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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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Effect of peptides derived from p24 proteins on ArfGAP1 activity. (A and B) Activity was assayed on Arf-1 preloaded with [γ-32P]GTP in the presence of liposomes and in the absence or presence of 0.2 mM of respective peptides, and the release of [γ-32P] was monitored. (C and D) Activity was assayed on Golgi membrane-bound Arf-1 preloaded with [γ-32P]GTP. At time zero, 50 nM ArfGAP1 were added to each incubation except to the control, in the presence or absence of peptides at a concentration of 0.2 mM unless otherwise indicated. At different time points, the amount of [γ-32P]GTP, which remained bound to membranes, was determined.
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fig5: Effect of peptides derived from p24 proteins on ArfGAP1 activity. (A and B) Activity was assayed on Arf-1 preloaded with [γ-32P]GTP in the presence of liposomes and in the absence or presence of 0.2 mM of respective peptides, and the release of [γ-32P] was monitored. (C and D) Activity was assayed on Golgi membrane-bound Arf-1 preloaded with [γ-32P]GTP. At time zero, 50 nM ArfGAP1 were added to each incubation except to the control, in the presence or absence of peptides at a concentration of 0.2 mM unless otherwise indicated. At different time points, the amount of [γ-32P]GTP, which remained bound to membranes, was determined.

Mentions: Since GTP hydrolysis by Arf-1 is required, the observed inhibition by BeFx and AlFx suggests a possible role for ArfGAP1, a Golgi-localized Arf GAP, in the sorting process. How is this then reconciled with the proposed need for an inhibition of GAP activity (and hence GTP hydrolysis) to form priming complexes (Springer et al., 1999)? Since evidence exists in favor of cargo modulation of ArfGAP1 activity by the cytoplasmic domain of p24β1, we tested for the ability of p24 cytoplasmic domain peptides to inhibit ArfGAP1 activity. In solution using Δ17Arf-1 in the presence of coatomer, we observed a strong inhibition in the presence of the p24β1 peptide but not with the p24δ1 peptide, essentially confirming previous observations (Goldberg, 2000; unpublished data). When using full-length myristoylated Arf-1 bound to phospholipid vesicles, coatomer stimulation of GAP activity is not observed (Szafer et al., 2000), whereas on Golgi membranes coatomer approximately doubles ArfGAP1 activity (Szafer et al., 2001). Therefore, it was of interest to examine the effect of p24 peptides under conditions that more closely resemble those prevailing in the cells. The result of an experiment where GAP activity on Arf-1 was monitored on liposomes in the presence of 0.2 mM p24α2, p24β1, or p24δ1 cytoplasmic domain peptides is shown in Fig. 5 A. As can be seen, addition of the p24α2 peptide had little if any effect on GAP activity, whereas both p24β1 and p24δ1 caused inhibition. Strongest inhibition was obtained with the p24β1 peptide. To examine which amino acid residues in the p24β1 peptide are important for inhibitory effect, we substituted two amino acids at the time throughout the cytoplasmic domain (Table I). The effect of each peptide except for the p24β1 (RR) peptide, which could not be solubilized, is shown in Fig. 5 B. Strikingly, the p24β1 (FF) peptide showed no inhibitory effect. Similarly, the p24β1 (YL) peptide showed only a minor inhibitory effect, whereas p24β1 (EV) revealed partial inhibition. No loss of inhibitory effect was seen with p24β1 LK, VV, and the +A peptide, which contained an additional alanine residue at the COOH terminus. A similar pattern was observed when using Golgi membranes in place of the liposomes (Fig. 5 C). In this experiment, Golgi membranes were preincubated with myristoylated Arf-1 and γ32-P–GTP to allow for the binding of the nucleotide to Arf-1. Nucleotide exchange was then stopped by the addition of BFA followed by addition of ArfGAP1. The decrease in the amount of membrane-bound GTP was monitored as a measure for GTP hydrolysis on Arf-1 (Szafer et al., 2001). A strong inhibition was obtained using the p24β1 peptide at 0.2 mM. Under these conditions, neither p24α2 nor p24δ1 revealed significant inhibition (unpublished data), suggesting that the inhibition on Golgi membranes was p24β1 specific. We also examined selected p24β1 substitution peptides. As seen with liposomes, substituting YL or FF to AA resulted in a loss of inhibitory activity (Fig. 5 D). Again, the p24β1 (LK) peptide was comparable to the p24β1 wild-type peptide and showed strong inhibition.


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)

Effect of peptides derived from p24 proteins on ArfGAP1 activity. (A and B) Activity was assayed on Arf-1 preloaded with [γ-32P]GTP in the presence of liposomes and in the absence or presence of 0.2 mM of respective peptides, and the release of [γ-32P] was monitored. (C and D) Activity was assayed on Golgi membrane-bound Arf-1 preloaded with [γ-32P]GTP. At time zero, 50 nM ArfGAP1 were added to each incubation except to the control, in the presence or absence of peptides at a concentration of 0.2 mM unless otherwise indicated. At different time points, the amount of [γ-32P]GTP, which remained bound to membranes, was determined.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199348&req=5

fig5: Effect of peptides derived from p24 proteins on ArfGAP1 activity. (A and B) Activity was assayed on Arf-1 preloaded with [γ-32P]GTP in the presence of liposomes and in the absence or presence of 0.2 mM of respective peptides, and the release of [γ-32P] was monitored. (C and D) Activity was assayed on Golgi membrane-bound Arf-1 preloaded with [γ-32P]GTP. At time zero, 50 nM ArfGAP1 were added to each incubation except to the control, in the presence or absence of peptides at a concentration of 0.2 mM unless otherwise indicated. At different time points, the amount of [γ-32P]GTP, which remained bound to membranes, was determined.
Mentions: Since GTP hydrolysis by Arf-1 is required, the observed inhibition by BeFx and AlFx suggests a possible role for ArfGAP1, a Golgi-localized Arf GAP, in the sorting process. How is this then reconciled with the proposed need for an inhibition of GAP activity (and hence GTP hydrolysis) to form priming complexes (Springer et al., 1999)? Since evidence exists in favor of cargo modulation of ArfGAP1 activity by the cytoplasmic domain of p24β1, we tested for the ability of p24 cytoplasmic domain peptides to inhibit ArfGAP1 activity. In solution using Δ17Arf-1 in the presence of coatomer, we observed a strong inhibition in the presence of the p24β1 peptide but not with the p24δ1 peptide, essentially confirming previous observations (Goldberg, 2000; unpublished data). When using full-length myristoylated Arf-1 bound to phospholipid vesicles, coatomer stimulation of GAP activity is not observed (Szafer et al., 2000), whereas on Golgi membranes coatomer approximately doubles ArfGAP1 activity (Szafer et al., 2001). Therefore, it was of interest to examine the effect of p24 peptides under conditions that more closely resemble those prevailing in the cells. The result of an experiment where GAP activity on Arf-1 was monitored on liposomes in the presence of 0.2 mM p24α2, p24β1, or p24δ1 cytoplasmic domain peptides is shown in Fig. 5 A. As can be seen, addition of the p24α2 peptide had little if any effect on GAP activity, whereas both p24β1 and p24δ1 caused inhibition. Strongest inhibition was obtained with the p24β1 peptide. To examine which amino acid residues in the p24β1 peptide are important for inhibitory effect, we substituted two amino acids at the time throughout the cytoplasmic domain (Table I). The effect of each peptide except for the p24β1 (RR) peptide, which could not be solubilized, is shown in Fig. 5 B. Strikingly, the p24β1 (FF) peptide showed no inhibitory effect. Similarly, the p24β1 (YL) peptide showed only a minor inhibitory effect, whereas p24β1 (EV) revealed partial inhibition. No loss of inhibitory effect was seen with p24β1 LK, VV, and the +A peptide, which contained an additional alanine residue at the COOH terminus. A similar pattern was observed when using Golgi membranes in place of the liposomes (Fig. 5 C). In this experiment, Golgi membranes were preincubated with myristoylated Arf-1 and γ32-P–GTP to allow for the binding of the nucleotide to Arf-1. Nucleotide exchange was then stopped by the addition of BFA followed by addition of ArfGAP1. The decrease in the amount of membrane-bound GTP was monitored as a measure for GTP hydrolysis on Arf-1 (Szafer et al., 2001). A strong inhibition was obtained using the p24β1 peptide at 0.2 mM. Under these conditions, neither p24α2 nor p24δ1 revealed significant inhibition (unpublished data), suggesting that the inhibition on Golgi membranes was p24β1 specific. We also examined selected p24β1 substitution peptides. As seen with liposomes, substituting YL or FF to AA resulted in a loss of inhibitory activity (Fig. 5 D). Again, the p24β1 (LK) peptide was comparable to the p24β1 wild-type peptide and showed strong inhibition.

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