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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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In vivo GAP activity of ArfGAP1-YFP. (A) Golgi and cytoplasmic distribution of ArfGAP1-YFP in a stable NRK cell line expressing ArfGAP1-YFP at low levels. (B) Golgi-associated pool of Arf1-CFP (expressed as a percentage of total cellular fluorescence) measured in NRK cells stably expressing Arf1-CFP alone (control) (n = 16) or after cotransfection with ArfGAP1-YFP (n = 16). Note that the steady-state Golgi pool of Arf1-CFP is reduced in cells coexpressing ArfGAP1-YFP. (C) Kinetics of dissociation of Arf1-CFP during BFA treatment (5 μg/ml−1) measured in NRK cells stably expressing Arf1-CFP alone (black Δ), with cotransfected ArfGAP1-YFP (red ○), or with cotransfected ArfGAP1Δ64N-YFP (green □). (D) NRK cells transiently expressing ArfGAP1-CFP and GalT-YFP were imaged 20 h after transfection. In cells with high levels of ArfGAP1-CFP expression (arrows), GalT-YFP was redistributed into the ER while ArfGAP1–YFP was found mainly in the cytoplasm. Bar, 5 μm.
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fig1: In vivo GAP activity of ArfGAP1-YFP. (A) Golgi and cytoplasmic distribution of ArfGAP1-YFP in a stable NRK cell line expressing ArfGAP1-YFP at low levels. (B) Golgi-associated pool of Arf1-CFP (expressed as a percentage of total cellular fluorescence) measured in NRK cells stably expressing Arf1-CFP alone (control) (n = 16) or after cotransfection with ArfGAP1-YFP (n = 16). Note that the steady-state Golgi pool of Arf1-CFP is reduced in cells coexpressing ArfGAP1-YFP. (C) Kinetics of dissociation of Arf1-CFP during BFA treatment (5 μg/ml−1) measured in NRK cells stably expressing Arf1-CFP alone (black Δ), with cotransfected ArfGAP1-YFP (red ○), or with cotransfected ArfGAP1Δ64N-YFP (green □). (D) NRK cells transiently expressing ArfGAP1-CFP and GalT-YFP were imaged 20 h after transfection. In cells with high levels of ArfGAP1-CFP expression (arrows), GalT-YFP was redistributed into the ER while ArfGAP1–YFP was found mainly in the cytoplasm. Bar, 5 μm.

Mentions: To study ArfGAP dynamics in living cells, we fused CFP or YFP to the carboxy terminus of ArfGAP1 (ArfGAP1-C/YFP) and generated a stable NRK cell line expressing the chimera at low levels. In these cells, ArfGAP1-YFP could be seen distributed on Golgi membranes (colocalizing with the Golgi marker, galactosyltransferase tagged with CFP) in the cytoplasm (Fig. 1 A) with approximately four times more ArfGAP1-YFP in the cytoplasm than on Golgi membranes. In addition to being present in the cytoplasm and on Golgi membranes, a small amount of ArfGAP1-YFP could be seen associated with peripheral structures containing ɛCOPI-YFP (i.e., ERGIC; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200410142/DC1). A similar ArfGAP1 distribution pattern has been reported by antibody labeling (Cukierman et al., 1995), indicating that ArfGAP1-YFP distributes in a similar manner to endogenous untagged ArfGAP1.


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)

In vivo GAP activity of ArfGAP1-YFP. (A) Golgi and cytoplasmic distribution of ArfGAP1-YFP in a stable NRK cell line expressing ArfGAP1-YFP at low levels. (B) Golgi-associated pool of Arf1-CFP (expressed as a percentage of total cellular fluorescence) measured in NRK cells stably expressing Arf1-CFP alone (control) (n = 16) or after cotransfection with ArfGAP1-YFP (n = 16). Note that the steady-state Golgi pool of Arf1-CFP is reduced in cells coexpressing ArfGAP1-YFP. (C) Kinetics of dissociation of Arf1-CFP during BFA treatment (5 μg/ml−1) measured in NRK cells stably expressing Arf1-CFP alone (black Δ), with cotransfected ArfGAP1-YFP (red ○), or with cotransfected ArfGAP1Δ64N-YFP (green □). (D) NRK cells transiently expressing ArfGAP1-CFP and GalT-YFP were imaged 20 h after transfection. In cells with high levels of ArfGAP1-CFP expression (arrows), GalT-YFP was redistributed into the ER while ArfGAP1–YFP was found mainly in the cytoplasm. Bar, 5 μm.
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Related In: Results  -  Collection

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fig1: In vivo GAP activity of ArfGAP1-YFP. (A) Golgi and cytoplasmic distribution of ArfGAP1-YFP in a stable NRK cell line expressing ArfGAP1-YFP at low levels. (B) Golgi-associated pool of Arf1-CFP (expressed as a percentage of total cellular fluorescence) measured in NRK cells stably expressing Arf1-CFP alone (control) (n = 16) or after cotransfection with ArfGAP1-YFP (n = 16). Note that the steady-state Golgi pool of Arf1-CFP is reduced in cells coexpressing ArfGAP1-YFP. (C) Kinetics of dissociation of Arf1-CFP during BFA treatment (5 μg/ml−1) measured in NRK cells stably expressing Arf1-CFP alone (black Δ), with cotransfected ArfGAP1-YFP (red ○), or with cotransfected ArfGAP1Δ64N-YFP (green □). (D) NRK cells transiently expressing ArfGAP1-CFP and GalT-YFP were imaged 20 h after transfection. In cells with high levels of ArfGAP1-CFP expression (arrows), GalT-YFP was redistributed into the ER while ArfGAP1–YFP was found mainly in the cytoplasm. Bar, 5 μm.
Mentions: To study ArfGAP dynamics in living cells, we fused CFP or YFP to the carboxy terminus of ArfGAP1 (ArfGAP1-C/YFP) and generated a stable NRK cell line expressing the chimera at low levels. In these cells, ArfGAP1-YFP could be seen distributed on Golgi membranes (colocalizing with the Golgi marker, galactosyltransferase tagged with CFP) in the cytoplasm (Fig. 1 A) with approximately four times more ArfGAP1-YFP in the cytoplasm than on Golgi membranes. In addition to being present in the cytoplasm and on Golgi membranes, a small amount of ArfGAP1-YFP could be seen associated with peripheral structures containing ɛCOPI-YFP (i.e., ERGIC; Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200410142/DC1). A similar ArfGAP1 distribution pattern has been reported by antibody labeling (Cukierman et al., 1995), indicating that ArfGAP1-YFP distributes in a similar manner to endogenous untagged ArfGAP1.

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