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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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Accumulation of COPI vesicles in the presence of the α-SNAPdn mutant. (A) Vesicles formed using salt-washed Golgi membranes were compared with vesicles formed using nonwashed membranes in the absence or presence of the α-SNAPdn mutant. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, p24γ3, p24α2, and p24β1 followed by ECL. Note the lower amount of Mann II in the absence of the α-SNAPdn mutant. Golgi membranes are shown in increasing amounts expressed as the percentage of starting material. (B) Vesicles were formed using salt-washed Golgi membranes in the presence or absence of exogenously added NSF/α-SNAPwt and/or the α-SNAPdn mutant. (C) Coatomer was depleted (Dp) from cytosol using specific monoclonal antibodies, recognizing native coatomer or mock (Mo) depleted using an irrelevant antibody and used in budding reactions with nonwashed membranes and the α-SNAPdn mutant. Depleted cytosol was rescued (Rs) subsequently by adding back exogenously purified coatomer and compared with vesicles formed using untreated cytosol (Ctr). (D) Vesicle budding was performed using nonwashed membranes in the presence of the α-SNAPdn mutant and tested for anterograde cargo incorporation (pIgR, RSA, and ApoE). Vesicles were also generated in the presence of an ATP depletion system (ATP−) for comparison. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis and revealed by the ECL method using antibodies.
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fig1: Accumulation of COPI vesicles in the presence of the α-SNAPdn mutant. (A) Vesicles formed using salt-washed Golgi membranes were compared with vesicles formed using nonwashed membranes in the absence or presence of the α-SNAPdn mutant. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, p24γ3, p24α2, and p24β1 followed by ECL. Note the lower amount of Mann II in the absence of the α-SNAPdn mutant. Golgi membranes are shown in increasing amounts expressed as the percentage of starting material. (B) Vesicles were formed using salt-washed Golgi membranes in the presence or absence of exogenously added NSF/α-SNAPwt and/or the α-SNAPdn mutant. (C) Coatomer was depleted (Dp) from cytosol using specific monoclonal antibodies, recognizing native coatomer or mock (Mo) depleted using an irrelevant antibody and used in budding reactions with nonwashed membranes and the α-SNAPdn mutant. Depleted cytosol was rescued (Rs) subsequently by adding back exogenously purified coatomer and compared with vesicles formed using untreated cytosol (Ctr). (D) Vesicle budding was performed using nonwashed membranes in the presence of the α-SNAPdn mutant and tested for anterograde cargo incorporation (pIgR, RSA, and ApoE). Vesicles were also generated in the presence of an ATP depletion system (ATP−) for comparison. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis and revealed by the ECL method using antibodies.

Mentions: To ensure a reproducible build-up of COPI-derived vesicles, we had previously subjected membranes to a salt wash before the budding event (Lanoix et al., 1999). A drawback of this was that fenestrated membranes such as the cis-Golgi network (CGN) and trans-Golgi network (TGN) tended to be lost during the washing procedure. However, performing the budding assay using nonwashed membranes often resulted in a low recovery of Mann II signal compared with that obtained when using salt-washed membranes. A likely explanation for this is that by removing peripheral components, such as NSF and α-SNAP, formed vesicles cannot readily fuse back once uncoated. To avoid vesicle fusion, we performed the budding reaction in the presence of a dominant negative mutant of α-SNAP, L294A (α-SNAPdn), which is known to effectively block NSF-dependent fusion (Barnard et al., 1997) by locking the 20S fusion complex on the membrane (McBride et al., 1999). The effect of the α-SNAPdn mutant on salt-washed and nonwashed membranes is shown in Fig. 1 A. Using salt-washed membranes, the level of Mann II in vesicles was high (∼20% of that observed in Golgi membranes), whereas the level of p24 proteins was very low in both the Golgi and the vesicle fraction, which suggests that most of the CGN had been lost due to the salt wash. In contrast, when using nonwashed membranes the level of Mann II obtained in vesicles was reduced greatly, whereas the amount of p24 proteins was now high. Upon addition of the α-SNAPdn mutant, the level of Mann II increased significantly, whereas the level of p24 proteins remained unaffected. The idea that the salt wash had removed peripheral NSF and α-SNAP, thereby preventing vesicle consumption, was supported by the lower Mann II signal upon addition of recombinant and complexed NSF/α-SNAP (Fig. 1 B). Addition of the α-SNAPdn mutant together with NSF/α-SNAPwt blocked reduction. From a technical standpoint, addition of the α-SNAPdn mutant clearly improved the recovery of resident proteins when using nonwashed membranes, permitting us to use membranes that more accurately represent the entire Golgi apparatus including the CGN and TGN. Therefore, we added the α-SNAPdn mutant into subsequent budding experiments.


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)

Accumulation of COPI vesicles in the presence of the α-SNAPdn mutant. (A) Vesicles formed using salt-washed Golgi membranes were compared with vesicles formed using nonwashed membranes in the absence or presence of the α-SNAPdn mutant. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, p24γ3, p24α2, and p24β1 followed by ECL. Note the lower amount of Mann II in the absence of the α-SNAPdn mutant. Golgi membranes are shown in increasing amounts expressed as the percentage of starting material. (B) Vesicles were formed using salt-washed Golgi membranes in the presence or absence of exogenously added NSF/α-SNAPwt and/or the α-SNAPdn mutant. (C) Coatomer was depleted (Dp) from cytosol using specific monoclonal antibodies, recognizing native coatomer or mock (Mo) depleted using an irrelevant antibody and used in budding reactions with nonwashed membranes and the α-SNAPdn mutant. Depleted cytosol was rescued (Rs) subsequently by adding back exogenously purified coatomer and compared with vesicles formed using untreated cytosol (Ctr). (D) Vesicle budding was performed using nonwashed membranes in the presence of the α-SNAPdn mutant and tested for anterograde cargo incorporation (pIgR, RSA, and ApoE). Vesicles were also generated in the presence of an ATP depletion system (ATP−) for comparison. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis and revealed by the ECL method using antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199348&req=5

fig1: Accumulation of COPI vesicles in the presence of the α-SNAPdn mutant. (A) Vesicles formed using salt-washed Golgi membranes were compared with vesicles formed using nonwashed membranes in the absence or presence of the α-SNAPdn mutant. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, p24γ3, p24α2, and p24β1 followed by ECL. Note the lower amount of Mann II in the absence of the α-SNAPdn mutant. Golgi membranes are shown in increasing amounts expressed as the percentage of starting material. (B) Vesicles were formed using salt-washed Golgi membranes in the presence or absence of exogenously added NSF/α-SNAPwt and/or the α-SNAPdn mutant. (C) Coatomer was depleted (Dp) from cytosol using specific monoclonal antibodies, recognizing native coatomer or mock (Mo) depleted using an irrelevant antibody and used in budding reactions with nonwashed membranes and the α-SNAPdn mutant. Depleted cytosol was rescued (Rs) subsequently by adding back exogenously purified coatomer and compared with vesicles formed using untreated cytosol (Ctr). (D) Vesicle budding was performed using nonwashed membranes in the presence of the α-SNAPdn mutant and tested for anterograde cargo incorporation (pIgR, RSA, and ApoE). Vesicles were also generated in the presence of an ATP depletion system (ATP−) for comparison. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis and revealed by the ECL method using antibodies.
Mentions: To ensure a reproducible build-up of COPI-derived vesicles, we had previously subjected membranes to a salt wash before the budding event (Lanoix et al., 1999). A drawback of this was that fenestrated membranes such as the cis-Golgi network (CGN) and trans-Golgi network (TGN) tended to be lost during the washing procedure. However, performing the budding assay using nonwashed membranes often resulted in a low recovery of Mann II signal compared with that obtained when using salt-washed membranes. A likely explanation for this is that by removing peripheral components, such as NSF and α-SNAP, formed vesicles cannot readily fuse back once uncoated. To avoid vesicle fusion, we performed the budding reaction in the presence of a dominant negative mutant of α-SNAP, L294A (α-SNAPdn), which is known to effectively block NSF-dependent fusion (Barnard et al., 1997) by locking the 20S fusion complex on the membrane (McBride et al., 1999). The effect of the α-SNAPdn mutant on salt-washed and nonwashed membranes is shown in Fig. 1 A. Using salt-washed membranes, the level of Mann II in vesicles was high (∼20% of that observed in Golgi membranes), whereas the level of p24 proteins was very low in both the Golgi and the vesicle fraction, which suggests that most of the CGN had been lost due to the salt wash. In contrast, when using nonwashed membranes the level of Mann II obtained in vesicles was reduced greatly, whereas the amount of p24 proteins was now high. Upon addition of the α-SNAPdn mutant, the level of Mann II increased significantly, whereas the level of p24 proteins remained unaffected. The idea that the salt wash had removed peripheral NSF and α-SNAP, thereby preventing vesicle consumption, was supported by the lower Mann II signal upon addition of recombinant and complexed NSF/α-SNAP (Fig. 1 B). Addition of the α-SNAPdn mutant together with NSF/α-SNAPwt blocked reduction. From a technical standpoint, addition of the α-SNAPdn mutant clearly improved the recovery of resident proteins when using nonwashed membranes, permitting us to use membranes that more accurately represent the entire Golgi apparatus including the CGN and TGN. Therefore, we added the α-SNAPdn mutant into subsequent budding experiments.

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