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Synaptotagmin 1 directs repetitive release by coupling vesicle exocytosis to the Rab3 cycle.

Cheng Y, Wang J, Wang Y, Ding M - Elife (2015)

Bottom Line: How this harmonization is achieved is not known.In the absence of Ca(2+), synaptotagmin 1 binds to Rab3 GTPase activating protein (GAP) and inhibits the GTP hydrolysis of Rab3 protein.In the presence of Ca(2+), synaptotagmin 1 releases Rab3 GAP and promotes membrane disassociation of Rab3.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
In response to Ca(2+) influx, a synapse needs to release neurotransmitters quickly while immediately preparing for repeat firing. How this harmonization is achieved is not known. In this study, we found that the Ca(2+) sensor synaptotagmin 1 orchestrates the membrane association/disassociation cycle of Rab3, which functions in activity-dependent recruitment of synaptic vesicles. In the absence of Ca(2+), synaptotagmin 1 binds to Rab3 GTPase activating protein (GAP) and inhibits the GTP hydrolysis of Rab3 protein. Rab3 GAP resides on synaptic vesicles, and synaptotagmin 1 is essential for the synaptic localization of Rab3 GAP. In the presence of Ca(2+), synaptotagmin 1 releases Rab3 GAP and promotes membrane disassociation of Rab3. Without synaptotagmin 1, the tight coupling between vesicle exocytosis and Rab3 membrane disassociation is disrupted. We uncovered the long-sought molecular apparatus linking vesicle exocytosis to Rab3 cycling and we also revealed the important function of synaptotagmin 1 in repetitive synaptic vesicle release.

No MeSH data available.


Related in: MedlinePlus

The GTP-bound form of RAB-3 is decreased in snt-1 mutants.(A) A GST-fused RBD domain of RIM2 binds active GTP-RAB-3. The amount of GTP-RAB-3 pulled down by RBD is decreased in both aex-3 and snt-1 animals. (B) Quantification of the GTP-RAB-3 level in wild type, aex-3, and snt-1. Data are presented as mean ± SD; **p < 0.01. (C) The amount of GFP-RAB-3 in the cytosolic fraction is increased in snt-1 mutants. (D) Localization of GFP::RAB-27 puncta is affected by mutation of aex-3, but not by mutation of snt-1. The white arrows indicate the cell bodies. (E) Over-expression of aex-3 does not rescue the snt-1 mutant phenotype. Yellow arrows indicate the cell bodies. (F) The AEX-3::GFP level is unchanged in snt-1 mutants compared to wild type. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.006
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fig3: The GTP-bound form of RAB-3 is decreased in snt-1 mutants.(A) A GST-fused RBD domain of RIM2 binds active GTP-RAB-3. The amount of GTP-RAB-3 pulled down by RBD is decreased in both aex-3 and snt-1 animals. (B) Quantification of the GTP-RAB-3 level in wild type, aex-3, and snt-1. Data are presented as mean ± SD; **p < 0.01. (C) The amount of GFP-RAB-3 in the cytosolic fraction is increased in snt-1 mutants. (D) Localization of GFP::RAB-27 puncta is affected by mutation of aex-3, but not by mutation of snt-1. The white arrows indicate the cell bodies. (E) Over-expression of aex-3 does not rescue the snt-1 mutant phenotype. Yellow arrows indicate the cell bodies. (F) The AEX-3::GFP level is unchanged in snt-1 mutants compared to wild type. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.006

Mentions: How does mutation of snt-1 affect the SV membrane association of RAB-3? Previous studies showed that the localization of RAB-3 on SV membranes is tightly associated with its GTP-bound state (Zerial and McBride, 2001). Therefore, we tested whether the loss of RAB-3 from SVs in snt-1 mutants is caused by reduction of GTP-bound RAB-3. The active GTP-Rab3 binds to the RBD domain of its effector RIM, while the inactive GDP-Rab3 does not. Previous reports demonstrated that the RBD domain of mammalian RIM2 could bind to the worm GTP-RAB-3 (Wang et al., 1997; Mahoney et al., 2006). Thus, we performed pull-down assays to examine the GTP-RAB-3 level in vivo. In wild-type worm lysates, the active GTP-bound form of RAB-3 protein was efficiently pulled down by GST-RBD (Figure 3A). In contrast, the amount of GTP-RAB-3 pulled down by RIM2 RBD was significantly reduced in snt-1 lysates (Figure 3A,B). In the absence of RAB-3 GEF, the GDP-bound RAB-3 cannot be converted to the GTP-bound RAB-3. Indeed, in aex-3 animals, the amount of RAB-3 that can be pulled down by GST-RBD is also greatly decreased (Figure 3A,B). Thus snt-1, similar to aex-3, affects the level of GTP-bound RAB-3 in vivo.10.7554/eLife.05118.006Figure 3.The GTP-bound form of RAB-3 is decreased in snt-1 mutants.


Synaptotagmin 1 directs repetitive release by coupling vesicle exocytosis to the Rab3 cycle.

Cheng Y, Wang J, Wang Y, Ding M - Elife (2015)

The GTP-bound form of RAB-3 is decreased in snt-1 mutants.(A) A GST-fused RBD domain of RIM2 binds active GTP-RAB-3. The amount of GTP-RAB-3 pulled down by RBD is decreased in both aex-3 and snt-1 animals. (B) Quantification of the GTP-RAB-3 level in wild type, aex-3, and snt-1. Data are presented as mean ± SD; **p < 0.01. (C) The amount of GFP-RAB-3 in the cytosolic fraction is increased in snt-1 mutants. (D) Localization of GFP::RAB-27 puncta is affected by mutation of aex-3, but not by mutation of snt-1. The white arrows indicate the cell bodies. (E) Over-expression of aex-3 does not rescue the snt-1 mutant phenotype. Yellow arrows indicate the cell bodies. (F) The AEX-3::GFP level is unchanged in snt-1 mutants compared to wild type. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.006
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fig3: The GTP-bound form of RAB-3 is decreased in snt-1 mutants.(A) A GST-fused RBD domain of RIM2 binds active GTP-RAB-3. The amount of GTP-RAB-3 pulled down by RBD is decreased in both aex-3 and snt-1 animals. (B) Quantification of the GTP-RAB-3 level in wild type, aex-3, and snt-1. Data are presented as mean ± SD; **p < 0.01. (C) The amount of GFP-RAB-3 in the cytosolic fraction is increased in snt-1 mutants. (D) Localization of GFP::RAB-27 puncta is affected by mutation of aex-3, but not by mutation of snt-1. The white arrows indicate the cell bodies. (E) Over-expression of aex-3 does not rescue the snt-1 mutant phenotype. Yellow arrows indicate the cell bodies. (F) The AEX-3::GFP level is unchanged in snt-1 mutants compared to wild type. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.006
Mentions: How does mutation of snt-1 affect the SV membrane association of RAB-3? Previous studies showed that the localization of RAB-3 on SV membranes is tightly associated with its GTP-bound state (Zerial and McBride, 2001). Therefore, we tested whether the loss of RAB-3 from SVs in snt-1 mutants is caused by reduction of GTP-bound RAB-3. The active GTP-Rab3 binds to the RBD domain of its effector RIM, while the inactive GDP-Rab3 does not. Previous reports demonstrated that the RBD domain of mammalian RIM2 could bind to the worm GTP-RAB-3 (Wang et al., 1997; Mahoney et al., 2006). Thus, we performed pull-down assays to examine the GTP-RAB-3 level in vivo. In wild-type worm lysates, the active GTP-bound form of RAB-3 protein was efficiently pulled down by GST-RBD (Figure 3A). In contrast, the amount of GTP-RAB-3 pulled down by RIM2 RBD was significantly reduced in snt-1 lysates (Figure 3A,B). In the absence of RAB-3 GEF, the GDP-bound RAB-3 cannot be converted to the GTP-bound RAB-3. Indeed, in aex-3 animals, the amount of RAB-3 that can be pulled down by GST-RBD is also greatly decreased (Figure 3A,B). Thus snt-1, similar to aex-3, affects the level of GTP-bound RAB-3 in vivo.10.7554/eLife.05118.006Figure 3.The GTP-bound form of RAB-3 is decreased in snt-1 mutants.

Bottom Line: How this harmonization is achieved is not known.In the absence of Ca(2+), synaptotagmin 1 binds to Rab3 GTPase activating protein (GAP) and inhibits the GTP hydrolysis of Rab3 protein.In the presence of Ca(2+), synaptotagmin 1 releases Rab3 GAP and promotes membrane disassociation of Rab3.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
In response to Ca(2+) influx, a synapse needs to release neurotransmitters quickly while immediately preparing for repeat firing. How this harmonization is achieved is not known. In this study, we found that the Ca(2+) sensor synaptotagmin 1 orchestrates the membrane association/disassociation cycle of Rab3, which functions in activity-dependent recruitment of synaptic vesicles. In the absence of Ca(2+), synaptotagmin 1 binds to Rab3 GTPase activating protein (GAP) and inhibits the GTP hydrolysis of Rab3 protein. Rab3 GAP resides on synaptic vesicles, and synaptotagmin 1 is essential for the synaptic localization of Rab3 GAP. In the presence of Ca(2+), synaptotagmin 1 releases Rab3 GAP and promotes membrane disassociation of Rab3. Without synaptotagmin 1, the tight coupling between vesicle exocytosis and Rab3 membrane disassociation is disrupted. We uncovered the long-sought molecular apparatus linking vesicle exocytosis to Rab3 cycling and we also revealed the important function of synaptotagmin 1 in repetitive synaptic vesicle release.

No MeSH data available.


Related in: MedlinePlus