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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 microinjected anti-EAGE on BFA induced relocation of Golgi enzymes. Vero cells were microinjected with Fabfragments of anti-EAGE. 30 min after injection 5 μg/ml BFA was added to the medium and cells were incubated for further 2.5 h at  37°C. Cells were then fixed and double stained for injected antibodies (b, d, and f) and marker proteins for (a) the ER (PDI), (c) the intermediate compartment (ERGIC 53), and (e) the Golgi complex (NAGT1). Microinjected antibodies accumulate in patches (arrowheads in d; arrows in f) which colocalize with NAGT1 (arrows in e) but not ERGIC 53 and PDI. The injected anti-EAGE thus affect the  BFA-induced relocalization of Golgi proteins to the ER. Bar, 10 μm.
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Figure 2: Effect of microinjected anti-EAGE on BFA induced relocation of Golgi enzymes. Vero cells were microinjected with Fabfragments of anti-EAGE. 30 min after injection 5 μg/ml BFA was added to the medium and cells were incubated for further 2.5 h at 37°C. Cells were then fixed and double stained for injected antibodies (b, d, and f) and marker proteins for (a) the ER (PDI), (c) the intermediate compartment (ERGIC 53), and (e) the Golgi complex (NAGT1). Microinjected antibodies accumulate in patches (arrowheads in d; arrows in f) which colocalize with NAGT1 (arrows in e) but not ERGIC 53 and PDI. The injected anti-EAGE thus affect the BFA-induced relocalization of Golgi proteins to the ER. Bar, 10 μm.

Mentions: The resident Golgi membrane proteins NAGT I (Fig. 2 e) and Man II (not shown), as well as p58, a protein associated with the cytoplasmic surface of Golgi membranes (Bloom and Brashear, 1989), accumulate in β-COP positive patches (Fig. 2 f) when virus-infected cells, microinjected with anti-EAGE, are treated with BFA while continuously kept at 39.5°C. These patches do not contain the “IC marker lectin” ERGIC53 (Schweizer et al., 1988; Schindler et al., 1993; Arar et al., 1995; Fig. 2, c and d), and the ER marker proteins, PDI and calnexin (not shown), maintain their reticular distributions and are not enriched in these structures either (Fig. 2, a and b). Furthermore, no ts-O45-G accumulates in these patches under nonpermissive conditions (Fig. 3, c and d). Interestingly, the normally predominantly cis-Golgi localized KDEL receptor accumulates in these BFA-treated cells in the patches containing ts-O45-G (Fig. 4, d and e) and ERGIC53, and not in those positive for Golgi enzymes or injected anti-EAGE (Fig. 4 c). This result indicates that injected anti-EAGE does not interfere with the BFA-induced relocation of KDEL receptor to the IC. Furthermore, the distribution of KDEL receptor (Fig. 4 a) appears not significantly changed in control cells microinjected with anti-EAGE (Fig. 4 b); occasionally, formation of tubular structures can be observed. The formation of these aggregates is specific for injected anti-EAGE, since neither microinjected anti110-12 nor anti–β′-COP (not shown; these antibodies when injected into cells bind to coatomer in vivo) lead to an accumulation of coat proteins in patches under these conditions (Fig. 3, e and f), and no significant number of the β-COP and Golgi protein containing aggregates form in the absence of BFA at nonpermissive temperature (Fig. 3, a and b). Furthermore, the effect of injected anti-EAGE is not dominant over the effect of BFA, since if the antibodies were injected after BFA treatment of infected cells, no patches could be seen (not shown). It is also an early effect of BFA action, since patches containing Golgi proteins and β-COP can already be observed ∼5 min after addition of the drug (data not shown). These results corroborate our biochemical data which suggest that ts-O45-G does not meet Golgi enzymes in BFA-treated cells which have been injected with anti-EAGE. Thus, injected anti-EAGE inhibits the BFA-induced transfer of resident Golgi proteins to the ER and leads to a rapid accumulation of Golgi proteins in COPI containing cytoplasmic aggregates distinct from the ER/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)

Effect of microinjected anti-EAGE on BFA induced relocation of Golgi enzymes. Vero cells were microinjected with Fabfragments of anti-EAGE. 30 min after injection 5 μg/ml BFA was added to the medium and cells were incubated for further 2.5 h at  37°C. Cells were then fixed and double stained for injected antibodies (b, d, and f) and marker proteins for (a) the ER (PDI), (c) the intermediate compartment (ERGIC 53), and (e) the Golgi complex (NAGT1). Microinjected antibodies accumulate in patches (arrowheads in d; arrows in f) which colocalize with NAGT1 (arrows in e) but not ERGIC 53 and PDI. The injected anti-EAGE thus affect the  BFA-induced relocalization of Golgi proteins to the ER. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Effect of microinjected anti-EAGE on BFA induced relocation of Golgi enzymes. Vero cells were microinjected with Fabfragments of anti-EAGE. 30 min after injection 5 μg/ml BFA was added to the medium and cells were incubated for further 2.5 h at 37°C. Cells were then fixed and double stained for injected antibodies (b, d, and f) and marker proteins for (a) the ER (PDI), (c) the intermediate compartment (ERGIC 53), and (e) the Golgi complex (NAGT1). Microinjected antibodies accumulate in patches (arrowheads in d; arrows in f) which colocalize with NAGT1 (arrows in e) but not ERGIC 53 and PDI. The injected anti-EAGE thus affect the BFA-induced relocalization of Golgi proteins to the ER. Bar, 10 μm.
Mentions: The resident Golgi membrane proteins NAGT I (Fig. 2 e) and Man II (not shown), as well as p58, a protein associated with the cytoplasmic surface of Golgi membranes (Bloom and Brashear, 1989), accumulate in β-COP positive patches (Fig. 2 f) when virus-infected cells, microinjected with anti-EAGE, are treated with BFA while continuously kept at 39.5°C. These patches do not contain the “IC marker lectin” ERGIC53 (Schweizer et al., 1988; Schindler et al., 1993; Arar et al., 1995; Fig. 2, c and d), and the ER marker proteins, PDI and calnexin (not shown), maintain their reticular distributions and are not enriched in these structures either (Fig. 2, a and b). Furthermore, no ts-O45-G accumulates in these patches under nonpermissive conditions (Fig. 3, c and d). Interestingly, the normally predominantly cis-Golgi localized KDEL receptor accumulates in these BFA-treated cells in the patches containing ts-O45-G (Fig. 4, d and e) and ERGIC53, and not in those positive for Golgi enzymes or injected anti-EAGE (Fig. 4 c). This result indicates that injected anti-EAGE does not interfere with the BFA-induced relocation of KDEL receptor to the IC. Furthermore, the distribution of KDEL receptor (Fig. 4 a) appears not significantly changed in control cells microinjected with anti-EAGE (Fig. 4 b); occasionally, formation of tubular structures can be observed. The formation of these aggregates is specific for injected anti-EAGE, since neither microinjected anti110-12 nor anti–β′-COP (not shown; these antibodies when injected into cells bind to coatomer in vivo) lead to an accumulation of coat proteins in patches under these conditions (Fig. 3, e and f), and no significant number of the β-COP and Golgi protein containing aggregates form in the absence of BFA at nonpermissive temperature (Fig. 3, a and b). Furthermore, the effect of injected anti-EAGE is not dominant over the effect of BFA, since if the antibodies were injected after BFA treatment of infected cells, no patches could be seen (not shown). It is also an early effect of BFA action, since patches containing Golgi proteins and β-COP can already be observed ∼5 min after addition of the drug (data not shown). These results corroborate our biochemical data which suggest that ts-O45-G does not meet Golgi enzymes in BFA-treated cells which have been injected with anti-EAGE. Thus, injected anti-EAGE inhibits the BFA-induced transfer of resident Golgi proteins to the ER and leads to a rapid accumulation of Golgi proteins in COPI containing cytoplasmic aggregates distinct from the ER/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