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Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M, Nilsson T - J. Cell Biol. (2001)

Bottom Line: Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride.Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes.We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69017 Heidelberg, Germany.

ABSTRACT
We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

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GTP hydrolysis–driven sorting coupled with ArfGAP1 modulation. The model is divided into four steps from left to right and then from bottom to top. (I) GDP bound to Arf-1 attached to the membrane is replaced with GTP by Arf-1 guanine exchange factor. Cytosolic coatomer binds Arf-1GTP but is quickly released upon GTP hydrolysis by Arf-1. This step requires ArfGAP1 and is denoted in green as having a high activity (HA) on Arf-1. Resident cargo proteins bind coatomer directly through cytoplasmic domain motifs (e.g., K[X]KXX). In order for coatomer to capture resident proteins, coatomer first needs to be released from the membrane. This “sorting step” requires GTP hydrolysis by Arf-1. If resident cargo proteins are in addition induced to cluster (Weiss and Nilsson, 2000), coatomer is predicted to bind more strongly, thus favoring these over other resident proteins. (II) The activity of ArfGAP1 is downmodulated by captured resident cargo proteins. The rate of GTP hydrolysis by Arf-1 is as a consequence slowed down, enabling coatomer to remain on the membrane. The presence of resident cargo proteins promotes polymerization of the coat leading up to vesicle formation and budding (III). Though downmodulated in its activity, residual ArfGAP1 activity will permit Arf-1 to hydrolyze its GTP, and coatomer is released back into the cytosol. For simplicity, the release of coatomer, Arf-1, and ArfGAP1 from the vesicle is shown as one event.
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fig7: GTP hydrolysis–driven sorting coupled with ArfGAP1 modulation. The model is divided into four steps from left to right and then from bottom to top. (I) GDP bound to Arf-1 attached to the membrane is replaced with GTP by Arf-1 guanine exchange factor. Cytosolic coatomer binds Arf-1GTP but is quickly released upon GTP hydrolysis by Arf-1. This step requires ArfGAP1 and is denoted in green as having a high activity (HA) on Arf-1. Resident cargo proteins bind coatomer directly through cytoplasmic domain motifs (e.g., K[X]KXX). In order for coatomer to capture resident proteins, coatomer first needs to be released from the membrane. This “sorting step” requires GTP hydrolysis by Arf-1. If resident cargo proteins are in addition induced to cluster (Weiss and Nilsson, 2000), coatomer is predicted to bind more strongly, thus favoring these over other resident proteins. (II) The activity of ArfGAP1 is downmodulated by captured resident cargo proteins. The rate of GTP hydrolysis by Arf-1 is as a consequence slowed down, enabling coatomer to remain on the membrane. The presence of resident cargo proteins promotes polymerization of the coat leading up to vesicle formation and budding (III). Though downmodulated in its activity, residual ArfGAP1 activity will permit Arf-1 to hydrolyze its GTP, and coatomer is released back into the cytosol. For simplicity, the release of coatomer, Arf-1, and ArfGAP1 from the vesicle is shown as one event.

Mentions: The need for GTP hydrolysis by Arf-1 and a downregulation of ArfGAP1 activity is best explained in a two-stage mechanism (Fig. 7, I and II). First, release of coatomer is required for rebinding to membrane patches occupied by resident proteins. Coatomer units are predicted here to detach and rebind multiple times in order to capture the resident proteins. This process requires GTP hydrolysis by Arf-1 and full activity provided by ArfGAP1 (denoted in green and HA for high activity). Wherever coatomer units successfully capture resident proteins, ArfGAP1 activity is lowered as a consequence of cargo modulation (denoted in red and LA for low activity). We presented evidence in favor of a direct interaction between ArfGAP1 and cytoplasmic domain peptides of p24β1 (and to a lesser extent, p24δ1) and their ability to downmodulate ArfGAP1 activity on membranes in the absence of coatomer. However, we do not rule out an involvement of coatomer also in this process. Thus, the cytoplasmic domain of the resident protein is shown as interacting both with ArfGAP1 and with coatomer, and the two latter are also shown as interacting. A binding release cycle (Fig. 7 I) coupled with downmodulation of ArfGAP1 by resident cargo (Fig. 7 II) will lead to the enrichment of “priming complexes.” Polymerization of the COPI coat (Fig. 7 III) is then promoted by the presence of high concentration of resident proteins (Reinhard et al., 1999): at least four cytoplasmic domains of p24δ1/coatomer unit were required for optimal coat polymerization. The vesicle can then bud and although downmodulated, ArfGAP1 will stimulate GTP hydrolysis by Arf-1, resulting in coat release (Fig. 7 IV).


Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M, Nilsson T - J. Cell Biol. (2001)

GTP hydrolysis–driven sorting coupled with ArfGAP1 modulation. The model is divided into four steps from left to right and then from bottom to top. (I) GDP bound to Arf-1 attached to the membrane is replaced with GTP by Arf-1 guanine exchange factor. Cytosolic coatomer binds Arf-1GTP but is quickly released upon GTP hydrolysis by Arf-1. This step requires ArfGAP1 and is denoted in green as having a high activity (HA) on Arf-1. Resident cargo proteins bind coatomer directly through cytoplasmic domain motifs (e.g., K[X]KXX). In order for coatomer to capture resident proteins, coatomer first needs to be released from the membrane. This “sorting step” requires GTP hydrolysis by Arf-1. If resident cargo proteins are in addition induced to cluster (Weiss and Nilsson, 2000), coatomer is predicted to bind more strongly, thus favoring these over other resident proteins. (II) The activity of ArfGAP1 is downmodulated by captured resident cargo proteins. The rate of GTP hydrolysis by Arf-1 is as a consequence slowed down, enabling coatomer to remain on the membrane. The presence of resident cargo proteins promotes polymerization of the coat leading up to vesicle formation and budding (III). Though downmodulated in its activity, residual ArfGAP1 activity will permit Arf-1 to hydrolyze its GTP, and coatomer is released back into the cytosol. For simplicity, the release of coatomer, Arf-1, and ArfGAP1 from the vesicle is shown as one event.
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Related In: Results  -  Collection

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fig7: GTP hydrolysis–driven sorting coupled with ArfGAP1 modulation. The model is divided into four steps from left to right and then from bottom to top. (I) GDP bound to Arf-1 attached to the membrane is replaced with GTP by Arf-1 guanine exchange factor. Cytosolic coatomer binds Arf-1GTP but is quickly released upon GTP hydrolysis by Arf-1. This step requires ArfGAP1 and is denoted in green as having a high activity (HA) on Arf-1. Resident cargo proteins bind coatomer directly through cytoplasmic domain motifs (e.g., K[X]KXX). In order for coatomer to capture resident proteins, coatomer first needs to be released from the membrane. This “sorting step” requires GTP hydrolysis by Arf-1. If resident cargo proteins are in addition induced to cluster (Weiss and Nilsson, 2000), coatomer is predicted to bind more strongly, thus favoring these over other resident proteins. (II) The activity of ArfGAP1 is downmodulated by captured resident cargo proteins. The rate of GTP hydrolysis by Arf-1 is as a consequence slowed down, enabling coatomer to remain on the membrane. The presence of resident cargo proteins promotes polymerization of the coat leading up to vesicle formation and budding (III). Though downmodulated in its activity, residual ArfGAP1 activity will permit Arf-1 to hydrolyze its GTP, and coatomer is released back into the cytosol. For simplicity, the release of coatomer, Arf-1, and ArfGAP1 from the vesicle is shown as one event.
Mentions: The need for GTP hydrolysis by Arf-1 and a downregulation of ArfGAP1 activity is best explained in a two-stage mechanism (Fig. 7, I and II). First, release of coatomer is required for rebinding to membrane patches occupied by resident proteins. Coatomer units are predicted here to detach and rebind multiple times in order to capture the resident proteins. This process requires GTP hydrolysis by Arf-1 and full activity provided by ArfGAP1 (denoted in green and HA for high activity). Wherever coatomer units successfully capture resident proteins, ArfGAP1 activity is lowered as a consequence of cargo modulation (denoted in red and LA for low activity). We presented evidence in favor of a direct interaction between ArfGAP1 and cytoplasmic domain peptides of p24β1 (and to a lesser extent, p24δ1) and their ability to downmodulate ArfGAP1 activity on membranes in the absence of coatomer. However, we do not rule out an involvement of coatomer also in this process. Thus, the cytoplasmic domain of the resident protein is shown as interacting both with ArfGAP1 and with coatomer, and the two latter are also shown as interacting. A binding release cycle (Fig. 7 I) coupled with downmodulation of ArfGAP1 by resident cargo (Fig. 7 II) will lead to the enrichment of “priming complexes.” Polymerization of the COPI coat (Fig. 7 III) is then promoted by the presence of high concentration of resident proteins (Reinhard et al., 1999): at least four cytoplasmic domains of p24δ1/coatomer unit were required for optimal coat polymerization. The vesicle can then bud and although downmodulated, ArfGAP1 will stimulate GTP hydrolysis by Arf-1, resulting in coat release (Fig. 7 IV).

Bottom Line: Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride.Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes.We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69017 Heidelberg, Germany.

ABSTRACT
We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Show MeSH
Related in: MedlinePlus