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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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Kinetics of ArfGAP1 binding to and dissociation from Golgi membranes. (A and C) NRK cells stably expressing ArfGAP1-YFP in A or transiently expressing ArfGAP1Δ64N-YFP in C were imaged before (prebleach) and after photobleaching the Golgi region (outlined in red) with high intensity laser light. Note the rapid fluorescence recovery into the Golgi. (B) Repeated photobleaching (FLIP) of the cytoplasm defined by the area between the two red lines caused all Golgi fluorescence within an NRK cell expressing ArfGAP1-YFP to disappear over time. (D) Quantification of the FRAP experiment from A and C, as well as from a similar FRAP experiment in NRK cells stably expressing ArfGAP1-YFP in which an Arf1[Q71L] plasmid was microinjected 9 h before microscopy. Golgi fluorescence in this as well as all other FRAP and BFA experiments was represented as the ratio of Golgi-to-total cell fluorescence divided by the initial ratio. Bars, 5 μm.
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fig2: Kinetics of ArfGAP1 binding to and dissociation from Golgi membranes. (A and C) NRK cells stably expressing ArfGAP1-YFP in A or transiently expressing ArfGAP1Δ64N-YFP in C were imaged before (prebleach) and after photobleaching the Golgi region (outlined in red) with high intensity laser light. Note the rapid fluorescence recovery into the Golgi. (B) Repeated photobleaching (FLIP) of the cytoplasm defined by the area between the two red lines caused all Golgi fluorescence within an NRK cell expressing ArfGAP1-YFP to disappear over time. (D) Quantification of the FRAP experiment from A and C, as well as from a similar FRAP experiment in NRK cells stably expressing ArfGAP1-YFP in which an Arf1[Q71L] plasmid was microinjected 9 h before microscopy. Golgi fluorescence in this as well as all other FRAP and BFA experiments was represented as the ratio of Golgi-to-total cell fluorescence divided by the initial ratio. Bars, 5 μm.

Mentions: Given that ArfGAP1-YFP appropriately targets and functions within cells, we used the chimera in experiments aimed at investigating the kinetic properties of ArfGAP1's association with Golgi membranes. These and subsequent experiments were performed in the stable NRK cell line that expresses ArfGAP1-YFP at low levels (2.5-fold above endogenous ArfGAP1), unless indicated otherwise. We first addressed whether ArfGAP1 resides stably or only transiently after being recruited to Golgi membranes. This was accomplished using FRAP. Upon photobleaching the Golgi pool of fluorescence, we observed rapid recovery from the nonbleached cytoplasmic pool, with the original, prebleach fraction of Golgi fluorescence (i.e., 20% of the total cellular pool) completely restored in just under 1 min (Fig. 2, A and D). As ArfGAP1-YFP in the cytoplasm recovered into a photobleached box with a half-time of 1 s (not depicted), the recovery rate of ArfGAP1-YFP observed after photobleaching the Golgi was determined primarily by membrane association–dissociation processes (and was not limited by ArfGAP1-YFP diffusion through the cytoplasm). We concluded, therefore, that Golgi-associated ArfGAP1-YFP molecules undergo rapid exchange with freely diffusing ArfGAP1-YFP molecules in the cytoplasm, and that an individual ArfGAP1-YFP molecule spends only a short period bound to Golgi membranes. Evidence that all Golgi-associated ArfGAP1-YFP molecules are dynamically associated with the Golgi in this manner was suggested by the fact that when the cytoplasm was repeatedly photobleached using the technique of fluorescence loss in photobleaching (FLIP; for review see Lippincott-Schwartz et al., 2001), virtually all Golgi-associated ArfGAP1-YFP fluorescence was lost over time (Fig. 2 B, FLIP). Hence, ArfGAP1 resides only transiently after being recruited 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)

Kinetics of ArfGAP1 binding to and dissociation from Golgi membranes. (A and C) NRK cells stably expressing ArfGAP1-YFP in A or transiently expressing ArfGAP1Δ64N-YFP in C were imaged before (prebleach) and after photobleaching the Golgi region (outlined in red) with high intensity laser light. Note the rapid fluorescence recovery into the Golgi. (B) Repeated photobleaching (FLIP) of the cytoplasm defined by the area between the two red lines caused all Golgi fluorescence within an NRK cell expressing ArfGAP1-YFP to disappear over time. (D) Quantification of the FRAP experiment from A and C, as well as from a similar FRAP experiment in NRK cells stably expressing ArfGAP1-YFP in which an Arf1[Q71L] plasmid was microinjected 9 h before microscopy. Golgi fluorescence in this as well as all other FRAP and BFA experiments was represented as the ratio of Golgi-to-total cell fluorescence divided by the initial ratio. Bars, 5 μm.
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Related In: Results  -  Collection

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fig2: Kinetics of ArfGAP1 binding to and dissociation from Golgi membranes. (A and C) NRK cells stably expressing ArfGAP1-YFP in A or transiently expressing ArfGAP1Δ64N-YFP in C were imaged before (prebleach) and after photobleaching the Golgi region (outlined in red) with high intensity laser light. Note the rapid fluorescence recovery into the Golgi. (B) Repeated photobleaching (FLIP) of the cytoplasm defined by the area between the two red lines caused all Golgi fluorescence within an NRK cell expressing ArfGAP1-YFP to disappear over time. (D) Quantification of the FRAP experiment from A and C, as well as from a similar FRAP experiment in NRK cells stably expressing ArfGAP1-YFP in which an Arf1[Q71L] plasmid was microinjected 9 h before microscopy. Golgi fluorescence in this as well as all other FRAP and BFA experiments was represented as the ratio of Golgi-to-total cell fluorescence divided by the initial ratio. Bars, 5 μm.
Mentions: Given that ArfGAP1-YFP appropriately targets and functions within cells, we used the chimera in experiments aimed at investigating the kinetic properties of ArfGAP1's association with Golgi membranes. These and subsequent experiments were performed in the stable NRK cell line that expresses ArfGAP1-YFP at low levels (2.5-fold above endogenous ArfGAP1), unless indicated otherwise. We first addressed whether ArfGAP1 resides stably or only transiently after being recruited to Golgi membranes. This was accomplished using FRAP. Upon photobleaching the Golgi pool of fluorescence, we observed rapid recovery from the nonbleached cytoplasmic pool, with the original, prebleach fraction of Golgi fluorescence (i.e., 20% of the total cellular pool) completely restored in just under 1 min (Fig. 2, A and D). As ArfGAP1-YFP in the cytoplasm recovered into a photobleached box with a half-time of 1 s (not depicted), the recovery rate of ArfGAP1-YFP observed after photobleaching the Golgi was determined primarily by membrane association–dissociation processes (and was not limited by ArfGAP1-YFP diffusion through the cytoplasm). We concluded, therefore, that Golgi-associated ArfGAP1-YFP molecules undergo rapid exchange with freely diffusing ArfGAP1-YFP molecules in the cytoplasm, and that an individual ArfGAP1-YFP molecule spends only a short period bound to Golgi membranes. Evidence that all Golgi-associated ArfGAP1-YFP molecules are dynamically associated with the Golgi in this manner was suggested by the fact that when the cytoplasm was repeatedly photobleached using the technique of fluorescence loss in photobleaching (FLIP; for review see Lippincott-Schwartz et al., 2001), virtually all Golgi-associated ArfGAP1-YFP fluorescence was lost over time (Fig. 2 B, FLIP). Hence, ArfGAP1 resides only transiently after being recruited 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