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Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Scheel J, Pepperkok R, Lowe M, Griffiths G, Kreis TE - J. Cell Biol. (1997)

Bottom Line: These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor.Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC.These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Sciences III, University of Geneva, Switzerland.

ABSTRACT
Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

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Morphological EM analysis of the effect of injected antibodies against β-COP on the BFA-induced reorganization of the  Golgi complex. Typical examples for the structures identified and quantified in Fig. 5 in BFA-treated ts-O45 VSV-infected cells injected  with Fab-fragments of anti-EAGE (a–c and f) or with anti-110-12 (d, e, and g) are shown. Cells in a–e have been treated for 1 h with 5  μg/ml BFA 30 min after injection with antibodies, while BFA has been washed-out for 1 h from cells shown in f and g. a shows a low magnification and b a higher magnification micrograph of tubulo-vesicular clusters that typically accumulate in BFA-treated cells injected with  anti-EAGE; electron dense spherical aggregates are illustrated in c. In d and e tubulo-vesicular clusters are shown at low and high magnification in control cells injected with anti-110-12. Reassembled stacks of Golgi cisternae are shown in cells injected with anti-EAGE (f)  or anti-110-12 (g). C, cisterna; CE, centriole pair; G, stacks of Golgi cisternae; N, nucleus; RER, rough ER; asterisks in a and d indicate  tubulo-vesicular clusters, and in c, dense spherical aggregates. Arrowheads indicate possible COP-coated buds or vesicles. Bars: (b, c,  and e) 100 nm; (a, d, f, and g) 200 nm.
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Figure 7: Morphological EM analysis of the effect of injected antibodies against β-COP on the BFA-induced reorganization of the Golgi complex. Typical examples for the structures identified and quantified in Fig. 5 in BFA-treated ts-O45 VSV-infected cells injected with Fab-fragments of anti-EAGE (a–c and f) or with anti-110-12 (d, e, and g) are shown. Cells in a–e have been treated for 1 h with 5 μg/ml BFA 30 min after injection with antibodies, while BFA has been washed-out for 1 h from cells shown in f and g. a shows a low magnification and b a higher magnification micrograph of tubulo-vesicular clusters that typically accumulate in BFA-treated cells injected with anti-EAGE; electron dense spherical aggregates are illustrated in c. In d and e tubulo-vesicular clusters are shown at low and high magnification in control cells injected with anti-110-12. Reassembled stacks of Golgi cisternae are shown in cells injected with anti-EAGE (f) or anti-110-12 (g). C, cisterna; CE, centriole pair; G, stacks of Golgi cisternae; N, nucleus; RER, rough ER; asterisks in a and d indicate tubulo-vesicular clusters, and in c, dense spherical aggregates. Arrowheads indicate possible COP-coated buds or vesicles. Bars: (b, c, and e) 100 nm; (a, d, f, and g) 200 nm.

Mentions: Quantitative EM analysis of thin sections revealed that virtually no identifiable Golgi stacks remained after a 1-h treatment with BFA at 39.5°C in control (anti-110-12) or anti–EAGE-injected cells (Fig. 6 a). Thus, the scattered patches of Golgi membranes positive for β-COP seen by immunofluorescence microscopy in cells injected with antiEAGE and after BFA treatment are unlikely to represent Golgi mini-stacks. In contrast, numerous dense spherical aggregates (Fig. 7 c) were found in the BFA-treated cells microinjected with anti-EAGE but not anti-110-12 (Fig. 6 c). The number of these spherical aggregates appears to correspond roughly with the number of β-COP–positive patches expected from light microscopy observations. Similar structures shown to contain β-COP and membranes have previously been observed in different cell types under various conditions of treatment (Oprins et al., 1993; Orci et al., 1993; Pepperkok et al., 1993; Hendricks et al., 1993). Furthermore, a significant fraction of tubulo-vesicular structures (Fig. 7, a, d, and e) appear in the injected cells; although more of these clusters are found in cells injected with anti-EAGE, a significant number is also present in control injected cells (Fig. 6 b). Thus, it is possible that in cells injected with anti-EAGE, BFA-induced relocation of resident Golgi proteins is arrested in these spherical aggregates. Since the tubulo-vesicular clusters are present in cells injected with either antibodies, they probably represent the structures where ts-O45-G, ERGIC53, and Golgi markers accumulate in BFA-treated cells, i.e., the IC.


Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Scheel J, Pepperkok R, Lowe M, Griffiths G, Kreis TE - J. Cell Biol. (1997)

Morphological EM analysis of the effect of injected antibodies against β-COP on the BFA-induced reorganization of the  Golgi complex. Typical examples for the structures identified and quantified in Fig. 5 in BFA-treated ts-O45 VSV-infected cells injected  with Fab-fragments of anti-EAGE (a–c and f) or with anti-110-12 (d, e, and g) are shown. Cells in a–e have been treated for 1 h with 5  μg/ml BFA 30 min after injection with antibodies, while BFA has been washed-out for 1 h from cells shown in f and g. a shows a low magnification and b a higher magnification micrograph of tubulo-vesicular clusters that typically accumulate in BFA-treated cells injected with  anti-EAGE; electron dense spherical aggregates are illustrated in c. In d and e tubulo-vesicular clusters are shown at low and high magnification in control cells injected with anti-110-12. Reassembled stacks of Golgi cisternae are shown in cells injected with anti-EAGE (f)  or anti-110-12 (g). C, cisterna; CE, centriole pair; G, stacks of Golgi cisternae; N, nucleus; RER, rough ER; asterisks in a and d indicate  tubulo-vesicular clusters, and in c, dense spherical aggregates. Arrowheads indicate possible COP-coated buds or vesicles. Bars: (b, c,  and e) 100 nm; (a, d, f, and g) 200 nm.
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Figure 7: Morphological EM analysis of the effect of injected antibodies against β-COP on the BFA-induced reorganization of the Golgi complex. Typical examples for the structures identified and quantified in Fig. 5 in BFA-treated ts-O45 VSV-infected cells injected with Fab-fragments of anti-EAGE (a–c and f) or with anti-110-12 (d, e, and g) are shown. Cells in a–e have been treated for 1 h with 5 μg/ml BFA 30 min after injection with antibodies, while BFA has been washed-out for 1 h from cells shown in f and g. a shows a low magnification and b a higher magnification micrograph of tubulo-vesicular clusters that typically accumulate in BFA-treated cells injected with anti-EAGE; electron dense spherical aggregates are illustrated in c. In d and e tubulo-vesicular clusters are shown at low and high magnification in control cells injected with anti-110-12. Reassembled stacks of Golgi cisternae are shown in cells injected with anti-EAGE (f) or anti-110-12 (g). C, cisterna; CE, centriole pair; G, stacks of Golgi cisternae; N, nucleus; RER, rough ER; asterisks in a and d indicate tubulo-vesicular clusters, and in c, dense spherical aggregates. Arrowheads indicate possible COP-coated buds or vesicles. Bars: (b, c, and e) 100 nm; (a, d, f, and g) 200 nm.
Mentions: Quantitative EM analysis of thin sections revealed that virtually no identifiable Golgi stacks remained after a 1-h treatment with BFA at 39.5°C in control (anti-110-12) or anti–EAGE-injected cells (Fig. 6 a). Thus, the scattered patches of Golgi membranes positive for β-COP seen by immunofluorescence microscopy in cells injected with antiEAGE and after BFA treatment are unlikely to represent Golgi mini-stacks. In contrast, numerous dense spherical aggregates (Fig. 7 c) were found in the BFA-treated cells microinjected with anti-EAGE but not anti-110-12 (Fig. 6 c). The number of these spherical aggregates appears to correspond roughly with the number of β-COP–positive patches expected from light microscopy observations. Similar structures shown to contain β-COP and membranes have previously been observed in different cell types under various conditions of treatment (Oprins et al., 1993; Orci et al., 1993; Pepperkok et al., 1993; Hendricks et al., 1993). Furthermore, a significant fraction of tubulo-vesicular structures (Fig. 7, a, d, and e) appear in the injected cells; although more of these clusters are found in cells injected with anti-EAGE, a significant number is also present in control injected cells (Fig. 6 b). Thus, it is possible that in cells injected with anti-EAGE, BFA-induced relocation of resident Golgi proteins is arrested in these spherical aggregates. Since the tubulo-vesicular clusters are present in cells injected with either antibodies, they probably represent the structures where ts-O45-G, ERGIC53, and Golgi markers accumulate in BFA-treated cells, i.e., the IC.

Bottom Line: These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor.Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC.These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Sciences III, University of Geneva, Switzerland.

ABSTRACT
Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

Show MeSH
Related in: MedlinePlus