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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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125I-Transferrin recycling back to the cell surface is not inhibited by BFA.  PC12/N49A endosomes were  labeled with 125I-rat transferrin (0.2 μg/ml) at 15°C for 40  min. The free and surface-bound ligands were removed  by repeated washing followed by mild acidic treatment of the cell surface. The  cells were incubated in the  absence (○) or presence of  BFA (5 μg/ml) (⋄) at 0°C in  a media containing 100 μg/ml  of cold rat transferrin and  chased for different times at  37°C. Transferrin in the media (•, ♦) and in the cells (○, ⋄) was determined as described.  Error bars represent standard errors of triplicate points of one  representative experiment from three independent ones.
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Figure 6: 125I-Transferrin recycling back to the cell surface is not inhibited by BFA. PC12/N49A endosomes were labeled with 125I-rat transferrin (0.2 μg/ml) at 15°C for 40 min. The free and surface-bound ligands were removed by repeated washing followed by mild acidic treatment of the cell surface. The cells were incubated in the absence (○) or presence of BFA (5 μg/ml) (⋄) at 0°C in a media containing 100 μg/ml of cold rat transferrin and chased for different times at 37°C. Transferrin in the media (•, ♦) and in the cells (○, ⋄) was determined as described. Error bars represent standard errors of triplicate points of one representative experiment from three independent ones.

Mentions: Although formation of synaptic vesicles from the 15°C compartment was sensitive to BFA, other parts of the endocytotic pathway were not. Internalization of VAMP-TAg from the cell surface was assessed as acid-resistant bound 125I-KT3 antibody and found to be similar in the control and BFA-treated cells (Fig. 5 a). Insensitivity of internalization to BFA was also shown by immunofluorescence (Fig. 5 b). Recycling of transferrin from endosomes to the cell surface was also relatively unaffected by BFA, as shown by the kinetics of 125I-labeled rat transferrin recycling from endosomes to the plasma membrane after BFA treatment (Fig. 6). To measure recycling, 125I-labeled rat transferrin was internalized at 15°C, cells chilled to 4°C, and plasma membrane–associated ligand removed by washing. Cells were then incubated in the absence or presence of BFA for different times at 37°C. As previously reported in K562 and NRK cells (Lippincott-Schwartz et al., 1991a; Schonhorn and Wessling-Resnick, 1994), the change in the rate of transferrin externalization was not very dramatic after BFA treatment in N49A/PC12 cells, either evaluated as cell-associated radioactivity or released radioactivity.


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

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

125I-Transferrin recycling back to the cell surface is not inhibited by BFA.  PC12/N49A endosomes were  labeled with 125I-rat transferrin (0.2 μg/ml) at 15°C for 40  min. The free and surface-bound ligands were removed  by repeated washing followed by mild acidic treatment of the cell surface. The  cells were incubated in the  absence (○) or presence of  BFA (5 μg/ml) (⋄) at 0°C in  a media containing 100 μg/ml  of cold rat transferrin and  chased for different times at  37°C. Transferrin in the media (•, ♦) and in the cells (○, ⋄) was determined as described.  Error bars represent standard errors of triplicate points of one  representative experiment from three independent ones.
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Related In: Results  -  Collection

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Figure 6: 125I-Transferrin recycling back to the cell surface is not inhibited by BFA. PC12/N49A endosomes were labeled with 125I-rat transferrin (0.2 μg/ml) at 15°C for 40 min. The free and surface-bound ligands were removed by repeated washing followed by mild acidic treatment of the cell surface. The cells were incubated in the absence (○) or presence of BFA (5 μg/ml) (⋄) at 0°C in a media containing 100 μg/ml of cold rat transferrin and chased for different times at 37°C. Transferrin in the media (•, ♦) and in the cells (○, ⋄) was determined as described. Error bars represent standard errors of triplicate points of one representative experiment from three independent ones.
Mentions: Although formation of synaptic vesicles from the 15°C compartment was sensitive to BFA, other parts of the endocytotic pathway were not. Internalization of VAMP-TAg from the cell surface was assessed as acid-resistant bound 125I-KT3 antibody and found to be similar in the control and BFA-treated cells (Fig. 5 a). Insensitivity of internalization to BFA was also shown by immunofluorescence (Fig. 5 b). Recycling of transferrin from endosomes to the cell surface was also relatively unaffected by BFA, as shown by the kinetics of 125I-labeled rat transferrin recycling from endosomes to the plasma membrane after BFA treatment (Fig. 6). To measure recycling, 125I-labeled rat transferrin was internalized at 15°C, cells chilled to 4°C, and plasma membrane–associated ligand removed by washing. Cells were then incubated in the absence or presence of BFA for different times at 37°C. As previously reported in K562 and NRK cells (Lippincott-Schwartz et al., 1991a; Schonhorn and Wessling-Resnick, 1994), the change in the rate of transferrin externalization was not very dramatic after BFA treatment in N49A/PC12 cells, either evaluated as cell-associated radioactivity or released radioactivity.

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