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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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Recruitment of coat proteins onto Golgi membranes during cargo transport through the Golgi. (A) COS-7 cells transiently expressing VSVG-CFP only, VSVG-CFP and ArfGAP1-YFP, or VSVG-CFP and ArfGAP1Δ64N-YFP were incubated at 40°C shortly after transfection to retain VSVG-CFP in the ER. Cells were imaged over time upon shift from 40°C to 32°C. Shown are the distributions at 0 and 30 min. To observe the change on Golgi-bound coatomer, COS-7 cells were infected with ts045 VSV and were cultured at 40°C for 4 h. Then the cells were shifted to 32°C for 30 min, fixed, and followed by immunofluorescence staining with anti–β-COP antibody and Alexa Fluor 594–tagged second antibody. (B) To assess changes in ArfGAP1 and coatomer association with Golgi membranes during VSVG transport, the total ArfGAP1-YFP (n = 6), ArfGAP1Δ64N-YFP (n = 6), or β-COP (n = 6) fluorescence associated with the Golgi was measured under nonsaturating conditions, expressed as a fraction of the total cellular fluorescence, and normalized to the initial zero minute value. (C) After shifting from 40°C to 32°C for 30 min, ArfGAP1-YFP in the Golgi region in cells expressing both VSVG-CFP and ArfGAP1-YFP were photobleached, and the fluorescence recovery into the bleached area was monitored over time. The observed rapid recovery of ArfGAP1-YFP fluorescence into the Golgi region indicated that ArfGAP1-YFP molecules continued to cycle between Golgi membranes and the cytoplasm during VSVG-CFP transport through this organelle. Bars, 5 μm.
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fig7: Recruitment of coat proteins onto Golgi membranes during cargo transport through the Golgi. (A) COS-7 cells transiently expressing VSVG-CFP only, VSVG-CFP and ArfGAP1-YFP, or VSVG-CFP and ArfGAP1Δ64N-YFP were incubated at 40°C shortly after transfection to retain VSVG-CFP in the ER. Cells were imaged over time upon shift from 40°C to 32°C. Shown are the distributions at 0 and 30 min. To observe the change on Golgi-bound coatomer, COS-7 cells were infected with ts045 VSV and were cultured at 40°C for 4 h. Then the cells were shifted to 32°C for 30 min, fixed, and followed by immunofluorescence staining with anti–β-COP antibody and Alexa Fluor 594–tagged second antibody. (B) To assess changes in ArfGAP1 and coatomer association with Golgi membranes during VSVG transport, the total ArfGAP1-YFP (n = 6), ArfGAP1Δ64N-YFP (n = 6), or β-COP (n = 6) fluorescence associated with the Golgi was measured under nonsaturating conditions, expressed as a fraction of the total cellular fluorescence, and normalized to the initial zero minute value. (C) After shifting from 40°C to 32°C for 30 min, ArfGAP1-YFP in the Golgi region in cells expressing both VSVG-CFP and ArfGAP1-YFP were photobleached, and the fluorescence recovery into the bleached area was monitored over time. The observed rapid recovery of ArfGAP1-YFP fluorescence into the Golgi region indicated that ArfGAP1-YFP molecules continued to cycle between Golgi membranes and the cytoplasm during VSVG-CFP transport through this organelle. Bars, 5 μm.

Mentions: To investigate this possibility, we visualized the behavior of ArfGAP1-YFP during release of a bolus of VSVG-CFP into the secretory pathway by temperature shift from 40°C to 32°C (Bergmann, 1989; Presley et al., 1997; Scales et al., 1997). Notably, a dramatic increase in the amount of Golgi-associated ArfGAP1-YFP was observed at the time when VSVG-CFP molecules passed through the Golgi apparatus, with Golgi-associated ArfGAP1-YFP levels more than doubling (Fig. 6, A and B). When VSVG-CFP molecules reached the plasma membrane, Golgi-associated ArfGAP1-YFP levels dropped back down to the levels observed before release of VSVG cargo from the ER (not depicted). The increased size of the Golgi pool of ArfGAP1-YFP was not a general effect of increased cargo transport through the Golgi, but was specific and dependent on ArfGAP1's ability to interact with Arf1 and coatomer. This was demonstrated in cells expressing ArfGAP1Δ64N-YFP, which showed no change in the size of their Golgi pool during VSVG-CFP transport through the Golgi (Fig. 7, A and B). This was further confirmed by a similar increase in Golgi membrane-bound β-COP in response to VSVG flowing into the Golgi (Fig. 7, A and B). When the Golgi pool of ArfGAP1-YFP was photobleached at the time when VSVG-CFP passed through the Golgi, rapid and complete recovery of Golgi fluorescence occurred (Fig. 7 C). This indicated that the additional ArfGAP1-YFP molecules recruited onto the Golgi during this time period were not stably associated but underwent continuous binding and release, as observed for ArfGAP1-YFP molecules normally associated with the Golgi (Fig. 2 A). Together, these results indicated that ArfGAP1 and coatomer levels on the Golgi are modulated in response to changes in secretory cargo transport through the Golgi. The modulation occurs as ArfGAP1 continuously cycles on and off Golgi membranes and is dependent on ArfGAP1's ability to interact with Arf1.


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)

Recruitment of coat proteins onto Golgi membranes during cargo transport through the Golgi. (A) COS-7 cells transiently expressing VSVG-CFP only, VSVG-CFP and ArfGAP1-YFP, or VSVG-CFP and ArfGAP1Δ64N-YFP were incubated at 40°C shortly after transfection to retain VSVG-CFP in the ER. Cells were imaged over time upon shift from 40°C to 32°C. Shown are the distributions at 0 and 30 min. To observe the change on Golgi-bound coatomer, COS-7 cells were infected with ts045 VSV and were cultured at 40°C for 4 h. Then the cells were shifted to 32°C for 30 min, fixed, and followed by immunofluorescence staining with anti–β-COP antibody and Alexa Fluor 594–tagged second antibody. (B) To assess changes in ArfGAP1 and coatomer association with Golgi membranes during VSVG transport, the total ArfGAP1-YFP (n = 6), ArfGAP1Δ64N-YFP (n = 6), or β-COP (n = 6) fluorescence associated with the Golgi was measured under nonsaturating conditions, expressed as a fraction of the total cellular fluorescence, and normalized to the initial zero minute value. (C) After shifting from 40°C to 32°C for 30 min, ArfGAP1-YFP in the Golgi region in cells expressing both VSVG-CFP and ArfGAP1-YFP were photobleached, and the fluorescence recovery into the bleached area was monitored over time. The observed rapid recovery of ArfGAP1-YFP fluorescence into the Golgi region indicated that ArfGAP1-YFP molecules continued to cycle between Golgi membranes and the cytoplasm during VSVG-CFP transport through this organelle. Bars, 5 μm.
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fig7: Recruitment of coat proteins onto Golgi membranes during cargo transport through the Golgi. (A) COS-7 cells transiently expressing VSVG-CFP only, VSVG-CFP and ArfGAP1-YFP, or VSVG-CFP and ArfGAP1Δ64N-YFP were incubated at 40°C shortly after transfection to retain VSVG-CFP in the ER. Cells were imaged over time upon shift from 40°C to 32°C. Shown are the distributions at 0 and 30 min. To observe the change on Golgi-bound coatomer, COS-7 cells were infected with ts045 VSV and were cultured at 40°C for 4 h. Then the cells were shifted to 32°C for 30 min, fixed, and followed by immunofluorescence staining with anti–β-COP antibody and Alexa Fluor 594–tagged second antibody. (B) To assess changes in ArfGAP1 and coatomer association with Golgi membranes during VSVG transport, the total ArfGAP1-YFP (n = 6), ArfGAP1Δ64N-YFP (n = 6), or β-COP (n = 6) fluorescence associated with the Golgi was measured under nonsaturating conditions, expressed as a fraction of the total cellular fluorescence, and normalized to the initial zero minute value. (C) After shifting from 40°C to 32°C for 30 min, ArfGAP1-YFP in the Golgi region in cells expressing both VSVG-CFP and ArfGAP1-YFP were photobleached, and the fluorescence recovery into the bleached area was monitored over time. The observed rapid recovery of ArfGAP1-YFP fluorescence into the Golgi region indicated that ArfGAP1-YFP molecules continued to cycle between Golgi membranes and the cytoplasm during VSVG-CFP transport through this organelle. Bars, 5 μm.
Mentions: To investigate this possibility, we visualized the behavior of ArfGAP1-YFP during release of a bolus of VSVG-CFP into the secretory pathway by temperature shift from 40°C to 32°C (Bergmann, 1989; Presley et al., 1997; Scales et al., 1997). Notably, a dramatic increase in the amount of Golgi-associated ArfGAP1-YFP was observed at the time when VSVG-CFP molecules passed through the Golgi apparatus, with Golgi-associated ArfGAP1-YFP levels more than doubling (Fig. 6, A and B). When VSVG-CFP molecules reached the plasma membrane, Golgi-associated ArfGAP1-YFP levels dropped back down to the levels observed before release of VSVG cargo from the ER (not depicted). The increased size of the Golgi pool of ArfGAP1-YFP was not a general effect of increased cargo transport through the Golgi, but was specific and dependent on ArfGAP1's ability to interact with Arf1 and coatomer. This was demonstrated in cells expressing ArfGAP1Δ64N-YFP, which showed no change in the size of their Golgi pool during VSVG-CFP transport through the Golgi (Fig. 7, A and B). This was further confirmed by a similar increase in Golgi membrane-bound β-COP in response to VSVG flowing into the Golgi (Fig. 7, A and B). When the Golgi pool of ArfGAP1-YFP was photobleached at the time when VSVG-CFP passed through the Golgi, rapid and complete recovery of Golgi fluorescence occurred (Fig. 7 C). This indicated that the additional ArfGAP1-YFP molecules recruited onto the Golgi during this time period were not stably associated but underwent continuous binding and release, as observed for ArfGAP1-YFP molecules normally associated with the Golgi (Fig. 2 A). Together, these results indicated that ArfGAP1 and coatomer levels on the Golgi are modulated in response to changes in secretory cargo transport through the Golgi. The modulation occurs as ArfGAP1 continuously cycles on and off Golgi membranes and is dependent on ArfGAP1's ability to interact with Arf1.

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