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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

rbg-2 suppresses the snt-1 mutant phenotype.(A) Schematic representation of the rbg-1(ok1660) deletion mutation. (B) Schematic representation of the rbg-2(ok3195) deletion mutation. Solid boxes indicate exons and thin lines indicate introns. The bar below the gene indicates the deleted region. (C) The enlarged GFP::RAB-3 puncta phenotype in rbg-1 mutants is rescued by expressing a wild-type copy of the rbg-1 gene. (D) Quantification of the synaptic enrichment of GFP::RAB-3 signal in the genotypes shown in (C). Data are represented as mean ± SD; **p < 0.01. (E) Loss of rbg-2 function suppresses the snt-1 mutant phenotype. White arrows indicate the cell bodies. (F) RAB-3 is co-precipitated with RBG-1. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.008
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fig4s1: rbg-2 suppresses the snt-1 mutant phenotype.(A) Schematic representation of the rbg-1(ok1660) deletion mutation. (B) Schematic representation of the rbg-2(ok3195) deletion mutation. Solid boxes indicate exons and thin lines indicate introns. The bar below the gene indicates the deleted region. (C) The enlarged GFP::RAB-3 puncta phenotype in rbg-1 mutants is rescued by expressing a wild-type copy of the rbg-1 gene. (D) Quantification of the synaptic enrichment of GFP::RAB-3 signal in the genotypes shown in (C). Data are represented as mean ± SD; **p < 0.01. (E) Loss of rbg-2 function suppresses the snt-1 mutant phenotype. White arrows indicate the cell bodies. (F) RAB-3 is co-precipitated with RBG-1. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.008

Mentions: Alternatively, the decreased GTP-RAB-3 level may be caused by increased RAB-3 GTPase activity in snt-1 mutants. Rab3 GTPase activity is greatly facilitated by Rab3-specific GTPase-activating protein (GAP). Rab3 GAP is composed of the catalytic subunit Rab3GAP1 and the noncatalytic subunit Rab3GAP2. rbg-1 and rbg-2 encode Rab3GAP1 and Rab3GAP2, respectively in worms (Figure 4—figure supplement 1A,B) (Fukui et al., 1997; Nagano et al., 1998). In the absence of Rab3 GAP, the RAB-3 synaptic enrichment is enhanced, which is consistent with the role of Rab3 GAP in assisting GTP hydrolysis (Figure 4—figure supplement 1C,D). If RAB-3 GTP hydrolysis activity is indeed increased in snt-1 mutants, we would expect that loss of GAP function will suppress the snt-1 mutant phenotype. Indeed, in rbg-1;snt-1 double mutants, we found that the diffuse GFP::RAB-3 phenotype of snt-1 single mutants is significantly suppressed (Figure 4A,B). Furthermore, mutation of the rbg-2 gene also suppressed the diffuse RAB-3 phenotype in snt-1 mutants (Figure 4—figure supplement 1E). In contrast, the diffuse GFP::RAB-3 signal caused by aex-3 mutation could not be suppressed by rbg-1 (Figure 4A,B). We next performed RIM2-RBD pull-down assays to test whether the GTP-RAB-3 level was restored in rbg-1;snt-1 mutants. In contrast to the greatly reduced GTP-RAB-3 level in snt-1 lysates, the amount of GTP-bound RAB-3 is significantly increased in rbg-1;snt-1 samples (Figure 4C). Taken together, these results suggest that snt-1 indeed regulate the RAB-3/SV association specifically by inhibiting RAB-3 GTP hydrolysis.10.7554/eLife.05118.007Figure 4.RAB-3 GAP mutations suppress the snt-1 mutant phenotype.


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

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

rbg-2 suppresses the snt-1 mutant phenotype.(A) Schematic representation of the rbg-1(ok1660) deletion mutation. (B) Schematic representation of the rbg-2(ok3195) deletion mutation. Solid boxes indicate exons and thin lines indicate introns. The bar below the gene indicates the deleted region. (C) The enlarged GFP::RAB-3 puncta phenotype in rbg-1 mutants is rescued by expressing a wild-type copy of the rbg-1 gene. (D) Quantification of the synaptic enrichment of GFP::RAB-3 signal in the genotypes shown in (C). Data are represented as mean ± SD; **p < 0.01. (E) Loss of rbg-2 function suppresses the snt-1 mutant phenotype. White arrows indicate the cell bodies. (F) RAB-3 is co-precipitated with RBG-1. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.008
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Related In: Results  -  Collection

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fig4s1: rbg-2 suppresses the snt-1 mutant phenotype.(A) Schematic representation of the rbg-1(ok1660) deletion mutation. (B) Schematic representation of the rbg-2(ok3195) deletion mutation. Solid boxes indicate exons and thin lines indicate introns. The bar below the gene indicates the deleted region. (C) The enlarged GFP::RAB-3 puncta phenotype in rbg-1 mutants is rescued by expressing a wild-type copy of the rbg-1 gene. (D) Quantification of the synaptic enrichment of GFP::RAB-3 signal in the genotypes shown in (C). Data are represented as mean ± SD; **p < 0.01. (E) Loss of rbg-2 function suppresses the snt-1 mutant phenotype. White arrows indicate the cell bodies. (F) RAB-3 is co-precipitated with RBG-1. Scale bars, 5 µm.DOI:http://dx.doi.org/10.7554/eLife.05118.008
Mentions: Alternatively, the decreased GTP-RAB-3 level may be caused by increased RAB-3 GTPase activity in snt-1 mutants. Rab3 GTPase activity is greatly facilitated by Rab3-specific GTPase-activating protein (GAP). Rab3 GAP is composed of the catalytic subunit Rab3GAP1 and the noncatalytic subunit Rab3GAP2. rbg-1 and rbg-2 encode Rab3GAP1 and Rab3GAP2, respectively in worms (Figure 4—figure supplement 1A,B) (Fukui et al., 1997; Nagano et al., 1998). In the absence of Rab3 GAP, the RAB-3 synaptic enrichment is enhanced, which is consistent with the role of Rab3 GAP in assisting GTP hydrolysis (Figure 4—figure supplement 1C,D). If RAB-3 GTP hydrolysis activity is indeed increased in snt-1 mutants, we would expect that loss of GAP function will suppress the snt-1 mutant phenotype. Indeed, in rbg-1;snt-1 double mutants, we found that the diffuse GFP::RAB-3 phenotype of snt-1 single mutants is significantly suppressed (Figure 4A,B). Furthermore, mutation of the rbg-2 gene also suppressed the diffuse RAB-3 phenotype in snt-1 mutants (Figure 4—figure supplement 1E). In contrast, the diffuse GFP::RAB-3 signal caused by aex-3 mutation could not be suppressed by rbg-1 (Figure 4A,B). We next performed RIM2-RBD pull-down assays to test whether the GTP-RAB-3 level was restored in rbg-1;snt-1 mutants. In contrast to the greatly reduced GTP-RAB-3 level in snt-1 lysates, the amount of GTP-bound RAB-3 is significantly increased in rbg-1;snt-1 samples (Figure 4C). Taken together, these results suggest that snt-1 indeed regulate the RAB-3/SV association specifically by inhibiting RAB-3 GTP hydrolysis.10.7554/eLife.05118.007Figure 4.RAB-3 GAP mutations suppress the snt-1 mutant phenotype.

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