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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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Dominant-positive and -negative mutant ARF1 inhibit  the appearance of mature synaptic vesicles in a cell-free system.  Standard reaction mixtures similar to those described in Fig. 8  were performed in the absence or presence of recombinant  myristoylated wild-type, Q71L (dominant positive), and T31N  (dominant negative) proteins. The ARF1 proteins incubated with  the reaction mixes for 15 min at 0°C and then warmed to 37°C for  30 min. (a) At 50 μM, both Q71L (▵) and T31N (---○---) inhibited the appearance of matured vesicles as assessed in velocity  sedimentation compared to control (—○—). The addition of  wild-type ARF1 (⋄) was without effect on the vesicle production.  Vesicle production did not occur at 4°C (◫̶ ). In b dominant-negative and -positive mutant forms of ARF1 were added at concentrations ranging from 1 to 50 μM. The maximal inhibitory effect  was reached by 5 μM for both of them (0% inhibition corresponds to 8,157 cpm).
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Figure 9: Dominant-positive and -negative mutant ARF1 inhibit the appearance of mature synaptic vesicles in a cell-free system. Standard reaction mixtures similar to those described in Fig. 8 were performed in the absence or presence of recombinant myristoylated wild-type, Q71L (dominant positive), and T31N (dominant negative) proteins. The ARF1 proteins incubated with the reaction mixes for 15 min at 0°C and then warmed to 37°C for 30 min. (a) At 50 μM, both Q71L (▵) and T31N (---○---) inhibited the appearance of matured vesicles as assessed in velocity sedimentation compared to control (—○—). The addition of wild-type ARF1 (⋄) was without effect on the vesicle production. Vesicle production did not occur at 4°C (◫̶ ). In b dominant-negative and -positive mutant forms of ARF1 were added at concentrations ranging from 1 to 50 μM. The maximal inhibitory effect was reached by 5 μM for both of them (0% inhibition corresponds to 8,157 cpm).

Mentions: To further address ARF1 involvement, we tested the effects of purified ARF1 mutants Q71L, a GTPase , and T31N (a GTP-binding defective mutant) on the formation of properly sized SVs, assessed by velocity gradient sedimentation. The amount of labeled SVs was unchanged by supplementing the standard reaction mixtures with 50 μM wild-type ARF1 (Fig. 9 a). In contrast, in standard reactions supplemented with 50 μM of either dominant-positive (Q71L) or dominant-negative (T31N) mutants, there was a drastic reduction in the amount of labeled vesicles detected in velocity gradients. Both recombinant mutant proteins were effective at concentrations as low as 1 μM (Fig. 9 b). This concentration is similar to the amount of ARF1 estimated to be present in the rat brain cytosol used in the standard reaction mixtures (data not shown; Kahn et al., 1988). Consistently Q71L was more potent when compared with T31N. Both mutants were also able to induce a reduction in the production of labeled SVs in the suboptimal conditions when only PC12 cell cytosol is present without added brain cytosol (Desnos et al., 1995). Even under these conditions, however, additional wild-type ARF1 had no stimulatory effect (data not shown), indicating that the endogenous ARF1 protein present in the PC12 extracts was sufficient for the vesicle budding reaction.


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

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

Dominant-positive and -negative mutant ARF1 inhibit  the appearance of mature synaptic vesicles in a cell-free system.  Standard reaction mixtures similar to those described in Fig. 8  were performed in the absence or presence of recombinant  myristoylated wild-type, Q71L (dominant positive), and T31N  (dominant negative) proteins. The ARF1 proteins incubated with  the reaction mixes for 15 min at 0°C and then warmed to 37°C for  30 min. (a) At 50 μM, both Q71L (▵) and T31N (---○---) inhibited the appearance of matured vesicles as assessed in velocity  sedimentation compared to control (—○—). The addition of  wild-type ARF1 (⋄) was without effect on the vesicle production.  Vesicle production did not occur at 4°C (◫̶ ). In b dominant-negative and -positive mutant forms of ARF1 were added at concentrations ranging from 1 to 50 μM. The maximal inhibitory effect  was reached by 5 μM for both of them (0% inhibition corresponds to 8,157 cpm).
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Related In: Results  -  Collection

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Figure 9: Dominant-positive and -negative mutant ARF1 inhibit the appearance of mature synaptic vesicles in a cell-free system. Standard reaction mixtures similar to those described in Fig. 8 were performed in the absence or presence of recombinant myristoylated wild-type, Q71L (dominant positive), and T31N (dominant negative) proteins. The ARF1 proteins incubated with the reaction mixes for 15 min at 0°C and then warmed to 37°C for 30 min. (a) At 50 μM, both Q71L (▵) and T31N (---○---) inhibited the appearance of matured vesicles as assessed in velocity sedimentation compared to control (—○—). The addition of wild-type ARF1 (⋄) was without effect on the vesicle production. Vesicle production did not occur at 4°C (◫̶ ). In b dominant-negative and -positive mutant forms of ARF1 were added at concentrations ranging from 1 to 50 μM. The maximal inhibitory effect was reached by 5 μM for both of them (0% inhibition corresponds to 8,157 cpm).
Mentions: To further address ARF1 involvement, we tested the effects of purified ARF1 mutants Q71L, a GTPase , and T31N (a GTP-binding defective mutant) on the formation of properly sized SVs, assessed by velocity gradient sedimentation. The amount of labeled SVs was unchanged by supplementing the standard reaction mixtures with 50 μM wild-type ARF1 (Fig. 9 a). In contrast, in standard reactions supplemented with 50 μM of either dominant-positive (Q71L) or dominant-negative (T31N) mutants, there was a drastic reduction in the amount of labeled vesicles detected in velocity gradients. Both recombinant mutant proteins were effective at concentrations as low as 1 μM (Fig. 9 b). This concentration is similar to the amount of ARF1 estimated to be present in the rat brain cytosol used in the standard reaction mixtures (data not shown; Kahn et al., 1988). Consistently Q71L was more potent when compared with T31N. Both mutants were also able to induce a reduction in the production of labeled SVs in the suboptimal conditions when only PC12 cell cytosol is present without added brain cytosol (Desnos et al., 1995). Even under these conditions, however, additional wild-type ARF1 had no stimulatory effect (data not shown), indicating that the endogenous ARF1 protein present in the PC12 extracts was sufficient for the vesicle budding reaction.

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