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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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Characterization  of the in vitro budding reaction from 4°C-labeled membranes. (A) Effect of rat  brain cytosol concentration.  Standard reactions were performed using Percoll-washed  membranes with different  concentrations of rat brain  cytosol: 0 mg/ml (open circles), 0.5 mg/ml (closed diamonds), 1 mg/ml (open diamonds), and 2 mg/ml (closed  circles). The high speed supernatants (S2) of each reaction were centrifuged on a  5–25% glycerol velocity gradient. The radioactivity of  each fraction was plotted  against fraction numbers. (B)  Kinetics of the in vitro budding reaction. The amount  of 125I-KT3 recovered in the SV peak (pool of fractions 8–12) after a complete  budding reaction was measured as a function of time. (C) Nucleotide dependence  of the budding reactions. Standard in vitro budding reactions were carried out  under normal budding conditions (Percoll-washed membranes, rat brain cytosol,  and ATP at 37°C), or with 20 μM GTPγS, or in the absence of the ATP regeneration system. (Note that nucleotides in rat brain cytosol were removed by dialysis  against intracellular buffer.) Vesicle production (radioactivity in fractions 8–12)  was normalized as a percentage of the yield obtained under normal conditions  (Control). Reactions at 4°C were subtracted as backgrounds. Error bars represent  the range of the results for two independent experiments. The cytosol concentration in all experiments was 1.5 mg/ml.
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Figure 3: Characterization of the in vitro budding reaction from 4°C-labeled membranes. (A) Effect of rat brain cytosol concentration. Standard reactions were performed using Percoll-washed membranes with different concentrations of rat brain cytosol: 0 mg/ml (open circles), 0.5 mg/ml (closed diamonds), 1 mg/ml (open diamonds), and 2 mg/ml (closed circles). The high speed supernatants (S2) of each reaction were centrifuged on a 5–25% glycerol velocity gradient. The radioactivity of each fraction was plotted against fraction numbers. (B) Kinetics of the in vitro budding reaction. The amount of 125I-KT3 recovered in the SV peak (pool of fractions 8–12) after a complete budding reaction was measured as a function of time. (C) Nucleotide dependence of the budding reactions. Standard in vitro budding reactions were carried out under normal budding conditions (Percoll-washed membranes, rat brain cytosol, and ATP at 37°C), or with 20 μM GTPγS, or in the absence of the ATP regeneration system. (Note that nucleotides in rat brain cytosol were removed by dialysis against intracellular buffer.) Vesicle production (radioactivity in fractions 8–12) was normalized as a percentage of the yield obtained under normal conditions (Control). Reactions at 4°C were subtracted as backgrounds. Error bars represent the range of the results for two independent experiments. The cytosol concentration in all experiments was 1.5 mg/ml.

Mentions: In vitro vesicle budding is increased in efficiency by the use of cytosol. As shown in Fig. 3 A, the amount of vesicles generated in the budding reaction depended on the concentration of rat brain cytosol, suggesting that cytosolic factors are required for vesicle formation from the plasma membrane. A small vesicle peak was often observed in control reactions with no added rat brain cytosol. Since this background reaction is seen with washed membranes it may be due to coating proteins that remain bound to the plasma membrane after homogenization. As indicated by Western blot, clathrin and α-adaptin can be detected on the washed membranes (not shown). Immunofluorescence also supports the association of dynamin (Estes et al., 1996; Roos and Kelly, 1998) and AP2 (Gonzales-Gaitan and Jackle, 1997) with the plasma membrane at endocytotic “hot spots.”


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)

Characterization  of the in vitro budding reaction from 4°C-labeled membranes. (A) Effect of rat  brain cytosol concentration.  Standard reactions were performed using Percoll-washed  membranes with different  concentrations of rat brain  cytosol: 0 mg/ml (open circles), 0.5 mg/ml (closed diamonds), 1 mg/ml (open diamonds), and 2 mg/ml (closed  circles). The high speed supernatants (S2) of each reaction were centrifuged on a  5–25% glycerol velocity gradient. The radioactivity of  each fraction was plotted  against fraction numbers. (B)  Kinetics of the in vitro budding reaction. The amount  of 125I-KT3 recovered in the SV peak (pool of fractions 8–12) after a complete  budding reaction was measured as a function of time. (C) Nucleotide dependence  of the budding reactions. Standard in vitro budding reactions were carried out  under normal budding conditions (Percoll-washed membranes, rat brain cytosol,  and ATP at 37°C), or with 20 μM GTPγS, or in the absence of the ATP regeneration system. (Note that nucleotides in rat brain cytosol were removed by dialysis  against intracellular buffer.) Vesicle production (radioactivity in fractions 8–12)  was normalized as a percentage of the yield obtained under normal conditions  (Control). Reactions at 4°C were subtracted as backgrounds. Error bars represent  the range of the results for two independent experiments. The cytosol concentration in all experiments was 1.5 mg/ml.
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Related In: Results  -  Collection

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Figure 3: Characterization of the in vitro budding reaction from 4°C-labeled membranes. (A) Effect of rat brain cytosol concentration. Standard reactions were performed using Percoll-washed membranes with different concentrations of rat brain cytosol: 0 mg/ml (open circles), 0.5 mg/ml (closed diamonds), 1 mg/ml (open diamonds), and 2 mg/ml (closed circles). The high speed supernatants (S2) of each reaction were centrifuged on a 5–25% glycerol velocity gradient. The radioactivity of each fraction was plotted against fraction numbers. (B) Kinetics of the in vitro budding reaction. The amount of 125I-KT3 recovered in the SV peak (pool of fractions 8–12) after a complete budding reaction was measured as a function of time. (C) Nucleotide dependence of the budding reactions. Standard in vitro budding reactions were carried out under normal budding conditions (Percoll-washed membranes, rat brain cytosol, and ATP at 37°C), or with 20 μM GTPγS, or in the absence of the ATP regeneration system. (Note that nucleotides in rat brain cytosol were removed by dialysis against intracellular buffer.) Vesicle production (radioactivity in fractions 8–12) was normalized as a percentage of the yield obtained under normal conditions (Control). Reactions at 4°C were subtracted as backgrounds. Error bars represent the range of the results for two independent experiments. The cytosol concentration in all experiments was 1.5 mg/ml.
Mentions: In vitro vesicle budding is increased in efficiency by the use of cytosol. As shown in Fig. 3 A, the amount of vesicles generated in the budding reaction depended on the concentration of rat brain cytosol, suggesting that cytosolic factors are required for vesicle formation from the plasma membrane. A small vesicle peak was often observed in control reactions with no added rat brain cytosol. Since this background reaction is seen with washed membranes it may be due to coating proteins that remain bound to the plasma membrane after homogenization. As indicated by Western blot, clathrin and α-adaptin can be detected on the washed membranes (not shown). Immunofluorescence also supports the association of dynamin (Estes et al., 1996; Roos and Kelly, 1998) and AP2 (Gonzales-Gaitan and Jackle, 1997) with the plasma membrane at endocytotic “hot spots.”

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