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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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Immunoisolation of p24-containing vesicles reveals subpopulations of COPI vesicles. (A–D) Magnetic beads precoated with either an irrelevant control antibody (Ctr) or specific (Sp) antibodies to the cytoplasmic domain of p24γ3 were incubated with nonwashed Golgi membranes (A, left, and B) or with COPI vesicles formed from such membranes in the presence of the α-SNAPdn mutant (A, right, and C and D). Isolated membranes and vesicles were pelleted, fixed, embedded, and processed for EM or solubilized and subjected to SDS-PAGE. After transfer to nitrocellulose, Mann II, p24γ3, p24α2, p24δ1, and p24β1 were detected using specific antibodies followed by ECL. Vesicles remaining in the supernatant (Supn) after immunoisolation were also monitored in terms of their protein content (C, left lanes). (D) Immunoisolated vesicles were probed for the anterograde cargo marker, RSA, and compared with p24γ3, p24α2, p24δ1, and p24β1. Lanes containing total Golgi (Gt) and 20% of total vesicles (Vtot) used for immunoisolation are included for comparison.
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fig2: Immunoisolation of p24-containing vesicles reveals subpopulations of COPI vesicles. (A–D) Magnetic beads precoated with either an irrelevant control antibody (Ctr) or specific (Sp) antibodies to the cytoplasmic domain of p24γ3 were incubated with nonwashed Golgi membranes (A, left, and B) or with COPI vesicles formed from such membranes in the presence of the α-SNAPdn mutant (A, right, and C and D). Isolated membranes and vesicles were pelleted, fixed, embedded, and processed for EM or solubilized and subjected to SDS-PAGE. After transfer to nitrocellulose, Mann II, p24γ3, p24α2, p24δ1, and p24β1 were detected using specific antibodies followed by ECL. Vesicles remaining in the supernatant (Supn) after immunoisolation were also monitored in terms of their protein content (C, left lanes). (D) Immunoisolated vesicles were probed for the anterograde cargo marker, RSA, and compared with p24γ3, p24α2, p24δ1, and p24β1. Lanes containing total Golgi (Gt) and 20% of total vesicles (Vtot) used for immunoisolation are included for comparison.

Mentions: Since p24 proteins reside in the early part of the secretory pathway and Mann II in the Golgi stack centered around the medial cisternae (Velasco et al., 1993; Rabouille et al., 1995; Sohn et al., 1996; Rojo et al., 1997; Dominguez et al., 1998; Füllekrug et al., 1999), one would predict only a small overlap between these proteins in recycling COPI vesicles. This is reflected in the budding assay by the differential response to the α-SNAPdn mutant, improving a selective recovery of Mann II but not p24 proteins (Fig. 1 A, right). To examine this more directly, we immunoisolated p24-containing vesicles using magnetic beads coated with a cytoplasmic domain antibody to p24γ3 (Füllekrug et al., 1999). Affinity purified antibody attached to magnetic beads was first tested for its ability to bind Golgi membranes (Fig. 2 A, left). After incubation, membranes bound to beads were analyzed at the ultrastructural level and for protein content by SDS-PAGE followed by Western blotting. As can be seen, large membrane structures were observed on beads only when using the p24γ3 antibody (Sp) but not on beads coated with an irrelevant control antibody (Ctr). Addition of magnetic beads to the vesicle fraction (Fig. 2 A, right) revealed binding of vesicular structures with a diameter of 50–60 nm, the expected size of uncoated COPI vesicles using the p24γ3 antibody (Sp). No vesicles were seen binding to beads coated with the control antibody (Ctr). Western blots probed with antibodies to Mann II, p24α2, β1, δ1, and γ3 showed that no preferential parts of the Golgi (Gt) had been selected for (Fig. 2 B, Gt compared with Sp). No signal could be detected when using beads coated with the control antibody (Ctr).


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)

Immunoisolation of p24-containing vesicles reveals subpopulations of COPI vesicles. (A–D) Magnetic beads precoated with either an irrelevant control antibody (Ctr) or specific (Sp) antibodies to the cytoplasmic domain of p24γ3 were incubated with nonwashed Golgi membranes (A, left, and B) or with COPI vesicles formed from such membranes in the presence of the α-SNAPdn mutant (A, right, and C and D). Isolated membranes and vesicles were pelleted, fixed, embedded, and processed for EM or solubilized and subjected to SDS-PAGE. After transfer to nitrocellulose, Mann II, p24γ3, p24α2, p24δ1, and p24β1 were detected using specific antibodies followed by ECL. Vesicles remaining in the supernatant (Supn) after immunoisolation were also monitored in terms of their protein content (C, left lanes). (D) Immunoisolated vesicles were probed for the anterograde cargo marker, RSA, and compared with p24γ3, p24α2, p24δ1, and p24β1. Lanes containing total Golgi (Gt) and 20% of total vesicles (Vtot) used for immunoisolation are included for comparison.
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fig2: Immunoisolation of p24-containing vesicles reveals subpopulations of COPI vesicles. (A–D) Magnetic beads precoated with either an irrelevant control antibody (Ctr) or specific (Sp) antibodies to the cytoplasmic domain of p24γ3 were incubated with nonwashed Golgi membranes (A, left, and B) or with COPI vesicles formed from such membranes in the presence of the α-SNAPdn mutant (A, right, and C and D). Isolated membranes and vesicles were pelleted, fixed, embedded, and processed for EM or solubilized and subjected to SDS-PAGE. After transfer to nitrocellulose, Mann II, p24γ3, p24α2, p24δ1, and p24β1 were detected using specific antibodies followed by ECL. Vesicles remaining in the supernatant (Supn) after immunoisolation were also monitored in terms of their protein content (C, left lanes). (D) Immunoisolated vesicles were probed for the anterograde cargo marker, RSA, and compared with p24γ3, p24α2, p24δ1, and p24β1. Lanes containing total Golgi (Gt) and 20% of total vesicles (Vtot) used for immunoisolation are included for comparison.
Mentions: Since p24 proteins reside in the early part of the secretory pathway and Mann II in the Golgi stack centered around the medial cisternae (Velasco et al., 1993; Rabouille et al., 1995; Sohn et al., 1996; Rojo et al., 1997; Dominguez et al., 1998; Füllekrug et al., 1999), one would predict only a small overlap between these proteins in recycling COPI vesicles. This is reflected in the budding assay by the differential response to the α-SNAPdn mutant, improving a selective recovery of Mann II but not p24 proteins (Fig. 1 A, right). To examine this more directly, we immunoisolated p24-containing vesicles using magnetic beads coated with a cytoplasmic domain antibody to p24γ3 (Füllekrug et al., 1999). Affinity purified antibody attached to magnetic beads was first tested for its ability to bind Golgi membranes (Fig. 2 A, left). After incubation, membranes bound to beads were analyzed at the ultrastructural level and for protein content by SDS-PAGE followed by Western blotting. As can be seen, large membrane structures were observed on beads only when using the p24γ3 antibody (Sp) but not on beads coated with an irrelevant control antibody (Ctr). Addition of magnetic beads to the vesicle fraction (Fig. 2 A, right) revealed binding of vesicular structures with a diameter of 50–60 nm, the expected size of uncoated COPI vesicles using the p24γ3 antibody (Sp). No vesicles were seen binding to beads coated with the control antibody (Ctr). Western blots probed with antibodies to Mann II, p24α2, β1, δ1, and γ3 showed that no preferential parts of the Golgi (Gt) had been selected for (Fig. 2 B, Gt compared with Sp). No signal could be detected when using beads coated with the control antibody (Ctr).

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