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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 in the vesicle budding assay and binding of ArfGAP1 to cytoplasmic domain peptides. (A and B) Vesicles were formed in the absence or presence of different p24 cytoplasmic domain antibodies. (A) Peptides were added at the following concentrations: 0.1 and 0.01 mM. The effect of each peptide was evaluated by SDS PAGE and Western blotting using specific antibodies and compared with control conditions (C) or to a mock control (HCl) containing the same amount of HCl as in experiments where peptides were added. (C and D) Synthetic peptides corresponding to the cytoplasmic domains of different p24 proteins and E19 and RER1 were attached to Sepharose beads and probed for their ability to recruit ArfGAP1 from cytosol or purified recombinant ArfGAP1. (C) Two different salt conditions were tested for 230 mM Na/40 mM KCl and 115 KOAc (KAc buffer). (D) High salt conditions were used (300 mM Na/90 mM KCl).
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fig6: Effect of peptides in the vesicle budding assay and binding of ArfGAP1 to cytoplasmic domain peptides. (A and B) Vesicles were formed in the absence or presence of different p24 cytoplasmic domain antibodies. (A) Peptides were added at the following concentrations: 0.1 and 0.01 mM. The effect of each peptide was evaluated by SDS PAGE and Western blotting using specific antibodies and compared with control conditions (C) or to a mock control (HCl) containing the same amount of HCl as in experiments where peptides were added. (C and D) Synthetic peptides corresponding to the cytoplasmic domains of different p24 proteins and E19 and RER1 were attached to Sepharose beads and probed for their ability to recruit ArfGAP1 from cytosol or purified recombinant ArfGAP1. (C) Two different salt conditions were tested for 230 mM Na/40 mM KCl and 115 KOAc (KAc buffer). (D) High salt conditions were used (300 mM Na/90 mM KCl).

Mentions: To test the functional consequence of the inhibition of GAP activity, we tested the effect of p24 peptides on sorting and vesicle formation in the vesicle-budding assay (Fig. 6 A). Vesicle formation was performed as above but in the presence of 0.1 or 0.01 mM p24δ1, β1, or α2 peptides. As controls, vesicle formation was performed in the absence of peptides (two GTP controls are shown) or in the presence of HCl, which was added to the same extent to the residual amounts present in the peptide solutions (of 0.1 mM solubilized peptides). Addition of 0.1 mM p24β1 cytoplasmic domain peptide gave rise to a strong decrease in p24α2, β1, δ1, and γ3. Incorporation of Mann II was decreased also, suggesting that the p24β1 peptide acted on ArfGAP1 regardless of whether vesicles formed from early or medial-Golgi membranes. A similar but less complete inhibition was seen with the p24δ1 peptide, whereas no inhibition was observed upon addition of the p24α2 peptide. At 0.01 mM concentration, the p24β1 cytoplasmic domain peptide gave rise to a similar decrease in p24 proteins, and Mann II recovered in the vesicle fraction, whereas now the inhibition observed with the p24δ1 peptide was less obvious. Selected p24β1 substitution peptides were also examined for their ability to affect sorting and vesicle formation (Fig. 6 B). Again, substituting FF or YL to AA removed the inhibitory effect seen with the p24β1 wild-type peptide. Furthermore, when using the EV to AA substitution peptide, only a minor inhibitory effect could be seen mirroring the partial activity seen when used in the liposome based assay (compare with Fig. 5 B). It is interesting to note that both the p24β1 wild-type peptide and the LK to AA substitution peptide showed strong inhibitory effect in the assay. The amount of coatomer (revealed by a mAb to the βCOP subunit) detected in the vesicle fraction was still comparable to that observed in the control (HCl but no peptide added), suggesting that vesicles still formed but that sorting had been affected. When adding the p24β1 and p24α2 wild-type peptides together, the amount of βCOP was reduced slightly, whereas sorting remained strongly inhibited. Taken together, this indicates that those p24β1 peptides that inhibit sorting in the vesicle budding assay and show inhibitory effects in ArfGAP1 activity assays (Fig. 5) do so by interacting with ArfGAP1 directly.


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 in the vesicle budding assay and binding of ArfGAP1 to cytoplasmic domain peptides. (A and B) Vesicles were formed in the absence or presence of different p24 cytoplasmic domain antibodies. (A) Peptides were added at the following concentrations: 0.1 and 0.01 mM. The effect of each peptide was evaluated by SDS PAGE and Western blotting using specific antibodies and compared with control conditions (C) or to a mock control (HCl) containing the same amount of HCl as in experiments where peptides were added. (C and D) Synthetic peptides corresponding to the cytoplasmic domains of different p24 proteins and E19 and RER1 were attached to Sepharose beads and probed for their ability to recruit ArfGAP1 from cytosol or purified recombinant ArfGAP1. (C) Two different salt conditions were tested for 230 mM Na/40 mM KCl and 115 KOAc (KAc buffer). (D) High salt conditions were used (300 mM Na/90 mM KCl).
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fig6: Effect of peptides in the vesicle budding assay and binding of ArfGAP1 to cytoplasmic domain peptides. (A and B) Vesicles were formed in the absence or presence of different p24 cytoplasmic domain antibodies. (A) Peptides were added at the following concentrations: 0.1 and 0.01 mM. The effect of each peptide was evaluated by SDS PAGE and Western blotting using specific antibodies and compared with control conditions (C) or to a mock control (HCl) containing the same amount of HCl as in experiments where peptides were added. (C and D) Synthetic peptides corresponding to the cytoplasmic domains of different p24 proteins and E19 and RER1 were attached to Sepharose beads and probed for their ability to recruit ArfGAP1 from cytosol or purified recombinant ArfGAP1. (C) Two different salt conditions were tested for 230 mM Na/40 mM KCl and 115 KOAc (KAc buffer). (D) High salt conditions were used (300 mM Na/90 mM KCl).
Mentions: To test the functional consequence of the inhibition of GAP activity, we tested the effect of p24 peptides on sorting and vesicle formation in the vesicle-budding assay (Fig. 6 A). Vesicle formation was performed as above but in the presence of 0.1 or 0.01 mM p24δ1, β1, or α2 peptides. As controls, vesicle formation was performed in the absence of peptides (two GTP controls are shown) or in the presence of HCl, which was added to the same extent to the residual amounts present in the peptide solutions (of 0.1 mM solubilized peptides). Addition of 0.1 mM p24β1 cytoplasmic domain peptide gave rise to a strong decrease in p24α2, β1, δ1, and γ3. Incorporation of Mann II was decreased also, suggesting that the p24β1 peptide acted on ArfGAP1 regardless of whether vesicles formed from early or medial-Golgi membranes. A similar but less complete inhibition was seen with the p24δ1 peptide, whereas no inhibition was observed upon addition of the p24α2 peptide. At 0.01 mM concentration, the p24β1 cytoplasmic domain peptide gave rise to a similar decrease in p24 proteins, and Mann II recovered in the vesicle fraction, whereas now the inhibition observed with the p24δ1 peptide was less obvious. Selected p24β1 substitution peptides were also examined for their ability to affect sorting and vesicle formation (Fig. 6 B). Again, substituting FF or YL to AA removed the inhibitory effect seen with the p24β1 wild-type peptide. Furthermore, when using the EV to AA substitution peptide, only a minor inhibitory effect could be seen mirroring the partial activity seen when used in the liposome based assay (compare with Fig. 5 B). It is interesting to note that both the p24β1 wild-type peptide and the LK to AA substitution peptide showed strong inhibitory effect in the assay. The amount of coatomer (revealed by a mAb to the βCOP subunit) detected in the vesicle fraction was still comparable to that observed in the control (HCl but no peptide added), suggesting that vesicles still formed but that sorting had been affected. When adding the p24β1 and p24α2 wild-type peptides together, the amount of βCOP was reduced slightly, whereas sorting remained strongly inhibited. Taken together, this indicates that those p24β1 peptides that inhibit sorting in the vesicle budding assay and show inhibitory effects in ArfGAP1 activity assays (Fig. 5) do so by interacting with ArfGAP1 directly.

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