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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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Membrane association–dissociation kinetics of ArfGAP1-YFP at 4°C. (A) Prebleach and recovery images of NRK cell stably expressing ArfGAP1-YFP whose Golgi pool was photobleached after incubation at 4°C for 1 h or more. Bar, 5 μm. (B) Quantification of FRAP experiment shown in A and from a parallel experiment in which cells at 4°C expressing ArfGAP1-YFP were treated with AlF for 10 min before photobleaching the Golgi. Note the appearance of an immobile pool in the cells treated with AlF, which suggested that both Arf1-independent and Arf1-dependent pathways operate at 4°C.
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fig6: Membrane association–dissociation kinetics of ArfGAP1-YFP at 4°C. (A) Prebleach and recovery images of NRK cell stably expressing ArfGAP1-YFP whose Golgi pool was photobleached after incubation at 4°C for 1 h or more. Bar, 5 μm. (B) Quantification of FRAP experiment shown in A and from a parallel experiment in which cells at 4°C expressing ArfGAP1-YFP were treated with AlF for 10 min before photobleaching the Golgi. Note the appearance of an immobile pool in the cells treated with AlF, which suggested that both Arf1-independent and Arf1-dependent pathways operate at 4°C.

Mentions: Cells expressing ArfGAP1-YFP were incubated at 4°C and their Golgi pool of fluorescence was photobleached. Significantly, complete recovery into the Golgi region (with kinetics similar to that in cells incubated at 37°C) was observed, indicating ArfGAP1 still cycled on and off Golgi membranes at 4°C (Fig. 6 A). To determine whether such cycling involved movement of ArfGAP1 through the Arf1-dependent pathway (e.g., requiring Arf1-dependent GTP hydrolysis catalyzed by ArfGAP1), we tested by photobleaching whether AlF treatment for 10 min could induce an irreversibly bound pool of ArfGAP1 (corresponding to stabilized complexes of fluoride–Arf1–ArfGAP1–coatomer) at this temperature. Notably, ∼40% of the fluorescent Golgi pool showed no recovery after photobleaching (Fig. 6 B), indicating this percentage of ArfGAP1 on the Golgi had become immobilized during the AlF treatment. These data, combined with previous data showing GTP hydrolysis–dependent release of Arf1 and coatomer from membrane at 4°C (Presley et al., 2002), indicated that ArfGAP1-YFP molecules could enter and pass through the Arf1-dependent pathway at 4°C. Hence, ArfGAP1 catalytic activity within the coat lattice is not coupled to or dependent on vesicle budding. The findings thus favored a model in which ArfGAP1 is a stoichiometric component of the coat whose catalytic activity is necessary both for coat lattice assembly and disassembly (Yang et al., 2002; Bigay et al., 2003).


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)

Membrane association–dissociation kinetics of ArfGAP1-YFP at 4°C. (A) Prebleach and recovery images of NRK cell stably expressing ArfGAP1-YFP whose Golgi pool was photobleached after incubation at 4°C for 1 h or more. Bar, 5 μm. (B) Quantification of FRAP experiment shown in A and from a parallel experiment in which cells at 4°C expressing ArfGAP1-YFP were treated with AlF for 10 min before photobleaching the Golgi. Note the appearance of an immobile pool in the cells treated with AlF, which suggested that both Arf1-independent and Arf1-dependent pathways operate at 4°C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171832&req=5

fig6: Membrane association–dissociation kinetics of ArfGAP1-YFP at 4°C. (A) Prebleach and recovery images of NRK cell stably expressing ArfGAP1-YFP whose Golgi pool was photobleached after incubation at 4°C for 1 h or more. Bar, 5 μm. (B) Quantification of FRAP experiment shown in A and from a parallel experiment in which cells at 4°C expressing ArfGAP1-YFP were treated with AlF for 10 min before photobleaching the Golgi. Note the appearance of an immobile pool in the cells treated with AlF, which suggested that both Arf1-independent and Arf1-dependent pathways operate at 4°C.
Mentions: Cells expressing ArfGAP1-YFP were incubated at 4°C and their Golgi pool of fluorescence was photobleached. Significantly, complete recovery into the Golgi region (with kinetics similar to that in cells incubated at 37°C) was observed, indicating ArfGAP1 still cycled on and off Golgi membranes at 4°C (Fig. 6 A). To determine whether such cycling involved movement of ArfGAP1 through the Arf1-dependent pathway (e.g., requiring Arf1-dependent GTP hydrolysis catalyzed by ArfGAP1), we tested by photobleaching whether AlF treatment for 10 min could induce an irreversibly bound pool of ArfGAP1 (corresponding to stabilized complexes of fluoride–Arf1–ArfGAP1–coatomer) at this temperature. Notably, ∼40% of the fluorescent Golgi pool showed no recovery after photobleaching (Fig. 6 B), indicating this percentage of ArfGAP1 on the Golgi had become immobilized during the AlF treatment. These data, combined with previous data showing GTP hydrolysis–dependent release of Arf1 and coatomer from membrane at 4°C (Presley et al., 2002), indicated that ArfGAP1-YFP molecules could enter and pass through the Arf1-dependent pathway at 4°C. Hence, ArfGAP1 catalytic activity within the coat lattice is not coupled to or dependent on vesicle budding. The findings thus favored a model in which ArfGAP1 is a stoichiometric component of the coat whose catalytic activity is necessary both for coat lattice assembly and disassembly (Yang et al., 2002; Bigay et al., 2003).

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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