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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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Effect of injected antibodies against β−COP on the localization of NAGT1 after BFA wash-out in VSV-infected cells. ts-O45  VSV-infected Vero cells were incubated for 1 h at 39.5°C with 5 μg/ml BFA and subsequently microinjected at 39.5°C with Fab-fragments of anti-EAGE or with anti-110-12 in the presence of BFA. Cells were transferred into normal medium immediately after injection and BFA washed-out at 31°C for 1 h. Cells were then fixed and stained for injected antibodies (b and d) and NAGT1 (a and c). Control  cells were double labeled for NAGT1 (e) and β-COP (f) for comparison. In many cells anti-EAGE interfere with the proper reassembly  of a compact Golgi complex in a juxtanuclear region (e.g., lower injected cells in d). Injected anti-EAGE also appears to interfere with the  distribution of coatomer; β-COP and NAGT1 are more tightly colocalized in these injected cells (c and d) than in cells injected with anti110-12 or in control cells where β-COP is “wrapped-around” the NAGT1 positive membranes (arrows in a, b, e, and f). Bar, 10 μm.
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Figure 9: Effect of injected antibodies against β−COP on the localization of NAGT1 after BFA wash-out in VSV-infected cells. ts-O45 VSV-infected Vero cells were incubated for 1 h at 39.5°C with 5 μg/ml BFA and subsequently microinjected at 39.5°C with Fab-fragments of anti-EAGE or with anti-110-12 in the presence of BFA. Cells were transferred into normal medium immediately after injection and BFA washed-out at 31°C for 1 h. Cells were then fixed and stained for injected antibodies (b and d) and NAGT1 (a and c). Control cells were double labeled for NAGT1 (e) and β-COP (f) for comparison. In many cells anti-EAGE interfere with the proper reassembly of a compact Golgi complex in a juxtanuclear region (e.g., lower injected cells in d). Injected anti-EAGE also appears to interfere with the distribution of coatomer; β-COP and NAGT1 are more tightly colocalized in these injected cells (c and d) than in cells injected with anti110-12 or in control cells where β-COP is “wrapped-around” the NAGT1 positive membranes (arrows in a, b, e, and f). Bar, 10 μm.

Mentions: Virus-infected Vero cells were treated with BFA for 1 h, microinjected with antibodies in the presence of BFA at 39.5°C, and subsequently transferred to medium at permissive temperature lacking the drug. Initially, ts-O45-G is accumulated in ERGIC53 positive patches (Fig. 8, a and b). In uninjected cells or cells injected with anti-110-12, tsO45-G is transported normally to the plasma membrane through the reforming Golgi apparatus (data not shown). However, in cells microinjected with anti-EAGE, ts-O45-G is arrested in tubular structures (Fig. 8 c) positive for ERGIC53 (Fig. 8 d), suggesting that under these conditions anterograde protein transport is blocked in the IC, apparently in identical fashion to injected cells shifted to permissive temperature that had not been treated with BFA (Pepperkok et al., 1993). Similarly to the anti–EAGE– injected cells not treated with BFA, β-COP accumulates in patches which were often found on or at the ends of tubules containing ERGIC53 and ts-O45-G (Fig. 8, e and f). Interestingly, NAGT1 (myc-tagged and stably expressed in Vero cells) and p58 (not shown) resumed an apparently normal Golgi distribution in about half of the cells injected with anti-EAGE within 1 h after removal of BFA (Fig. 9, c and d); in the other cells, NAGT1 remained in perinuclear clusters (e.g., lower injected cell in Fig. 9 c). No significant effect on Golgi reassembly was found in cells injected with anti-110-12 (Fig. 9, a and b). It appears that coatomer does not accumulate in a vesicular pattern at the periphery of the Golgi complex demarcated by a medial-Golgi resident protein (NAGT1) in cells injected with anti-EAGE (Fig. 9, c and d), as is typically observed in control cells or cells injected with anti-110-12 (see arrows in Fig. 9, a, b, e, and f).


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)

Effect of injected antibodies against β−COP on the localization of NAGT1 after BFA wash-out in VSV-infected cells. ts-O45  VSV-infected Vero cells were incubated for 1 h at 39.5°C with 5 μg/ml BFA and subsequently microinjected at 39.5°C with Fab-fragments of anti-EAGE or with anti-110-12 in the presence of BFA. Cells were transferred into normal medium immediately after injection and BFA washed-out at 31°C for 1 h. Cells were then fixed and stained for injected antibodies (b and d) and NAGT1 (a and c). Control  cells were double labeled for NAGT1 (e) and β-COP (f) for comparison. In many cells anti-EAGE interfere with the proper reassembly  of a compact Golgi complex in a juxtanuclear region (e.g., lower injected cells in d). Injected anti-EAGE also appears to interfere with the  distribution of coatomer; β-COP and NAGT1 are more tightly colocalized in these injected cells (c and d) than in cells injected with anti110-12 or in control cells where β-COP is “wrapped-around” the NAGT1 positive membranes (arrows in a, b, e, and f). Bar, 10 μm.
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Figure 9: Effect of injected antibodies against β−COP on the localization of NAGT1 after BFA wash-out in VSV-infected cells. ts-O45 VSV-infected Vero cells were incubated for 1 h at 39.5°C with 5 μg/ml BFA and subsequently microinjected at 39.5°C with Fab-fragments of anti-EAGE or with anti-110-12 in the presence of BFA. Cells were transferred into normal medium immediately after injection and BFA washed-out at 31°C for 1 h. Cells were then fixed and stained for injected antibodies (b and d) and NAGT1 (a and c). Control cells were double labeled for NAGT1 (e) and β-COP (f) for comparison. In many cells anti-EAGE interfere with the proper reassembly of a compact Golgi complex in a juxtanuclear region (e.g., lower injected cells in d). Injected anti-EAGE also appears to interfere with the distribution of coatomer; β-COP and NAGT1 are more tightly colocalized in these injected cells (c and d) than in cells injected with anti110-12 or in control cells where β-COP is “wrapped-around” the NAGT1 positive membranes (arrows in a, b, e, and f). Bar, 10 μm.
Mentions: Virus-infected Vero cells were treated with BFA for 1 h, microinjected with antibodies in the presence of BFA at 39.5°C, and subsequently transferred to medium at permissive temperature lacking the drug. Initially, ts-O45-G is accumulated in ERGIC53 positive patches (Fig. 8, a and b). In uninjected cells or cells injected with anti-110-12, tsO45-G is transported normally to the plasma membrane through the reforming Golgi apparatus (data not shown). However, in cells microinjected with anti-EAGE, ts-O45-G is arrested in tubular structures (Fig. 8 c) positive for ERGIC53 (Fig. 8 d), suggesting that under these conditions anterograde protein transport is blocked in the IC, apparently in identical fashion to injected cells shifted to permissive temperature that had not been treated with BFA (Pepperkok et al., 1993). Similarly to the anti–EAGE– injected cells not treated with BFA, β-COP accumulates in patches which were often found on or at the ends of tubules containing ERGIC53 and ts-O45-G (Fig. 8, e and f). Interestingly, NAGT1 (myc-tagged and stably expressed in Vero cells) and p58 (not shown) resumed an apparently normal Golgi distribution in about half of the cells injected with anti-EAGE within 1 h after removal of BFA (Fig. 9, c and d); in the other cells, NAGT1 remained in perinuclear clusters (e.g., lower injected cell in Fig. 9 c). No significant effect on Golgi reassembly was found in cells injected with anti-110-12 (Fig. 9, a and b). It appears that coatomer does not accumulate in a vesicular pattern at the periphery of the Golgi complex demarcated by a medial-Golgi resident protein (NAGT1) in cells injected with anti-EAGE (Fig. 9, c and d), as is typically observed in control cells or cells injected with anti-110-12 (see arrows in Fig. 9, a, b, e, and f).

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