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ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

Faúndez V, Horng JT, Kelly RB - J. Cell Biol. (1997)

Bottom Line: A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size.Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1.Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, The Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACT
Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

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In vivo synaptic vesicle biogenesis is inhibited by BFA  in a concentration dependent and reversible way. Stably transfected PC12 cells bearing the N49A VAMP-TAg construct were  labeled at 15°C for 40 min in the presence of 125I-KT3 antibody,  and the unbound antibody washed extensively. (a) After washing  out the free antibody, cells were incubated at 0°C for 15 min either in the absence (○) or presence (⋄) of BFA (5 μg/ml). Control and BFA-treated cells were warmed for 15 min. To measure  reversibility, one of the BFA-containing plates was washed thoroughly at 0°C and then reincubated at 37°C for another 15 min in  the absence of BFA (•). The reactions were stopped, cells homogenized, and high speed supernatants processed for velocity  sedimentation analysis as described in Methods. The data are one  example of two independent experiments. (b) Cells were incubated as described in a with 0.1–10 μg/ml of BFA, and then transferred at 37°C for 15 min and processed as described. BFA inhibited vesicle production in a dose-dependent way (0% inhibition  corresponds to 3550 cpm in the SV peak).
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Figure 1: In vivo synaptic vesicle biogenesis is inhibited by BFA in a concentration dependent and reversible way. Stably transfected PC12 cells bearing the N49A VAMP-TAg construct were labeled at 15°C for 40 min in the presence of 125I-KT3 antibody, and the unbound antibody washed extensively. (a) After washing out the free antibody, cells were incubated at 0°C for 15 min either in the absence (○) or presence (⋄) of BFA (5 μg/ml). Control and BFA-treated cells were warmed for 15 min. To measure reversibility, one of the BFA-containing plates was washed thoroughly at 0°C and then reincubated at 37°C for another 15 min in the absence of BFA (•). The reactions were stopped, cells homogenized, and high speed supernatants processed for velocity sedimentation analysis as described in Methods. The data are one example of two independent experiments. (b) Cells were incubated as described in a with 0.1–10 μg/ml of BFA, and then transferred at 37°C for 15 min and processed as described. BFA inhibited vesicle production in a dose-dependent way (0% inhibition corresponds to 3550 cpm in the SV peak).

Mentions: To determine whether SV biogenesis is an ARF-mediated process, the effect of BFA upon synaptic vesicle biogenesis in PC12 cells was examined in vivo. The PC12 cell line was stably transfected with a luminally tagged VAMP construct bearing a point mutation in the cytoplasmic tail, N49A (N49A/PC12). This VAMP derivative shows increased targeting to SV compared with wild type (Grote et al., 1995). It shows even more specific targeting to SVs than the del61-70 mutation used in a previous study (Desnos et al., 1995). Incubating intact cells at 15°C with iodinated antibodies (125I-KT3) against the lumenal epitope labels plasma membrane and intracellular compartments without labeling synaptic vesicles (Desnos et al., 1995). Internalizing the antibody at 15°C, removing free antibody, and then incubating the cells at 37°C caused the appearance of antibody-labeled SVs, monitored by their migration on glycerol velocity gradients (Fig. 1 a, ○). Free antibody remains at the top (right) of the velocity gradients. The addition of BFA (5 μg/ml) inhibited the production of labeled vesicles upon warming to 37°C (Fig. 1 a, ⋄). After washing the drug out, the BFA-mediated block was completely reversed within 15 min (Fig. 1 a, •). The fast reversibility of the block argues against nonspecific toxicity effects.


ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

Faúndez V, Horng JT, Kelly RB - J. Cell Biol. (1997)

In vivo synaptic vesicle biogenesis is inhibited by BFA  in a concentration dependent and reversible way. Stably transfected PC12 cells bearing the N49A VAMP-TAg construct were  labeled at 15°C for 40 min in the presence of 125I-KT3 antibody,  and the unbound antibody washed extensively. (a) After washing  out the free antibody, cells were incubated at 0°C for 15 min either in the absence (○) or presence (⋄) of BFA (5 μg/ml). Control and BFA-treated cells were warmed for 15 min. To measure  reversibility, one of the BFA-containing plates was washed thoroughly at 0°C and then reincubated at 37°C for another 15 min in  the absence of BFA (•). The reactions were stopped, cells homogenized, and high speed supernatants processed for velocity  sedimentation analysis as described in Methods. The data are one  example of two independent experiments. (b) Cells were incubated as described in a with 0.1–10 μg/ml of BFA, and then transferred at 37°C for 15 min and processed as described. BFA inhibited vesicle production in a dose-dependent way (0% inhibition  corresponds to 3550 cpm in the SV peak).
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Related In: Results  -  Collection

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Figure 1: In vivo synaptic vesicle biogenesis is inhibited by BFA in a concentration dependent and reversible way. Stably transfected PC12 cells bearing the N49A VAMP-TAg construct were labeled at 15°C for 40 min in the presence of 125I-KT3 antibody, and the unbound antibody washed extensively. (a) After washing out the free antibody, cells were incubated at 0°C for 15 min either in the absence (○) or presence (⋄) of BFA (5 μg/ml). Control and BFA-treated cells were warmed for 15 min. To measure reversibility, one of the BFA-containing plates was washed thoroughly at 0°C and then reincubated at 37°C for another 15 min in the absence of BFA (•). The reactions were stopped, cells homogenized, and high speed supernatants processed for velocity sedimentation analysis as described in Methods. The data are one example of two independent experiments. (b) Cells were incubated as described in a with 0.1–10 μg/ml of BFA, and then transferred at 37°C for 15 min and processed as described. BFA inhibited vesicle production in a dose-dependent way (0% inhibition corresponds to 3550 cpm in the SV peak).
Mentions: To determine whether SV biogenesis is an ARF-mediated process, the effect of BFA upon synaptic vesicle biogenesis in PC12 cells was examined in vivo. The PC12 cell line was stably transfected with a luminally tagged VAMP construct bearing a point mutation in the cytoplasmic tail, N49A (N49A/PC12). This VAMP derivative shows increased targeting to SV compared with wild type (Grote et al., 1995). It shows even more specific targeting to SVs than the del61-70 mutation used in a previous study (Desnos et al., 1995). Incubating intact cells at 15°C with iodinated antibodies (125I-KT3) against the lumenal epitope labels plasma membrane and intracellular compartments without labeling synaptic vesicles (Desnos et al., 1995). Internalizing the antibody at 15°C, removing free antibody, and then incubating the cells at 37°C caused the appearance of antibody-labeled SVs, monitored by their migration on glycerol velocity gradients (Fig. 1 a, ○). Free antibody remains at the top (right) of the velocity gradients. The addition of BFA (5 μg/ml) inhibited the production of labeled vesicles upon warming to 37°C (Fig. 1 a, ⋄). After washing the drug out, the BFA-mediated block was completely reversed within 15 min (Fig. 1 a, •). The fast reversibility of the block argues against nonspecific toxicity effects.

Bottom Line: A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size.Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1.Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, The Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACT
Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Show MeSH
Related in: MedlinePlus