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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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Golgi association–dissociation kinetics of ArfGAP1-YFP in AlF-treated cells. (A, C, E, and F) FRAP of Golgi fluorescence in NRK cells expressing respectively ArfGAP1-YFP (as a stable cell line) in A, ArfGAP1Δ64N-YFP (as a transient transfectant) in C, ɛCOP-YFP (as a stable cell line) in E, or GGA-YFP (as a transient transfectant) in F with or without AlF (50 μM AlCl3, 20 mM NaF) treatment for 10 min. (B) Release kinetics of ArfGAP1-YFP from Golgi membranes after addition of BFA (5 μg/ml−1) with or without AlF pretreatment for 10 min in NRK cells stably expressing ArfGAP1-YFP. (D) ldlF cells were transfected with ArfGAP1-YFP and incubated at 32°C or at 40°C for 3 h to deplete COPI from Golgi membranes. AlF was added for 10 min or not. ArfGAP1-YFP in the Golgi region was then photo-bleached and recovery of Golgi fluorescence was measured over time.
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fig4: Golgi association–dissociation kinetics of ArfGAP1-YFP in AlF-treated cells. (A, C, E, and F) FRAP of Golgi fluorescence in NRK cells expressing respectively ArfGAP1-YFP (as a stable cell line) in A, ArfGAP1Δ64N-YFP (as a transient transfectant) in C, ɛCOP-YFP (as a stable cell line) in E, or GGA-YFP (as a transient transfectant) in F with or without AlF (50 μM AlCl3, 20 mM NaF) treatment for 10 min. (B) Release kinetics of ArfGAP1-YFP from Golgi membranes after addition of BFA (5 μg/ml−1) with or without AlF pretreatment for 10 min in NRK cells stably expressing ArfGAP1-YFP. (D) ldlF cells were transfected with ArfGAP1-YFP and incubated at 32°C or at 40°C for 3 h to deplete COPI from Golgi membranes. AlF was added for 10 min or not. ArfGAP1-YFP in the Golgi region was then photo-bleached and recovery of Golgi fluorescence was measured over time.

Mentions: Addition of AlF to cells expressing ArfGAP1-YFP resulted in ∼40% of ArfGAP1-YFP on the Golgi becoming irreversibly bound, as assessed by photobleaching (Fig. 4 A). Because no stable Golgi pool of ArfGAP1-YFP was observed in untreated cells, we concluded that the irreversibly bound pool represented complexes of fluoride–Arf1–ArfGAP1. The size of the stabilized pool was similar to the ArfGAP1 pool populating the Arf1-dependent pathway, assessed by either BFA treatment or Arf1[Q71L] expression (Fig. 3 C and Fig. 2 D). This suggested that the complexes of fluoride–Arf1–ArfGAP1 were formed within the Arf1-dependent pathway.


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)

Golgi association–dissociation kinetics of ArfGAP1-YFP in AlF-treated cells. (A, C, E, and F) FRAP of Golgi fluorescence in NRK cells expressing respectively ArfGAP1-YFP (as a stable cell line) in A, ArfGAP1Δ64N-YFP (as a transient transfectant) in C, ɛCOP-YFP (as a stable cell line) in E, or GGA-YFP (as a transient transfectant) in F with or without AlF (50 μM AlCl3, 20 mM NaF) treatment for 10 min. (B) Release kinetics of ArfGAP1-YFP from Golgi membranes after addition of BFA (5 μg/ml−1) with or without AlF pretreatment for 10 min in NRK cells stably expressing ArfGAP1-YFP. (D) ldlF cells were transfected with ArfGAP1-YFP and incubated at 32°C or at 40°C for 3 h to deplete COPI from Golgi membranes. AlF was added for 10 min or not. ArfGAP1-YFP in the Golgi region was then photo-bleached and recovery of Golgi fluorescence was measured over time.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171832&req=5

fig4: Golgi association–dissociation kinetics of ArfGAP1-YFP in AlF-treated cells. (A, C, E, and F) FRAP of Golgi fluorescence in NRK cells expressing respectively ArfGAP1-YFP (as a stable cell line) in A, ArfGAP1Δ64N-YFP (as a transient transfectant) in C, ɛCOP-YFP (as a stable cell line) in E, or GGA-YFP (as a transient transfectant) in F with or without AlF (50 μM AlCl3, 20 mM NaF) treatment for 10 min. (B) Release kinetics of ArfGAP1-YFP from Golgi membranes after addition of BFA (5 μg/ml−1) with or without AlF pretreatment for 10 min in NRK cells stably expressing ArfGAP1-YFP. (D) ldlF cells were transfected with ArfGAP1-YFP and incubated at 32°C or at 40°C for 3 h to deplete COPI from Golgi membranes. AlF was added for 10 min or not. ArfGAP1-YFP in the Golgi region was then photo-bleached and recovery of Golgi fluorescence was measured over time.
Mentions: Addition of AlF to cells expressing ArfGAP1-YFP resulted in ∼40% of ArfGAP1-YFP on the Golgi becoming irreversibly bound, as assessed by photobleaching (Fig. 4 A). Because no stable Golgi pool of ArfGAP1-YFP was observed in untreated cells, we concluded that the irreversibly bound pool represented complexes of fluoride–Arf1–ArfGAP1. The size of the stabilized pool was similar to the ArfGAP1 pool populating the Arf1-dependent pathway, assessed by either BFA treatment or Arf1[Q71L] expression (Fig. 3 C and Fig. 2 D). This suggested that the complexes of fluoride–Arf1–ArfGAP1 were formed within the Arf1-dependent pathway.

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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