Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we ...found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca2+-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca2+-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca2+-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca2+-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca2+-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.
Synaptophysin and synaptobrevin/VAMP are abundant synaptic vesicle proteins that form homo- and heterooligomers. We now use chemical cross-linking in synaptosomes, pinched-off nerve terminals that ...are capable of stimulus-dependent neurotransmitter release, to investigate whether these complexes are regulated. We show that in synaptosomes treated with three stimuli that induce exocytosis (a depolarizing K+ solution, the excitatory neurotoxin α-latrotoxin, or the Ca2+-ionophore ionomycin), the homo- and heteromultimerization of synaptophysin and synaptobrevin is increased up to 6-fold. Whereas at rest less than 10% of the total synaptobrevin and synaptophysin could be chemically cross-linked into homo- and heteromeric complexes, after stimulation up to 25% of synaptobrevin and synaptophysin are present in homo- and heteromultimers, suggesting that a large fraction of these synaptic vesicle proteins physiologically participate in such complexes. The increase in multimerization of synaptophysin and synaptobrevin was only observed in intact but not in lysed synaptosomes and could not be inhibited by general kinase or phosphatase inhibitors. The stimulus dependence of synaptophysin and synaptobrevin multimers indicates that the complexes are not composed of a fixed multisubunit structure, for example, as an ion channel, but represent distinct functional states of synaptobrevin and synaptophysin that are modulated in parallel with synaptic vesicle exo- and endocytosis.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of delta-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not
Using PC12 cells that express transfected human growth hormone (hGH) as a secreted reporter protein, we have searched for Rab proteins that function in exocytosis. Among the Rab proteins tested, we ...found that besides the previously described Rab3 proteins, only members of the Rab11 family (Rab11a, 11b, and 25) impaired Ca
2+
-induced exocytosis. Rab11b, which is enriched in brain, had the strongest effect. Consistent with a role in exocytosis, Rab11 and Rab3 proteins were colocalized with other vesicle proteins on secretory vesicles in PC12 cells and on mature synaptic vesicles in brain. Rab11b mutants that fix Rab11b in the GTP- or GDP-bound state both effectively inhibited Ca
2+
-induced exocytosis but seemed to act by distinct mechanisms: whereas GDP-bound Rab11b greatly stimulated constitutive secretion of hGH and depleted hGH stores in secretory vesicles, GTP-bound Rab11b only had a moderate effect on constitutive secretion and no effect on vesicular hGH stores. These results suggest that, consistent with a GTP-dependent regulation of Rab function, GDP-bound Rab11b indirectly inhibits Ca
2+
-triggered exocytosis by causing the loss of hGH from the PC12 cells, whereas GTP-bound Rab11b directly impairs Ca
2+
-triggered exocytosis. In contrast to neuroendocrine PC12 cells in which GTP- and GDP-bound Rab11b inhibited Ca
2+
-induced, but not constitutive, exocytosis, in non-neuronal cells GTP- and GDP-bound Rab11b inhibited constitutive exocytosis and caused an accumulation of cellular hGH. Viewed together, our data suggest that, in addition to other functions, Rab11 has a specific role in neuronal and neuroendocrine but not in non-neuronal cells as a GTP-dependent switch between regulated and constitutive secretory pathways.
The venom of the black widow spider (BWSV) ( Latrodectus mactans tredecimguttatus ) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific
manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown,
and they have not been functionally expressed. This study reports on the primary structure of -latroinsectotoxin ( -LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. -LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree
of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain
organization of -LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted
molecular mass and apparent mobility of the protein ( 130 kDa) encoded in the -LIT gene differs from that of native -LIT purified from BWSV ( 110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native -LIT revealed a molecular ion with m/z + of 110916 ± 100, indicating that the native -LIT is 991 amino acids in length. When the full-length -LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing
991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar
concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native -LIT have a high Ca permeability, whereas those formed by truncated, recombinant protein do not.
The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a ...phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of δ-latroinsectotoxin (δ-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. δ-LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of δ-LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (~130 kDa) encoded in the δ-LIT gene differs from that of native δ-LIT purified from BWSV (~110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native δ-LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native δ-LIT is 991 amino acids in length. When the full-length δ-LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native δ-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.
α-Latrotoxin is a potent neurotoxin from black widow spider venom that binds to presynaptic receptors and causes massive neurotransmitter release. A surprising finding was the biochemical description ...of two distinct cell surface proteins that bind α-latrotoxin with nanomolar affinities; Neurexin Iα binds α-latrotoxin in a Ca2+-dependent manner, and CIRL/latrophilin binds in a Ca2+-independent manner. We have now generated and analyzed mice that lack neurexin Iα to test its importance in α-latrotoxin action. α-Latrotoxin binding to brain membranes from mutant mice was decreased by almost 50% compared with wild type membranes; the decrease was almost entirely due to a loss of Ca2+-dependent α-latrotoxin binding sites. In cultured hippocampal neurons, α-latrotoxin was still capable of activating neurotransmission in the absence of neurexin Iα. Direct measurements of 3Hglutamate release from synaptosomes, however, showed a major decrease in the amount of release triggered by α-latrotoxin in the presence of Ca2+. Thus neurexin Iα is not essential for α-latrotoxin action but contributes to α-latrotoxin action when Ca2+ is present. Viewed as a whole, our results show that mice contain two distinct types of α-latrotoxin receptors with similar affinities and abundance but different properties and functions. The action of α-latrotoxin may therefore be mediated by independent parallel pathways, of which the CIRL/latrophilin pathway is sufficient for neurotransmitter release, whereas the neurexin Iα pathway contributes to the Ca2+-dependent action of α-latrotoxin.
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