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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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GST-Dyn-PRD inhibits SV formation from the  plasma membrane but not  that from endosomes. Standard in vitro budding reactions with Percoll-washed  N49A/PC12 cell membranes  from cells labeled at 4°C  (plasma membrane) or 15°C  (endosome) were performed  with 300 μg/ml of GST-Dyn-PRD or 800 μg/ml GST control protein. SV production  (fractions 8–12) with added  GST-Dyn-PRD was normalized as a percentage of that with GST  control proteins. PM, plasma membrane; ES, endosome.
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Figure 8: GST-Dyn-PRD inhibits SV formation from the plasma membrane but not that from endosomes. Standard in vitro budding reactions with Percoll-washed N49A/PC12 cell membranes from cells labeled at 4°C (plasma membrane) or 15°C (endosome) were performed with 300 μg/ml of GST-Dyn-PRD or 800 μg/ml GST control protein. SV production (fractions 8–12) with added GST-Dyn-PRD was normalized as a percentage of that with GST control proteins. PM, plasma membrane; ES, endosome.

Mentions: The GTPase dynamin has been implicated in endocytosis from the plasma membrane (van der Bliek et al., 1993; Vallee and Okamoto, 1995; De Camilli and Takei, 1996). To explore how dynamin might function in SV formation, a fusion of GST to the proline-rich domain (PRD) of dynamin (GST-Dyn-PRD) was made and used to screen for brain-specific proteins that bound to it (Roos and Kelly, 1998). Four major PRD-binding proteins were found in brain extracts. To determine if dynamin or SH3-containing proteins that bind to the Dyn-PRD were involved in SV biogenesis from PC12 membranes, in vitro budding reactions were incubated with either GST (control) or GST-Dyn-PRD fusion proteins. The GST-Dyn-PRD protein inhibited vesicle formation from plasma membranes by >50%, but had no effect on budding from endosomes (Fig. 8). We conclude that the proteins that bind dynamin PRDs are likely to be involved in the AP2-mediated pathway of vesicle biogenesis, but not the endosomal, AP3-mediated pathway.


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)

GST-Dyn-PRD inhibits SV formation from the  plasma membrane but not  that from endosomes. Standard in vitro budding reactions with Percoll-washed  N49A/PC12 cell membranes  from cells labeled at 4°C  (plasma membrane) or 15°C  (endosome) were performed  with 300 μg/ml of GST-Dyn-PRD or 800 μg/ml GST control protein. SV production  (fractions 8–12) with added  GST-Dyn-PRD was normalized as a percentage of that with GST  control proteins. PM, plasma membrane; ES, endosome.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: GST-Dyn-PRD inhibits SV formation from the plasma membrane but not that from endosomes. Standard in vitro budding reactions with Percoll-washed N49A/PC12 cell membranes from cells labeled at 4°C (plasma membrane) or 15°C (endosome) were performed with 300 μg/ml of GST-Dyn-PRD or 800 μg/ml GST control protein. SV production (fractions 8–12) with added GST-Dyn-PRD was normalized as a percentage of that with GST control proteins. PM, plasma membrane; ES, endosome.
Mentions: The GTPase dynamin has been implicated in endocytosis from the plasma membrane (van der Bliek et al., 1993; Vallee and Okamoto, 1995; De Camilli and Takei, 1996). To explore how dynamin might function in SV formation, a fusion of GST to the proline-rich domain (PRD) of dynamin (GST-Dyn-PRD) was made and used to screen for brain-specific proteins that bound to it (Roos and Kelly, 1998). Four major PRD-binding proteins were found in brain extracts. To determine if dynamin or SH3-containing proteins that bind to the Dyn-PRD were involved in SV biogenesis from PC12 membranes, in vitro budding reactions were incubated with either GST (control) or GST-Dyn-PRD fusion proteins. The GST-Dyn-PRD protein inhibited vesicle formation from plasma membranes by >50%, but had no effect on budding from endosomes (Fig. 8). We conclude that the proteins that bind dynamin PRDs are likely to be involved in the AP2-mediated pathway of vesicle biogenesis, but not the endosomal, AP3-mediated pathway.

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