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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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The putative appendage domain of β3 subunits of AP3  complex inhibits SV production from plasma membrane but not  that from endosome. Standard in vitro budding reactions were  performed with 4°C-labeled (PM) or 15°C-labeled (ES), Percoll-washed N49A/PC12 membranes in the presence or absence of  150 μM GST fusion proteins bearing appendage domain of β3A,  β3B, or β3Amt. The amount of SV production was normalized to  the control reaction incubated with added 150 μM GST.
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Figure 7: The putative appendage domain of β3 subunits of AP3 complex inhibits SV production from plasma membrane but not that from endosome. Standard in vitro budding reactions were performed with 4°C-labeled (PM) or 15°C-labeled (ES), Percoll-washed N49A/PC12 membranes in the presence or absence of 150 μM GST fusion proteins bearing appendage domain of β3A, β3B, or β3Amt. The amount of SV production was normalized to the control reaction incubated with added 150 μM GST.

Mentions: Additional clathrin would not be needed in our in vitro assay if there were already sufficient clathrin on the donor membranes to generate budding. If that clathrin were recruited to the COOH-terminal appendage domains of AP3, then inhibition of budding might be seen in the presence of excess appendage domain. Incubating labeled endosomes with excess appendage domain, however, gave no inhibition of SV formation (Fig. 7 ES).


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)

The putative appendage domain of β3 subunits of AP3  complex inhibits SV production from plasma membrane but not  that from endosome. Standard in vitro budding reactions were  performed with 4°C-labeled (PM) or 15°C-labeled (ES), Percoll-washed N49A/PC12 membranes in the presence or absence of  150 μM GST fusion proteins bearing appendage domain of β3A,  β3B, or β3Amt. The amount of SV production was normalized to  the control reaction incubated with added 150 μM GST.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132944&req=5

Figure 7: The putative appendage domain of β3 subunits of AP3 complex inhibits SV production from plasma membrane but not that from endosome. Standard in vitro budding reactions were performed with 4°C-labeled (PM) or 15°C-labeled (ES), Percoll-washed N49A/PC12 membranes in the presence or absence of 150 μM GST fusion proteins bearing appendage domain of β3A, β3B, or β3Amt. The amount of SV production was normalized to the control reaction incubated with added 150 μM GST.
Mentions: Additional clathrin would not be needed in our in vitro assay if there were already sufficient clathrin on the donor membranes to generate budding. If that clathrin were recruited to the COOH-terminal appendage domains of AP3, then inhibition of budding might be seen in the presence of excess appendage domain. Incubating labeled endosomes with excess appendage domain, however, gave no inhibition of SV formation (Fig. 7 ES).

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