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ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells.

Liu W, Duden R, Phair RD, Lippincott-Schwartz J - J. Cell Biol. (2005)

Bottom Line: The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear.Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner.These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

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Biochemical evidence for membrane coatomer–ArfGAP1 complex formation. (A) COS-7 and NRK cell lysates were immunoprecipitated with polyclonal anti-ArfGAP1 antibody, and the immunocomplexes were subjected to immunoblot assay with the monoclonal anti–β-COP antibody. (B and C) NRK cells expressing ArfGAP1-YFP were treated without or with BFA (5 μg/ml−1) for 30 min, AlF for 10 min, BFA 30 min then AlF 10 min, or AlF 10 min then BFA 30 min. The cell lysates were immunoprecipitated with monoclonal anti-GFP antibody, and were followed by Western blotting with polyclonal anti–β-COP (top) antibody or anti-GFP antibody (bottom).
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fig5: Biochemical evidence for membrane coatomer–ArfGAP1 complex formation. (A) COS-7 and NRK cell lysates were immunoprecipitated with polyclonal anti-ArfGAP1 antibody, and the immunocomplexes were subjected to immunoblot assay with the monoclonal anti–β-COP antibody. (B and C) NRK cells expressing ArfGAP1-YFP were treated without or with BFA (5 μg/ml−1) for 30 min, AlF for 10 min, BFA 30 min then AlF 10 min, or AlF 10 min then BFA 30 min. The cell lysates were immunoprecipitated with monoclonal anti-GFP antibody, and were followed by Western blotting with polyclonal anti–β-COP (top) antibody or anti-GFP antibody (bottom).

Mentions: To obtain biochemical evidence that ArfGAP1 interacts with coatomer, we examined whether ArfGAP1 and β-COP underwent coimmunoprecipitation. In both COS-7 cells and NRK cells, endogenous β-COP coimmunoprecipitated with endogenous ArfGAP1 (Fig. 5 A). And, in cells expressing ArfGAP1-GFP, immunoprecipitation using GFP antibodies resulted in coimmunoprecipitation of β-COP. The complex between ArfGAP and β-COP was dependent on Arf1-GTP on membranes as BFA treatment, which inactivates Arf1, prevented ArfGAP1 and β-COP from interacting (Fig. 5 B). AlF treatment increased the association between ArfGAP and β-COP, as predicted if AlF stabilizes complexes of fluoride–Arf1–ArfGAP1–coatomer on membranes. This stabilization was dependent on Arf1 being present on membranes because treating cells with BFA before AlF treatment prevented ArfGAP1 and β-COP from associating, whereas treating cells with AlF before BFA treatment did not (Fig. 5 C). The data thus supported our morphological findings suggesting that ArfGAP1–coatomer–Arf1 ternary complexes form within cells and are affected by treatments with BFA or AlF.


ArfGAP1 dynamics and its role in COPI coat assembly on Golgi membranes of living cells.

Liu W, Duden R, Phair RD, Lippincott-Schwartz J - J. Cell Biol. (2005)

Biochemical evidence for membrane coatomer–ArfGAP1 complex formation. (A) COS-7 and NRK cell lysates were immunoprecipitated with polyclonal anti-ArfGAP1 antibody, and the immunocomplexes were subjected to immunoblot assay with the monoclonal anti–β-COP antibody. (B and C) NRK cells expressing ArfGAP1-YFP were treated without or with BFA (5 μg/ml−1) for 30 min, AlF for 10 min, BFA 30 min then AlF 10 min, or AlF 10 min then BFA 30 min. The cell lysates were immunoprecipitated with monoclonal anti-GFP antibody, and were followed by Western blotting with polyclonal anti–β-COP (top) antibody or anti-GFP antibody (bottom).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171832&req=5

fig5: Biochemical evidence for membrane coatomer–ArfGAP1 complex formation. (A) COS-7 and NRK cell lysates were immunoprecipitated with polyclonal anti-ArfGAP1 antibody, and the immunocomplexes were subjected to immunoblot assay with the monoclonal anti–β-COP antibody. (B and C) NRK cells expressing ArfGAP1-YFP were treated without or with BFA (5 μg/ml−1) for 30 min, AlF for 10 min, BFA 30 min then AlF 10 min, or AlF 10 min then BFA 30 min. The cell lysates were immunoprecipitated with monoclonal anti-GFP antibody, and were followed by Western blotting with polyclonal anti–β-COP (top) antibody or anti-GFP antibody (bottom).
Mentions: To obtain biochemical evidence that ArfGAP1 interacts with coatomer, we examined whether ArfGAP1 and β-COP underwent coimmunoprecipitation. In both COS-7 cells and NRK cells, endogenous β-COP coimmunoprecipitated with endogenous ArfGAP1 (Fig. 5 A). And, in cells expressing ArfGAP1-GFP, immunoprecipitation using GFP antibodies resulted in coimmunoprecipitation of β-COP. The complex between ArfGAP and β-COP was dependent on Arf1-GTP on membranes as BFA treatment, which inactivates Arf1, prevented ArfGAP1 and β-COP from interacting (Fig. 5 B). AlF treatment increased the association between ArfGAP and β-COP, as predicted if AlF stabilizes complexes of fluoride–Arf1–ArfGAP1–coatomer on membranes. This stabilization was dependent on Arf1 being present on membranes because treating cells with BFA before AlF treatment prevented ArfGAP1 and β-COP from associating, whereas treating cells with AlF before BFA treatment did not (Fig. 5 C). The data thus supported our morphological findings suggesting that ArfGAP1–coatomer–Arf1 ternary complexes form within cells and are affected by treatments with BFA or AlF.

Bottom Line: The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear.Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner.These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Secretory protein trafficking relies on the COPI coat, which by assembling into a lattice on Golgi membranes concentrates cargo at specific sites and deforms the membranes at these sites into coated buds and carriers. The GTPase-activating protein (GAP) responsible for catalyzing Arf1 GTP hydrolysis is an important part of this system, but the mechanism whereby ArfGAP is recruited to the coat, its stability within the coat, and its role in maintenance of the coat are unclear. Here, we use FRAP to monitor the membrane turnover of GFP-tagged versions of ArfGAP1, Arf1, and coatomer in living cells. ArfGAP1 underwent fast cytosol/Golgi exchange with approximately 40% of the exchange dependent on engagement of ArfGAP1 with coatomer and Arf1, and affected by secretory cargo load. Permanent activation of Arf1 resulted in ArfGAP1 being trapped on the Golgi in a coatomer-dependent manner. These data suggest that ArfGAP1, coatomer and Arf1 play interdependent roles in the assembly-disassembly cycle of the COPI coat in vivo.

Show MeSH