well as on plasma membrane and recycle together with other synaptic vesicle markers between both types of membranes [3]. Since csp's lack a transmembrane domain their association with membranes is undefined.…”

“…This result suggested that csp's may be an essential subunit or a modulator of presynaptic calcium channels and so should be localized to presynaptic membrane. Instead, csp's were found to copurify with synaptic vesicles in both Torpedo [2] and Drosophila [3]. Although the function of csp's is still not understood, their importance in the nervous system becomes obvious from an analysis of null alleles in Drosophila [4].…”

The Zinc Transporter ZnT3 Interacts with AP-3 and It Is Preferentially Targeted to a Distinct Synaptic Vesicle Subpopulation

“…Brains were pulverized in liquid nitrogen. Extracts were thawed at 4°C in buffer A (150 mM NaCl, 20 mM HEPES, pH 7.4, 5 mM EGTA, 5 mM MgCl 2 0.5) plus Complete antiproatease mixture and fractionated as described previously (van de Goor et al, 1995). Synaptic vesicles were resolved in glycerol velocity gradients as mentioned above.…”