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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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Proposed model for ArfGAP1's behavior on Golgi membranes and its role in coat lattice assembly. Upon binding to membranes, ArfGAP1 follows either an Arf1-independent pathway that is nonproductive for coat assembly or an Arf1-dependent pathway that is productive for coat assembly. ArfGAP1 molecules following either pathway persist on Golgi membranes for only short periods. Recruitment of ArfGAP1 into the Arf1-dependent pathway is mediated by coatomer, which by forming a ternary complex with ArfGAP1 and Arf1 generates the basic coat unit. These units diffuse in the membrane and nucleate into small aggregates. Upon assembly of the aggregates into basket-like lattices, the underlying membrane begins to bend, which stimulates ArfGAP1 activity (Bigay et al., 2003). This leads to the release of Arf1 from the lattice, with ArfGAP1 and coatomer becoming destabilized and releasing soon thereafter. Hence, there is a constant flux of Arf1, coatomer, and ArfGAP1 through the lattice during the assembly of the lattice whether or not the lattice pinches off as a coated vesicle.
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fig8: Proposed model for ArfGAP1's behavior on Golgi membranes and its role in coat lattice assembly. Upon binding to membranes, ArfGAP1 follows either an Arf1-independent pathway that is nonproductive for coat assembly or an Arf1-dependent pathway that is productive for coat assembly. ArfGAP1 molecules following either pathway persist on Golgi membranes for only short periods. Recruitment of ArfGAP1 into the Arf1-dependent pathway is mediated by coatomer, which by forming a ternary complex with ArfGAP1 and Arf1 generates the basic coat unit. These units diffuse in the membrane and nucleate into small aggregates. Upon assembly of the aggregates into basket-like lattices, the underlying membrane begins to bend, which stimulates ArfGAP1 activity (Bigay et al., 2003). This leads to the release of Arf1 from the lattice, with ArfGAP1 and coatomer becoming destabilized and releasing soon thereafter. Hence, there is a constant flux of Arf1, coatomer, and ArfGAP1 through the lattice during the assembly of the lattice whether or not the lattice pinches off as a coated vesicle.

Mentions: In this study, we have used fluorescence imaging and FRAP techniques to characterize the membrane association–dissociation kinetics of ArfGAP1 in single living cells. Building on models from in vitro reconstitution systems, we have then asked what role these kinetics play in the assembly and disassembly of the COPI coat lattice on Golgi membranes. We found that ArfGAP1 underwent fast exchange between Golgi membranes and cytosol. Once bound to membranes, ArfGAP1 followed either an Arf1-dependent pathway (productive for coat lattice assembly) or was released directly into the cytoplasm in an Arf1-independent manner (nonproductive for coat lattice assembly; Fig. 8, model). The extent of ArfGAP1's cytosol/Golgi exchange could be modulated by secretory cargo load, was independent of vesicle budding, and could be blocked in a coatomer-dependent fashion when Arf1 was permanently activated. These findings lead us to a model, discussed below, in which ArfGAP1, coatomer, and Arf1 play interdependent roles in the assembly–disassembly cycle of the COPI coat.


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)

Proposed model for ArfGAP1's behavior on Golgi membranes and its role in coat lattice assembly. Upon binding to membranes, ArfGAP1 follows either an Arf1-independent pathway that is nonproductive for coat assembly or an Arf1-dependent pathway that is productive for coat assembly. ArfGAP1 molecules following either pathway persist on Golgi membranes for only short periods. Recruitment of ArfGAP1 into the Arf1-dependent pathway is mediated by coatomer, which by forming a ternary complex with ArfGAP1 and Arf1 generates the basic coat unit. These units diffuse in the membrane and nucleate into small aggregates. Upon assembly of the aggregates into basket-like lattices, the underlying membrane begins to bend, which stimulates ArfGAP1 activity (Bigay et al., 2003). This leads to the release of Arf1 from the lattice, with ArfGAP1 and coatomer becoming destabilized and releasing soon thereafter. Hence, there is a constant flux of Arf1, coatomer, and ArfGAP1 through the lattice during the assembly of the lattice whether or not the lattice pinches off as a coated vesicle.
© Copyright Policy
Related In: Results  -  Collection

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

fig8: Proposed model for ArfGAP1's behavior on Golgi membranes and its role in coat lattice assembly. Upon binding to membranes, ArfGAP1 follows either an Arf1-independent pathway that is nonproductive for coat assembly or an Arf1-dependent pathway that is productive for coat assembly. ArfGAP1 molecules following either pathway persist on Golgi membranes for only short periods. Recruitment of ArfGAP1 into the Arf1-dependent pathway is mediated by coatomer, which by forming a ternary complex with ArfGAP1 and Arf1 generates the basic coat unit. These units diffuse in the membrane and nucleate into small aggregates. Upon assembly of the aggregates into basket-like lattices, the underlying membrane begins to bend, which stimulates ArfGAP1 activity (Bigay et al., 2003). This leads to the release of Arf1 from the lattice, with ArfGAP1 and coatomer becoming destabilized and releasing soon thereafter. Hence, there is a constant flux of Arf1, coatomer, and ArfGAP1 through the lattice during the assembly of the lattice whether or not the lattice pinches off as a coated vesicle.
Mentions: In this study, we have used fluorescence imaging and FRAP techniques to characterize the membrane association–dissociation kinetics of ArfGAP1 in single living cells. Building on models from in vitro reconstitution systems, we have then asked what role these kinetics play in the assembly and disassembly of the COPI coat lattice on Golgi membranes. We found that ArfGAP1 underwent fast exchange between Golgi membranes and cytosol. Once bound to membranes, ArfGAP1 followed either an Arf1-dependent pathway (productive for coat lattice assembly) or was released directly into the cytoplasm in an Arf1-independent manner (nonproductive for coat lattice assembly; Fig. 8, model). The extent of ArfGAP1's cytosol/Golgi exchange could be modulated by secretory cargo load, was independent of vesicle budding, and could be blocked in a coatomer-dependent fashion when Arf1 was permanently activated. These findings lead us to a model, discussed below, in which ArfGAP1, coatomer, and Arf1 play interdependent roles in the assembly–disassembly cycle of the COPI coat.

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