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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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In vitro budding  from cell homogenate labeled  at 4°C or 15°C. Cell homogenate (1 mg) was prepared  from N49A/PC12 cells labeled with 125I-KT3 at 4°C for  2 h. The homogenate was  then incubated with rat brain  cytosol (1.5 mg/ml) and ATP  at 37°C (closed circles) or 4°C  (open circles) for 30 min followed by centrifugation of  a high speed supernatant  (S2) on a 5–25% glycerol velocity gradient. A significant  amount of 125I-KT3 was recovered in fractions 8–12 (closed circles), cosedimenting with the  SVs (closed diamonds) generated by an in vitro reaction using  homogenate from 15°C-labeled N49A PC12 cells (Desnos et al.,  1995). No labeled KT3 was recovered in the SV peak when the in  vitro reactions were performed at 4°C (15°C cell labeling, open  diamonds; 4°C cell labeling, open circles). The label at the top  (right-hand side) of the gradients is free antibody.
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Figure 2: In vitro budding from cell homogenate labeled at 4°C or 15°C. Cell homogenate (1 mg) was prepared from N49A/PC12 cells labeled with 125I-KT3 at 4°C for 2 h. The homogenate was then incubated with rat brain cytosol (1.5 mg/ml) and ATP at 37°C (closed circles) or 4°C (open circles) for 30 min followed by centrifugation of a high speed supernatant (S2) on a 5–25% glycerol velocity gradient. A significant amount of 125I-KT3 was recovered in fractions 8–12 (closed circles), cosedimenting with the SVs (closed diamonds) generated by an in vitro reaction using homogenate from 15°C-labeled N49A PC12 cells (Desnos et al., 1995). No labeled KT3 was recovered in the SV peak when the in vitro reactions were performed at 4°C (15°C cell labeling, open diamonds; 4°C cell labeling, open circles). The label at the top (right-hand side) of the gradients is free antibody.

Mentions: To reconstitute in vitro vesicle budding from the plasma membrane, we labeled intact N49A cells with iodinated KT3 at 4°C for 2 h. After washing away the unbound antibody, the cells were homogenized and the homogenate incubated at 37°C in the presence of rat brain cytosol and an ATP-regenerating system. After the incubation, the reaction mix was centrifuged to remove large membranes and the supernatant was analyzed by glycerol velocity sedimentation. As shown in Fig. 2 (circles), we observed radioactivity in the SV-containing fractions (8–12) of the glycerol gradient, as well as in free antibody at the top of the gradient (14 and above). SV generated by labeling N49A cells at 15°C followed by an in vitro reaction at 37°C (Desnos et al., 1995) also migrate to the same glycerol fractions (Fig. 2, diamonds). The amount of radioactivity recovered in the SV peak was always about five times less when labeled plasma membrane was used, compared with labeled endosomes. The relatively weak signal was probably due to the limited amount of VAMP on the cell surface compared with the amount internalized at 15°C. The small size of the signal, the shorter incubation time, and the use of a less advantageous mutant VAMP (del61-70) may explain why no budding from plasma membranes was observed in earlier experiments (Desnos et al., 1995). Nonetheless, when expressed as recovery of membrane-bound radioactivity in SVs, the plasma membrane and the endosomal assays have comparable efficiency.


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)

In vitro budding  from cell homogenate labeled  at 4°C or 15°C. Cell homogenate (1 mg) was prepared  from N49A/PC12 cells labeled with 125I-KT3 at 4°C for  2 h. The homogenate was  then incubated with rat brain  cytosol (1.5 mg/ml) and ATP  at 37°C (closed circles) or 4°C  (open circles) for 30 min followed by centrifugation of  a high speed supernatant  (S2) on a 5–25% glycerol velocity gradient. A significant  amount of 125I-KT3 was recovered in fractions 8–12 (closed circles), cosedimenting with the  SVs (closed diamonds) generated by an in vitro reaction using  homogenate from 15°C-labeled N49A PC12 cells (Desnos et al.,  1995). No labeled KT3 was recovered in the SV peak when the in  vitro reactions were performed at 4°C (15°C cell labeling, open  diamonds; 4°C cell labeling, open circles). The label at the top  (right-hand side) of the gradients is free antibody.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: In vitro budding from cell homogenate labeled at 4°C or 15°C. Cell homogenate (1 mg) was prepared from N49A/PC12 cells labeled with 125I-KT3 at 4°C for 2 h. The homogenate was then incubated with rat brain cytosol (1.5 mg/ml) and ATP at 37°C (closed circles) or 4°C (open circles) for 30 min followed by centrifugation of a high speed supernatant (S2) on a 5–25% glycerol velocity gradient. A significant amount of 125I-KT3 was recovered in fractions 8–12 (closed circles), cosedimenting with the SVs (closed diamonds) generated by an in vitro reaction using homogenate from 15°C-labeled N49A PC12 cells (Desnos et al., 1995). No labeled KT3 was recovered in the SV peak when the in vitro reactions were performed at 4°C (15°C cell labeling, open diamonds; 4°C cell labeling, open circles). The label at the top (right-hand side) of the gradients is free antibody.
Mentions: To reconstitute in vitro vesicle budding from the plasma membrane, we labeled intact N49A cells with iodinated KT3 at 4°C for 2 h. After washing away the unbound antibody, the cells were homogenized and the homogenate incubated at 37°C in the presence of rat brain cytosol and an ATP-regenerating system. After the incubation, the reaction mix was centrifuged to remove large membranes and the supernatant was analyzed by glycerol velocity sedimentation. As shown in Fig. 2 (circles), we observed radioactivity in the SV-containing fractions (8–12) of the glycerol gradient, as well as in free antibody at the top of the gradient (14 and above). SV generated by labeling N49A cells at 15°C followed by an in vitro reaction at 37°C (Desnos et al., 1995) also migrate to the same glycerol fractions (Fig. 2, diamonds). The amount of radioactivity recovered in the SV peak was always about five times less when labeled plasma membrane was used, compared with labeled endosomes. The relatively weak signal was probably due to the limited amount of VAMP on the cell surface compared with the amount internalized at 15°C. The small size of the signal, the shorter incubation time, and the use of a less advantageous mutant VAMP (del61-70) may explain why no budding from plasma membranes was observed in earlier experiments (Desnos et al., 1995). Nonetheless, when expressed as recovery of membrane-bound radioactivity in SVs, the plasma membrane and the endosomal assays have comparable efficiency.

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