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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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(A) Immunoadsorption of SVs derived from the  plasma membrane. Aliquots of  SVs were incubated with M-450  Dynabeads (3 mg per reaction) coated with anti-VAMP  (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or  anti-syntaxin antibodies. Mouse  γ-globulin was used to determine non-specific binding.  After extensive wash, the radioactivity associated with  the beads was plotted as a  percentage of total radioactivity for each reaction. (B  and C) TfRs are sorted from  SVs from plasma membrane.  N49A/PC12 cells were labeled  with 125I-Tf (0.2 μg/ml; B) or  125I-KT3 (10 μg/ml; C) at 4°C  for 2 h. The cell homogenate  was incubated with rat brain cytosol and ATP at 37°C (closed  symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient  fraction was plotted against its sucrose concentration. Radioactivity from 125I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, 125I-Tf–labeled membranes were  recovered at 28% (0.5 sucrose; closed circles), well separated  from the SVs.
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Figure 4: (A) Immunoadsorption of SVs derived from the plasma membrane. Aliquots of SVs were incubated with M-450 Dynabeads (3 mg per reaction) coated with anti-VAMP (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or anti-syntaxin antibodies. Mouse γ-globulin was used to determine non-specific binding. After extensive wash, the radioactivity associated with the beads was plotted as a percentage of total radioactivity for each reaction. (B and C) TfRs are sorted from SVs from plasma membrane. N49A/PC12 cells were labeled with 125I-Tf (0.2 μg/ml; B) or 125I-KT3 (10 μg/ml; C) at 4°C for 2 h. The cell homogenate was incubated with rat brain cytosol and ATP at 37°C (closed symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient fraction was plotted against its sucrose concentration. Radioactivity from 125I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, 125I-Tf–labeled membranes were recovered at 28% (0.5 sucrose; closed circles), well separated from the SVs.

Mentions: Although neuroendocrine cells such as PC12 are not synapse-forming neurons, they have organelles that resemble SVs. Three criteria that are conventionally used to describe the SVs in PC12 cells are their homogeneous size, the presence of a full array of SV proteins, and the absence of other recycling plasma membrane proteins such as the TfR. To determine if SV markers other than VAMP are present on the small vesicles, M-450 Dynabeads (sheep anti–mouse IgG) coated with mAbs against SV proteins synaptophysin, synaptogyrin, or SV2 were used to immunoadsorb the vesicle fractions. As shown in Fig. 4 A, each of these antibodies adsorbed most of the radioactivity associated with the vesicle fractions. As expected, an almost complete adsorption was also achieved by an antibody against VAMP (synaptobrevin; Fig 4 A). However, antibodies against a t-SNARE protein, syntaxin, did not bind vesicles above the level of controls containing mouse γ-globulin confirming earlier observations (Salem et al., 1998). This result shows that the small vesicles formed from the plasma membrane, as expected for SVs, contain a spectrum of SV markers.


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)

(A) Immunoadsorption of SVs derived from the  plasma membrane. Aliquots of  SVs were incubated with M-450  Dynabeads (3 mg per reaction) coated with anti-VAMP  (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or  anti-syntaxin antibodies. Mouse  γ-globulin was used to determine non-specific binding.  After extensive wash, the radioactivity associated with  the beads was plotted as a  percentage of total radioactivity for each reaction. (B  and C) TfRs are sorted from  SVs from plasma membrane.  N49A/PC12 cells were labeled  with 125I-Tf (0.2 μg/ml; B) or  125I-KT3 (10 μg/ml; C) at 4°C  for 2 h. The cell homogenate  was incubated with rat brain cytosol and ATP at 37°C (closed  symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient  fraction was plotted against its sucrose concentration. Radioactivity from 125I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, 125I-Tf–labeled membranes were  recovered at 28% (0.5 sucrose; closed circles), well separated  from the SVs.
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Related In: Results  -  Collection

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Figure 4: (A) Immunoadsorption of SVs derived from the plasma membrane. Aliquots of SVs were incubated with M-450 Dynabeads (3 mg per reaction) coated with anti-VAMP (synaptobrevin), anti-synaptophysin (SY-38), anti-synaptogyrin (p29), anti-SV2, or anti-syntaxin antibodies. Mouse γ-globulin was used to determine non-specific binding. After extensive wash, the radioactivity associated with the beads was plotted as a percentage of total radioactivity for each reaction. (B and C) TfRs are sorted from SVs from plasma membrane. N49A/PC12 cells were labeled with 125I-Tf (0.2 μg/ml; B) or 125I-KT3 (10 μg/ml; C) at 4°C for 2 h. The cell homogenate was incubated with rat brain cytosol and ATP at 37°C (closed symbols) or 4°C (open symbols). A low speed (5,000 g min) supernatant was made from each reaction and centrifuged on a 10– 45% sucrose velocity gradient. Radioactivity of each gradient fraction was plotted against its sucrose concentration. Radioactivity from 125I-KT3 was recovered at 23% (1.5 sucrose; closed diamonds), similar to the SVs generated from endosomes (Lichtenstein et al., 1998). However, 125I-Tf–labeled membranes were recovered at 28% (0.5 sucrose; closed circles), well separated from the SVs.
Mentions: Although neuroendocrine cells such as PC12 are not synapse-forming neurons, they have organelles that resemble SVs. Three criteria that are conventionally used to describe the SVs in PC12 cells are their homogeneous size, the presence of a full array of SV proteins, and the absence of other recycling plasma membrane proteins such as the TfR. To determine if SV markers other than VAMP are present on the small vesicles, M-450 Dynabeads (sheep anti–mouse IgG) coated with mAbs against SV proteins synaptophysin, synaptogyrin, or SV2 were used to immunoadsorb the vesicle fractions. As shown in Fig. 4 A, each of these antibodies adsorbed most of the radioactivity associated with the vesicle fractions. As expected, an almost complete adsorption was also achieved by an antibody against VAMP (synaptobrevin; Fig 4 A). However, antibodies against a t-SNARE protein, syntaxin, did not bind vesicles above the level of controls containing mouse γ-globulin confirming earlier observations (Salem et al., 1998). This result shows that the small vesicles formed from the plasma membrane, as expected for SVs, contain a spectrum of SV markers.

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