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G Shiff

Publications and source records attributed to G Shiff.

5 recordsLinked to original sources
PubMed1998

Vacuolar H+-ATPase domains are transported separately in axons and assemble in Torpedo nerve endings.

Torpedo electric organ synaptosomes possess a typical vacuolar H+-ATPase (V-ATPase), inhibited by concanamycin A and insensitive to vanadate, made of the association of a catalytic soluble sector V1 to a membrane domain V0. In the electric nerves, the 57-kDa subunit B of the V1 sector was transported to the nerve endings by the slow axonal flow and did not accumulate upstream from an axonal block. In contrast, a 500% accumulation of the 15-kDa subunit c of the V0 membrane domain was observed, demonstrating that this subunit is conveyed by the fast axonal anterograde transport. After velocity sedimentation of solubilized nerve proteins, the 57- and 15-kDa subunits were recovered in different complexes corresponding, respectively, to the V1 and V0 domains. No fully assembled V-ATPase was detected. It is concluded that V1 and V0 domains of V-ATPase are transported separately in axons, at different rates, and that they only associate once arrived in nerve endings to form the active V-ATPase.

Animals↗
PubMed1997

Association of syntaxin with SNAP-25 and VAMP (synaptobrevin) during axonal transport.

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP-25, and a synaptic vesicle membrane protein, VAMP/synaptobrevin, form stable protein complexes that are involved in the docking and fusion of synaptic vesicles at the presynaptic membrane. These protein complexes have also been described in a homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ. In the present study, we performed similar experiments combining velocity sedimentation and immunoprecipitation on control or ligated electric nerves and found it was possible to distinguish syntaxin that is in the axonal plasma membrane from syntaxin that is transported by the fast anterograde axonal flow. Although syntaxin that is resident in axonal membranes is associated with SNAP-25 but not with VAMP, syntaxin that accumulates proximally to a ligature is associated with both SNAP-25 and VAMP in a stoichiometry very similar to that of the nerve terminal complex. In control nerves, lower amounts of syntaxin form a complex with VAMP, in proportion to syntaxin, which is conveyed by the axonal flow. Because added VAMP was unable to associate with syntaxin in solubilized control nerves and because neither solubilization by SDS nor dilution to the nanomolar range of syntaxin and VAMP concentrations before solubilization change the stoiechiometry between the immunoprecipitated proteins, this complex appears to be both formed prior to solubilization and stable thereafter. Hence, heterotrimeric complexes containing syntaxin, SNAP-25, and VAMP are already formed during fast anterograde axonal transport, before reaching the nerve endings.

Animals↗
PubMed1997

Rapid anterograde axonal transport of the syntaxin-SNAP 25-VAMP complex.

During the process of docking and fusion of synaptic vesicles to the presynaptic membrane, several presynaptic proteins bind sequentially to a core complex associating two proteins of the presynaptic membrane, syntaxin and SNAP 25, and a protein of synaptic vesicles, VAMP/synaptobrevin. We have immunoprecipitated this core complex after CHAPS solubilization of pure cholinergic synaptosomes of Torpedo electric organ, using anti-syntaxin or anti-VAMP immunobeads. In parallel, we studied syntaxin and VAMP, which are transported by the rapid axonal flow to the nerve endings. We found that syntaxin and VAMP accumulating at the proximal end of an electric nerve ligature were already engaged in complexes, as in synaptosomes. In unligated nerves also, significant amounts of VAMP associate with syntaxin. The possibility that these complexes form after solubilization was eliminated because added VAMP was unable to associate with syntaxin in solubilized control nerves and because similar amounts of complex were obtained after sodium dodecyl sulfate or CHAPS solubilization. Hence, syntaxin is already associated with SNAP 25 and VAMP during axonal transport, before reaching nerve endings.

Animals↗
PubMed1996

Association of syntaxin with SNAP 25 and VAMP (synaptobrevin) in Torpedo synaptosomes.

Two proteins of the presynaptic plasma membrane, syntaxin and SNAP 25, and VAMP/ synaptobrevin, a synaptic vesicle membrane protein, form stable protein complexes which are involved in the docking and fusion of synaptic vesicles at the mammalian brain presynaptic membrane. Similar protein complexes were revealed in an homogeneous population of cholinergic synaptosomes purified from Torpedo electric organ by combining velocity sedimentation and immunoprecipitation experiments. After CHAPS solubilization, virtually all the nerve terminal syntaxin was found in the form of large 16 S complexes, in association with 65% of SNAP 25 and 15% of VAMP. Upon Triton X100 solubilization, syntaxin was still recovered in association with SNAP 25 and VAMP but in smaller 8 S complexes. A small (2-5%) percentage of the nerve terminal 15 kDa proteolipid subunit of the v-H+ATPase and of mediatophore was copurified with syntaxin, using two different antisyntaxin monoclonal antibodies. The use of an homogeneous population of peripheral cholinergic nerve terminals allowed us to extend results on the composition of the brain presynaptic protein complexes to the Torpedo electric organ synapse, a model of the rapid neuromuscular synapses.

Acetylcholine↗