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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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ArfGAP1 follows both Arf1-dependent and –independent pathways on Golgi membrane. (A) Scheme for ArfGAP1 membrane binding and dissociation pathways. (B) Release of Arf1-CFP, ArfGAP1-YFP, and ArfGAP1Δ64N-YFP from Golgi membranes after BFA addition (5 μg/ml−1) in NRK cells stably expressing Arf1-CFP and cotransfected with ArfGAP1-YFP or ARFGAP1Δ64N-YFP. Notice the differences in the rates and extents of release of the three proteins from the Golgi during the BFA treatment. Bars, 5 μm. (C) Quantification of the release kinetics shown in B. (D) After 300 s of BFA treatment, the persisting Golgi pool of ArfGAP1-YFP in an NRK cell such as that shown in B, was photobleached and recovery into the Golgi region was quantified. The results indicated that the BFA-resistant Golgi pool of ArfGAP1-YFP continues to cycle on and off Golgi membranes before Golgi disassembly (which occurs sometime after 600 s in BFA).
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fig3: ArfGAP1 follows both Arf1-dependent and –independent pathways on Golgi membrane. (A) Scheme for ArfGAP1 membrane binding and dissociation pathways. (B) Release of Arf1-CFP, ArfGAP1-YFP, and ArfGAP1Δ64N-YFP from Golgi membranes after BFA addition (5 μg/ml−1) in NRK cells stably expressing Arf1-CFP and cotransfected with ArfGAP1-YFP or ARFGAP1Δ64N-YFP. Notice the differences in the rates and extents of release of the three proteins from the Golgi during the BFA treatment. Bars, 5 μm. (C) Quantification of the release kinetics shown in B. (D) After 300 s of BFA treatment, the persisting Golgi pool of ArfGAP1-YFP in an NRK cell such as that shown in B, was photobleached and recovery into the Golgi region was quantified. The results indicated that the BFA-resistant Golgi pool of ArfGAP1-YFP continues to cycle on and off Golgi membranes before Golgi disassembly (which occurs sometime after 600 s in BFA).

Mentions: The above results suggested that ArfGAP1 is recruited to membranes in an Arf1-independent fashion, but once on membranes, its fate differs depending on whether or not it interacts with Arf1 (Fig. 3 A, scheme). With no such interaction, ArfGAP1 releases from membranes via an Arf1-independent pathway, whereas with such an interaction, ArfGAP1 releases via an Arf1-dependent pathway that requires GTP hydrolysis on Arf1. Within either pathway, ArfGAP1 resides on membranes for only short periods (∼30 s) before releasing into the cytoplasm, whereupon it mixes with other ArfGAP1 molecules and then rebinds to Golgi membranes.


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)

ArfGAP1 follows both Arf1-dependent and –independent pathways on Golgi membrane. (A) Scheme for ArfGAP1 membrane binding and dissociation pathways. (B) Release of Arf1-CFP, ArfGAP1-YFP, and ArfGAP1Δ64N-YFP from Golgi membranes after BFA addition (5 μg/ml−1) in NRK cells stably expressing Arf1-CFP and cotransfected with ArfGAP1-YFP or ARFGAP1Δ64N-YFP. Notice the differences in the rates and extents of release of the three proteins from the Golgi during the BFA treatment. Bars, 5 μm. (C) Quantification of the release kinetics shown in B. (D) After 300 s of BFA treatment, the persisting Golgi pool of ArfGAP1-YFP in an NRK cell such as that shown in B, was photobleached and recovery into the Golgi region was quantified. The results indicated that the BFA-resistant Golgi pool of ArfGAP1-YFP continues to cycle on and off Golgi membranes before Golgi disassembly (which occurs sometime after 600 s in BFA).
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Related In: Results  -  Collection

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

fig3: ArfGAP1 follows both Arf1-dependent and –independent pathways on Golgi membrane. (A) Scheme for ArfGAP1 membrane binding and dissociation pathways. (B) Release of Arf1-CFP, ArfGAP1-YFP, and ArfGAP1Δ64N-YFP from Golgi membranes after BFA addition (5 μg/ml−1) in NRK cells stably expressing Arf1-CFP and cotransfected with ArfGAP1-YFP or ARFGAP1Δ64N-YFP. Notice the differences in the rates and extents of release of the three proteins from the Golgi during the BFA treatment. Bars, 5 μm. (C) Quantification of the release kinetics shown in B. (D) After 300 s of BFA treatment, the persisting Golgi pool of ArfGAP1-YFP in an NRK cell such as that shown in B, was photobleached and recovery into the Golgi region was quantified. The results indicated that the BFA-resistant Golgi pool of ArfGAP1-YFP continues to cycle on and off Golgi membranes before Golgi disassembly (which occurs sometime after 600 s in BFA).
Mentions: The above results suggested that ArfGAP1 is recruited to membranes in an Arf1-independent fashion, but once on membranes, its fate differs depending on whether or not it interacts with Arf1 (Fig. 3 A, scheme). With no such interaction, ArfGAP1 releases from membranes via an Arf1-independent pathway, whereas with such an interaction, ArfGAP1 releases via an Arf1-dependent pathway that requires GTP hydrolysis on Arf1. Within either pathway, ArfGAP1 resides on membranes for only short periods (∼30 s) before releasing into the cytoplasm, whereupon it mixes with other ArfGAP1 molecules and then rebinds to Golgi membranes.

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
Related in: MedlinePlus