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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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ARF1 stimulates  the SV budding activity in a  cell-free system in the presence of a HMW fraction  from rat brain cytosol. If  brain cytosol was omitted  from standard reaction mixtures that contained labeled  N49A/PC12 cell homogenates and ATP, little SV production was observed during  a 30 min incubation at 37°C  (○). The cytosol-free reaction was stimulated slightly  (◫̶ ) by the addition of  ARF1-depleted, HMW fractions (650 μg/assay) from Superose 6–fractionated rat  brain cytosol. Under these  conditions a direct stimulation of SV production by 15  μM of wild-type, myristoylated ARF1 could be observed (⋄) added before the cell-free budding reaction. Addition  of 15 μM T31N ARF1 (○) inhibited the reaction to approximately the cytosol-free level whereas Q71L ARF1 (▵) inhibited  the appearance of mature vesicles.
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Figure 10: ARF1 stimulates the SV budding activity in a cell-free system in the presence of a HMW fraction from rat brain cytosol. If brain cytosol was omitted from standard reaction mixtures that contained labeled N49A/PC12 cell homogenates and ATP, little SV production was observed during a 30 min incubation at 37°C (○). The cytosol-free reaction was stimulated slightly (◫̶ ) by the addition of ARF1-depleted, HMW fractions (650 μg/assay) from Superose 6–fractionated rat brain cytosol. Under these conditions a direct stimulation of SV production by 15 μM of wild-type, myristoylated ARF1 could be observed (⋄) added before the cell-free budding reaction. Addition of 15 μM T31N ARF1 (○) inhibited the reaction to approximately the cytosol-free level whereas Q71L ARF1 (▵) inhibited the appearance of mature vesicles.

Mentions: To demonstrate ARF dependence directly, the cell-free assay was changed by removing the ARF1 present in rat brain cytosol. This was done by passing the cytosol through a Superose 6 sizing column and taking high molecular weight (HMW) fractions reported to be enriched in coat complexes (Stamnes and Rothman, 1993; data not shown). These HMW fractions were pooled and added to the cell-free reaction in the absence of added rat brain cytosol. The addition of HMW fractions alone stimulated the formation of vesicles by 1.7 ± 0.15 times (n = 5). However the combined addition of HMW fractions plus myristoylated wild-type ARF1 increased the vesicle production 2.6 ± 0.15 times. Further, both mutant ARFs inhibited the appearance of vesicles in the presence of HMW (Fig. 10). These results show that ARF1 is required for SV biogenesis. A likely possibility is that it recruits coating molecules present in the HMW fraction.


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

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

ARF1 stimulates  the SV budding activity in a  cell-free system in the presence of a HMW fraction  from rat brain cytosol. If  brain cytosol was omitted  from standard reaction mixtures that contained labeled  N49A/PC12 cell homogenates and ATP, little SV production was observed during  a 30 min incubation at 37°C  (○). The cytosol-free reaction was stimulated slightly  (◫̶ ) by the addition of  ARF1-depleted, HMW fractions (650 μg/assay) from Superose 6–fractionated rat  brain cytosol. Under these  conditions a direct stimulation of SV production by 15  μM of wild-type, myristoylated ARF1 could be observed (⋄) added before the cell-free budding reaction. Addition  of 15 μM T31N ARF1 (○) inhibited the reaction to approximately the cytosol-free level whereas Q71L ARF1 (▵) inhibited  the appearance of mature vesicles.
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Related In: Results  -  Collection

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Figure 10: ARF1 stimulates the SV budding activity in a cell-free system in the presence of a HMW fraction from rat brain cytosol. If brain cytosol was omitted from standard reaction mixtures that contained labeled N49A/PC12 cell homogenates and ATP, little SV production was observed during a 30 min incubation at 37°C (○). The cytosol-free reaction was stimulated slightly (◫̶ ) by the addition of ARF1-depleted, HMW fractions (650 μg/assay) from Superose 6–fractionated rat brain cytosol. Under these conditions a direct stimulation of SV production by 15 μM of wild-type, myristoylated ARF1 could be observed (⋄) added before the cell-free budding reaction. Addition of 15 μM T31N ARF1 (○) inhibited the reaction to approximately the cytosol-free level whereas Q71L ARF1 (▵) inhibited the appearance of mature vesicles.
Mentions: To demonstrate ARF dependence directly, the cell-free assay was changed by removing the ARF1 present in rat brain cytosol. This was done by passing the cytosol through a Superose 6 sizing column and taking high molecular weight (HMW) fractions reported to be enriched in coat complexes (Stamnes and Rothman, 1993; data not shown). These HMW fractions were pooled and added to the cell-free reaction in the absence of added rat brain cytosol. The addition of HMW fractions alone stimulated the formation of vesicles by 1.7 ± 0.15 times (n = 5). However the combined addition of HMW fractions plus myristoylated wild-type ARF1 increased the vesicle production 2.6 ± 0.15 times. Further, both mutant ARFs inhibited the appearance of vesicles in the presence of HMW (Fig. 10). These results show that ARF1 is required for SV biogenesis. A likely possibility is that it recruits coating molecules present in the HMW fraction.

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