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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 plasma membrane  of N49A/PC12 cells is labeled  with KT3 mAb at 4°C. N49A/ PC12 cells were incubated in the  presence of KT3 mAb (10 μg/ ml) for 15 min at 4°C. Cells were  then warmed up to 37°C for 40  min (C and D) or kept at 4°C (A  and B). In B and D, cells were  treated with an acid wash (see  Materials and Methods) at 4°C  before fixation. The immunofluorescence was processed as described. After labeling at 4°C,  the label can be removed by an  acid wash, but not after warming  to 37°C. Bars, 10 μM.
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Figure 1: The plasma membrane of N49A/PC12 cells is labeled with KT3 mAb at 4°C. N49A/ PC12 cells were incubated in the presence of KT3 mAb (10 μg/ ml) for 15 min at 4°C. Cells were then warmed up to 37°C for 40 min (C and D) or kept at 4°C (A and B). In B and D, cells were treated with an acid wash (see Materials and Methods) at 4°C before fixation. The immunofluorescence was processed as described. After labeling at 4°C, the label can be removed by an acid wash, but not after warming to 37°C. Bars, 10 μM.

Mentions: To study vesicle formation from the plasma membrane, we generated homogenates of PC12 cells in which only SV proteins on the plasma membrane were labeled. The PC12 cell line used in the current study (N49A/PC12) was stably transfected with a luminally tagged VAMP construct that had a point mutation (N49A) in the cytoplasmic tail. This VAMP derivative shows increased targeting to SVs compared with wild type (Grote et al., 1995), and even more specific targeting to SVs than the del61-70 mutation used in a previous study (Desnos et al., 1995). To label the plasma membrane, N49A/PC12 cells were incubated at 4°C with antibodies (KT3) against the lumenal epitope. Immunofluorescent staining was detected around the cell periphery with no detectable staining of any intracellular structures (Fig. 1 A). The peripheral labeling could be stripped away with an acid wash (Fig. 1 B), indicating that, at 4°C, the antibodies only labeled epitopes exposed on the cell surface. In a control experiment, when N49A-transfected cells were labeled at 37°C for 40 min, the antibodies were internalized and an intense, acid strip–resistant intracellular staining could be observed (Fig. 1, C and D). Restriction of label to the cell surface was confirmed by biochemical subcellular fractionation experiments. Intact N49A cells were labeled at 4°C with iodinated KT3 followed by homogenization and fractionation on sucrose density gradients. A peak of radioactivity was recovered at 38% sucrose, which represents labeled plasma membranes (Clift-O'Grady et al., 1998). Thus, incubating cells with antibody at 4°C only labels the cell surface.


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 plasma membrane  of N49A/PC12 cells is labeled  with KT3 mAb at 4°C. N49A/ PC12 cells were incubated in the  presence of KT3 mAb (10 μg/ ml) for 15 min at 4°C. Cells were  then warmed up to 37°C for 40  min (C and D) or kept at 4°C (A  and B). In B and D, cells were  treated with an acid wash (see  Materials and Methods) at 4°C  before fixation. The immunofluorescence was processed as described. After labeling at 4°C,  the label can be removed by an  acid wash, but not after warming  to 37°C. Bars, 10 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132944&req=5

Figure 1: The plasma membrane of N49A/PC12 cells is labeled with KT3 mAb at 4°C. N49A/ PC12 cells were incubated in the presence of KT3 mAb (10 μg/ ml) for 15 min at 4°C. Cells were then warmed up to 37°C for 40 min (C and D) or kept at 4°C (A and B). In B and D, cells were treated with an acid wash (see Materials and Methods) at 4°C before fixation. The immunofluorescence was processed as described. After labeling at 4°C, the label can be removed by an acid wash, but not after warming to 37°C. Bars, 10 μM.
Mentions: To study vesicle formation from the plasma membrane, we generated homogenates of PC12 cells in which only SV proteins on the plasma membrane were labeled. The PC12 cell line used in the current study (N49A/PC12) was stably transfected with a luminally tagged VAMP construct that had a point mutation (N49A) in the cytoplasmic tail. This VAMP derivative shows increased targeting to SVs compared with wild type (Grote et al., 1995), and even more specific targeting to SVs than the del61-70 mutation used in a previous study (Desnos et al., 1995). To label the plasma membrane, N49A/PC12 cells were incubated at 4°C with antibodies (KT3) against the lumenal epitope. Immunofluorescent staining was detected around the cell periphery with no detectable staining of any intracellular structures (Fig. 1 A). The peripheral labeling could be stripped away with an acid wash (Fig. 1 B), indicating that, at 4°C, the antibodies only labeled epitopes exposed on the cell surface. In a control experiment, when N49A-transfected cells were labeled at 37°C for 40 min, the antibodies were internalized and an intense, acid strip–resistant intracellular staining could be observed (Fig. 1, C and D). Restriction of label to the cell surface was confirmed by biochemical subcellular fractionation experiments. Intact N49A cells were labeled at 4°C with iodinated KT3 followed by homogenization and fractionation on sucrose density gradients. A peak of radioactivity was recovered at 38% sucrose, which represents labeled plasma membranes (Clift-O'Grady et al., 1998). Thus, incubating cells with antibody at 4°C only labels the cell surface.

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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Related in: MedlinePlus