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Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.

Shi G, Faúndez V, Roos J, Dell'Angelica EC, Kelly RB - J. Cell Biol. (1998)

Bottom Line: The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive.The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not.Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACT
In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

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BFA inhibits in vitro SV formation from endosomes  but not from the plasma membrane. Cell homogenates from  N49A/PC12 cells labeled with 125I-KT3 at either 4°C for 2 h (open  circles) or 15°C for 40 min (closed circles) were incubated with rat  brain cytosol (1.5 mg/ml), ATP and the indicated amount of BFA  at 4°C for 15 min before warming to 37°C for 30 min. The amount  of SV production (radioactivity of fractions 8–12) was expressed  as a percentage of the yield obtained from reactions to which no  BFA was added.
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Figure 5: BFA inhibits in vitro SV formation from endosomes but not from the plasma membrane. Cell homogenates from N49A/PC12 cells labeled with 125I-KT3 at either 4°C for 2 h (open circles) or 15°C for 40 min (closed circles) were incubated with rat brain cytosol (1.5 mg/ml), ATP and the indicated amount of BFA at 4°C for 15 min before warming to 37°C for 30 min. The amount of SV production (radioactivity of fractions 8–12) was expressed as a percentage of the yield obtained from reactions to which no BFA was added.

Mentions: ARF1, a small GTP-binding protein, is required for SV biogenesis from endosomes (Faúndez et al., 1997). Its function is to recruit the AP3 adaptor complex as a coat to mediate vesicle budding (Faúndez et al., 1998). A hallmark of ARF1-mediated processes is their sensitivity to BFA, which inhibits GDP–GTP exchange activity of ARF1 (Donaldson et al., 1992). As shown in Fig. 5 (closed circles), SV biogenesis from endosomes was inhibited by BFA in a dose-dependent manner as described previously (Faúndez et al., 1997). However, the vesicle production from the plasma membranes was not inhibited in the presence of BFA ≤500 μg/ml. The difference in BFA sensitivity implies that coat recruitment to allow vesicle budding from the plasma membrane does not require a cytoplasmic ARF1. These observations are consistent with the previous reports showing that BFA inhibits vesicle formation from the TGN, but not from the cell surface (Robinson and Kreis, 1992).


Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.

Shi G, Faúndez V, Roos J, Dell'Angelica EC, Kelly RB - J. Cell Biol. (1998)

BFA inhibits in vitro SV formation from endosomes  but not from the plasma membrane. Cell homogenates from  N49A/PC12 cells labeled with 125I-KT3 at either 4°C for 2 h (open  circles) or 15°C for 40 min (closed circles) were incubated with rat  brain cytosol (1.5 mg/ml), ATP and the indicated amount of BFA  at 4°C for 15 min before warming to 37°C for 30 min. The amount  of SV production (radioactivity of fractions 8–12) was expressed  as a percentage of the yield obtained from reactions to which no  BFA was added.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132944&req=5

Figure 5: BFA inhibits in vitro SV formation from endosomes but not from the plasma membrane. Cell homogenates from N49A/PC12 cells labeled with 125I-KT3 at either 4°C for 2 h (open circles) or 15°C for 40 min (closed circles) were incubated with rat brain cytosol (1.5 mg/ml), ATP and the indicated amount of BFA at 4°C for 15 min before warming to 37°C for 30 min. The amount of SV production (radioactivity of fractions 8–12) was expressed as a percentage of the yield obtained from reactions to which no BFA was added.
Mentions: ARF1, a small GTP-binding protein, is required for SV biogenesis from endosomes (Faúndez et al., 1997). Its function is to recruit the AP3 adaptor complex as a coat to mediate vesicle budding (Faúndez et al., 1998). A hallmark of ARF1-mediated processes is their sensitivity to BFA, which inhibits GDP–GTP exchange activity of ARF1 (Donaldson et al., 1992). As shown in Fig. 5 (closed circles), SV biogenesis from endosomes was inhibited by BFA in a dose-dependent manner as described previously (Faúndez et al., 1997). However, the vesicle production from the plasma membranes was not inhibited in the presence of BFA ≤500 μg/ml. The difference in BFA sensitivity implies that coat recruitment to allow vesicle budding from the plasma membrane does not require a cytoplasmic ARF1. These observations are consistent with the previous reports showing that BFA inhibits vesicle formation from the TGN, but not from the cell surface (Robinson and Kreis, 1992).

Bottom Line: The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive.The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not.Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACT
In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

Show MeSH
Related in: MedlinePlus