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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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SVs generated from the plasma membrane require clathrin and AP2. (A) Standard  in vitro budding reactions from 4°C-labeled,  Percoll-washed N49A/PC12 membranes were  performed with clathrin-deficient cytosol,  with α-adaptin (AP2)-deficient cytosol, or  with normal cytosol containing 3 mg/ml clathrin hub fragment. SV production for each reaction was expressed as the percentage of  that from control reactions (with only rat  brain cytosol). The variability in results with  the hub fragment may be due to difficulties in  keeping it in solution. (B) Antibodies specific  for clathrin heavy chain (X22) or α-adaptin  (AP6), respectively, were used to deplete  clathrin and AP2 adapter complex from rat  brain cytosol. After depletion, 200 μg of rat  brain cytosol (supernatant) and immunoprecipitated product (pellet) were fractionated by SDS-PAGE followed by Western blotting  with an anti-clathrin heavy chain antibody and an anti–α-adaptin antibody. The remaining clathrin and α-adaptin in the cytosol were  plotted as a percentage of total clathrin and α-adaptin before depletion.
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Figure 6: SVs generated from the plasma membrane require clathrin and AP2. (A) Standard in vitro budding reactions from 4°C-labeled, Percoll-washed N49A/PC12 membranes were performed with clathrin-deficient cytosol, with α-adaptin (AP2)-deficient cytosol, or with normal cytosol containing 3 mg/ml clathrin hub fragment. SV production for each reaction was expressed as the percentage of that from control reactions (with only rat brain cytosol). The variability in results with the hub fragment may be due to difficulties in keeping it in solution. (B) Antibodies specific for clathrin heavy chain (X22) or α-adaptin (AP6), respectively, were used to deplete clathrin and AP2 adapter complex from rat brain cytosol. After depletion, 200 μg of rat brain cytosol (supernatant) and immunoprecipitated product (pellet) were fractionated by SDS-PAGE followed by Western blotting with an anti-clathrin heavy chain antibody and an anti–α-adaptin antibody. The remaining clathrin and α-adaptin in the cytosol were plotted as a percentage of total clathrin and α-adaptin before depletion.

Mentions: We were unable to obtain any evidence that clathrin (Faundez et al., 1997) or AP2 (Horng, J.-T., and R.B. Kelly, unpublished) were required for SV formation from endosomes. In contrast, SV formation from the plasma membrane needs clathrin and AP2. When the cell-free reconstitution system was supplied with rat brain cytosol depleted of clathrin heavy chain or AP2 (α-adaptin), the vesicle production was reduced (Fig. 6 A). The extent of depletion was confirmed by Western blotting of rat brain cytosol with clathrin and α-adaptin–specific antibodies (Fig. 6 B). The clathrin involvement was further tested using a bacterially expressed clathrin heavy chain fragment, often called the hub domain. The clathrin hub fragment is the minimal trimerization domain that can self-assemble into clathrin triskelions in vitro (Liu et al., 1995). It interferes with endogenous clathrin function by blocking clathrin polymerization into polyhedral vesicle coats (Liu et al., 1998). An inhibitory effect on vesicle biogenesis was observed upon the addition of the hub fragment (Fig. 6 A). These results suggest that the clathrin/AP2-coating complex is necessary to form KT3-containing vesicles from the plasma membrane.


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)

SVs generated from the plasma membrane require clathrin and AP2. (A) Standard  in vitro budding reactions from 4°C-labeled,  Percoll-washed N49A/PC12 membranes were  performed with clathrin-deficient cytosol,  with α-adaptin (AP2)-deficient cytosol, or  with normal cytosol containing 3 mg/ml clathrin hub fragment. SV production for each reaction was expressed as the percentage of  that from control reactions (with only rat  brain cytosol). The variability in results with  the hub fragment may be due to difficulties in  keeping it in solution. (B) Antibodies specific  for clathrin heavy chain (X22) or α-adaptin  (AP6), respectively, were used to deplete  clathrin and AP2 adapter complex from rat  brain cytosol. After depletion, 200 μg of rat  brain cytosol (supernatant) and immunoprecipitated product (pellet) were fractionated by SDS-PAGE followed by Western blotting  with an anti-clathrin heavy chain antibody and an anti–α-adaptin antibody. The remaining clathrin and α-adaptin in the cytosol were  plotted as a percentage of total clathrin and α-adaptin before depletion.
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Related In: Results  -  Collection

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Figure 6: SVs generated from the plasma membrane require clathrin and AP2. (A) Standard in vitro budding reactions from 4°C-labeled, Percoll-washed N49A/PC12 membranes were performed with clathrin-deficient cytosol, with α-adaptin (AP2)-deficient cytosol, or with normal cytosol containing 3 mg/ml clathrin hub fragment. SV production for each reaction was expressed as the percentage of that from control reactions (with only rat brain cytosol). The variability in results with the hub fragment may be due to difficulties in keeping it in solution. (B) Antibodies specific for clathrin heavy chain (X22) or α-adaptin (AP6), respectively, were used to deplete clathrin and AP2 adapter complex from rat brain cytosol. After depletion, 200 μg of rat brain cytosol (supernatant) and immunoprecipitated product (pellet) were fractionated by SDS-PAGE followed by Western blotting with an anti-clathrin heavy chain antibody and an anti–α-adaptin antibody. The remaining clathrin and α-adaptin in the cytosol were plotted as a percentage of total clathrin and α-adaptin before depletion.
Mentions: We were unable to obtain any evidence that clathrin (Faundez et al., 1997) or AP2 (Horng, J.-T., and R.B. Kelly, unpublished) were required for SV formation from endosomes. In contrast, SV formation from the plasma membrane needs clathrin and AP2. When the cell-free reconstitution system was supplied with rat brain cytosol depleted of clathrin heavy chain or AP2 (α-adaptin), the vesicle production was reduced (Fig. 6 A). The extent of depletion was confirmed by Western blotting of rat brain cytosol with clathrin and α-adaptin–specific antibodies (Fig. 6 B). The clathrin involvement was further tested using a bacterially expressed clathrin heavy chain fragment, often called the hub domain. The clathrin hub fragment is the minimal trimerization domain that can self-assemble into clathrin triskelions in vitro (Liu et al., 1995). It interferes with endogenous clathrin function by blocking clathrin polymerization into polyhedral vesicle coats (Liu et al., 1998). An inhibitory effect on vesicle biogenesis was observed upon the addition of the hub fragment (Fig. 6 A). These results suggest that the clathrin/AP2-coating complex is necessary to form KT3-containing vesicles from the plasma membrane.

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