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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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Immunolocalization of p24 proteins in cells and vesicles. (A–C) Subcellular localization of p24β1 (b, green) as determined by indirect immunofluorescence localization. For comparison, labeling of Mann II (red) is shown in A. The extent of overlap is shown in C. (D) Thin frozen sections of NRK cells were labeled with antibodies to p24β1 (10 nm gold). Gold particles were found at one side of the Golgi stack, codistributing with antibodies to p24γ3 (5 nm gold, arrows). (E and F) Vesicles bound to grids were labeled with antibodies against two different p24s and revealed by protein A gold conjugates. The majority of the labeled vesicles contained both p24 proteins of each combination. (G) Quantification of p24 proteins in pair-wise labeling experiments. Different antibodies and protein A gold sizes (pAG) were used. Vesicles labeled with only one type of gold particle (two or more of the same gold particle) are expressed as the percentage of single labeled (SL), and vesicles containing both types of gold particle (two or more different gold particles) are expressed as double labeled (DL).
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fig3: Immunolocalization of p24 proteins in cells and vesicles. (A–C) Subcellular localization of p24β1 (b, green) as determined by indirect immunofluorescence localization. For comparison, labeling of Mann II (red) is shown in A. The extent of overlap is shown in C. (D) Thin frozen sections of NRK cells were labeled with antibodies to p24β1 (10 nm gold). Gold particles were found at one side of the Golgi stack, codistributing with antibodies to p24γ3 (5 nm gold, arrows). (E and F) Vesicles bound to grids were labeled with antibodies against two different p24s and revealed by protein A gold conjugates. The majority of the labeled vesicles contained both p24 proteins of each combination. (G) Quantification of p24 proteins in pair-wise labeling experiments. Different antibodies and protein A gold sizes (pAG) were used. Vesicles labeled with only one type of gold particle (two or more of the same gold particle) are expressed as the percentage of single labeled (SL), and vesicles containing both types of gold particle (two or more different gold particles) are expressed as double labeled (DL).

Mentions: The immunoisolation data suggests that p24β1 is present in vesicles together with p24α2, δ1, and γ3. Whereas the steady-state localization of p24α2, δ1, and γ3 have been mapped to the cis side of the Golgi stack at the ultrastructural level (Sohn et al., 1996; Rojo et al., 1997; Dominguez et al., 1998; Füllekrug et al., 1999), Golgi localization of p24β1 has only been determined at the level of light microscopy (Gommel et al., 1999). Therefore, we determined the steady-state localization of p24β1 by immuno-EM. A rabbit peptide antibody was raised against the cytoplasmic domain of p24β1 and affinity purified. By indirect immunofluorescence, the antibody gave rise to a juxta nuclear Golgi-like staining pattern (Fig. 3, A–C) with extensive but not complete colocalization with the Golgi stack marker, Mann II. Peripheral punctate staining was also observed as seen previously when staining for p24α2, δ1, and γ3. In NRK cells at the ultrastructural level, p24β1 (10 nm) showed extensive colocalization with p24γ3 (5 nm, arrows) mapped previously to the cis side of the Golgi stack (Füllekrug et al., 1999). This completes the mapping of all four p24 proteins to the cis side of the Golgi stack.


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)

Immunolocalization of p24 proteins in cells and vesicles. (A–C) Subcellular localization of p24β1 (b, green) as determined by indirect immunofluorescence localization. For comparison, labeling of Mann II (red) is shown in A. The extent of overlap is shown in C. (D) Thin frozen sections of NRK cells were labeled with antibodies to p24β1 (10 nm gold). Gold particles were found at one side of the Golgi stack, codistributing with antibodies to p24γ3 (5 nm gold, arrows). (E and F) Vesicles bound to grids were labeled with antibodies against two different p24s and revealed by protein A gold conjugates. The majority of the labeled vesicles contained both p24 proteins of each combination. (G) Quantification of p24 proteins in pair-wise labeling experiments. Different antibodies and protein A gold sizes (pAG) were used. Vesicles labeled with only one type of gold particle (two or more of the same gold particle) are expressed as the percentage of single labeled (SL), and vesicles containing both types of gold particle (two or more different gold particles) are expressed as double labeled (DL).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199348&req=5

fig3: Immunolocalization of p24 proteins in cells and vesicles. (A–C) Subcellular localization of p24β1 (b, green) as determined by indirect immunofluorescence localization. For comparison, labeling of Mann II (red) is shown in A. The extent of overlap is shown in C. (D) Thin frozen sections of NRK cells were labeled with antibodies to p24β1 (10 nm gold). Gold particles were found at one side of the Golgi stack, codistributing with antibodies to p24γ3 (5 nm gold, arrows). (E and F) Vesicles bound to grids were labeled with antibodies against two different p24s and revealed by protein A gold conjugates. The majority of the labeled vesicles contained both p24 proteins of each combination. (G) Quantification of p24 proteins in pair-wise labeling experiments. Different antibodies and protein A gold sizes (pAG) were used. Vesicles labeled with only one type of gold particle (two or more of the same gold particle) are expressed as the percentage of single labeled (SL), and vesicles containing both types of gold particle (two or more different gold particles) are expressed as double labeled (DL).
Mentions: The immunoisolation data suggests that p24β1 is present in vesicles together with p24α2, δ1, and γ3. Whereas the steady-state localization of p24α2, δ1, and γ3 have been mapped to the cis side of the Golgi stack at the ultrastructural level (Sohn et al., 1996; Rojo et al., 1997; Dominguez et al., 1998; Füllekrug et al., 1999), Golgi localization of p24β1 has only been determined at the level of light microscopy (Gommel et al., 1999). Therefore, we determined the steady-state localization of p24β1 by immuno-EM. A rabbit peptide antibody was raised against the cytoplasmic domain of p24β1 and affinity purified. By indirect immunofluorescence, the antibody gave rise to a juxta nuclear Golgi-like staining pattern (Fig. 3, A–C) with extensive but not complete colocalization with the Golgi stack marker, Mann II. Peripheral punctate staining was also observed as seen previously when staining for p24α2, δ1, and γ3. In NRK cells at the ultrastructural level, p24β1 (10 nm) showed extensive colocalization with p24γ3 (5 nm, arrows) mapped previously to the cis side of the Golgi stack (Füllekrug et al., 1999). This completes the mapping of all four p24 proteins to the cis side of the Golgi stack.

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