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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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Anti-EAGE increases binding of β−COP to membranes in vitro. 14 μg of membranes enriched in IC and Golgi were  incubated for 10 min at 37°C with 160 μg rat liver cytosol in a reaction mixture containing either no additions (cytosol), 25 μM  GTPγS (GTPγS), 10 μg of anti-EAGE (EAGE), 10 μg Fab-fragments of anti-EAGE (EAGE-Fabs), 10 μg anti-110-12 (110-12),  anti-EAGE preincubated with the EAGE peptide (EAGE +  Peptide), or 30 μg EAGE peptide (Peptide). To block anti-EAGE  antibodies 10 μg IgG was incubated with 30 μg EAGE peptide in  PBS for 30 min on ice immediately before addition to the reaction. Membrane bound β−COP was detected by immunoblotting  with M3A5 as described in Materials and Methods. Shown is a  quantitation of the immunoblots from two duplicate experiments  (top) and a corresponding autoradiogram (bottom). Error bars  represent the standard mean error.
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Figure 10: Anti-EAGE increases binding of β−COP to membranes in vitro. 14 μg of membranes enriched in IC and Golgi were incubated for 10 min at 37°C with 160 μg rat liver cytosol in a reaction mixture containing either no additions (cytosol), 25 μM GTPγS (GTPγS), 10 μg of anti-EAGE (EAGE), 10 μg Fab-fragments of anti-EAGE (EAGE-Fabs), 10 μg anti-110-12 (110-12), anti-EAGE preincubated with the EAGE peptide (EAGE + Peptide), or 30 μg EAGE peptide (Peptide). To block anti-EAGE antibodies 10 μg IgG was incubated with 30 μg EAGE peptide in PBS for 30 min on ice immediately before addition to the reaction. Membrane bound β−COP was detected by immunoblotting with M3A5 as described in Materials and Methods. Shown is a quantitation of the immunoblots from two duplicate experiments (top) and a corresponding autoradiogram (bottom). Error bars represent the standard mean error.

Mentions: A small fraction of β-COP remains associated in vitro with membranes enriched in Golgi and IC after incubation with rat liver cytosol (Fig. 10). This binding is significantly increased (up to threefold) in the presence of GTPγS, in agreement with previous reports (Donaldson et al., 1991). A 2–3-fold enhanced binding of coatomer to Golgi membranes, similar to the levels obtained with GTPγS, can also be observed in the presence of anti-EAGE (Fig. 10). This increased binding occurs also with monovalent Fab-fragments, can be completely competed by pre-incubation of anti-EAGE with the EAGE peptide, and is specific since it does not occur with control antibodies anti-110-12 (Fig. 10). Thus, anti-EAGE significantly increases the fraction of membrane-bound coatomer. These results and the block of BFA's activity to remove β-COP from membranes in living cells microinjected with anti-EAGE suggest that anti-EAGE interferes with the regulation of dissociation of β-COP from membranes, most likely by locking it together with the other subunits of coatomer, in its membrane-bound form. We assume that this inhibition of membrane dissociation of coatomer by anti-EAGE interferes with membrane traffic between the ER/IC and the Golgi complex.


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)

Anti-EAGE increases binding of β−COP to membranes in vitro. 14 μg of membranes enriched in IC and Golgi were  incubated for 10 min at 37°C with 160 μg rat liver cytosol in a reaction mixture containing either no additions (cytosol), 25 μM  GTPγS (GTPγS), 10 μg of anti-EAGE (EAGE), 10 μg Fab-fragments of anti-EAGE (EAGE-Fabs), 10 μg anti-110-12 (110-12),  anti-EAGE preincubated with the EAGE peptide (EAGE +  Peptide), or 30 μg EAGE peptide (Peptide). To block anti-EAGE  antibodies 10 μg IgG was incubated with 30 μg EAGE peptide in  PBS for 30 min on ice immediately before addition to the reaction. Membrane bound β−COP was detected by immunoblotting  with M3A5 as described in Materials and Methods. Shown is a  quantitation of the immunoblots from two duplicate experiments  (top) and a corresponding autoradiogram (bottom). Error bars  represent the standard mean error.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139784&req=5

Figure 10: Anti-EAGE increases binding of β−COP to membranes in vitro. 14 μg of membranes enriched in IC and Golgi were incubated for 10 min at 37°C with 160 μg rat liver cytosol in a reaction mixture containing either no additions (cytosol), 25 μM GTPγS (GTPγS), 10 μg of anti-EAGE (EAGE), 10 μg Fab-fragments of anti-EAGE (EAGE-Fabs), 10 μg anti-110-12 (110-12), anti-EAGE preincubated with the EAGE peptide (EAGE + Peptide), or 30 μg EAGE peptide (Peptide). To block anti-EAGE antibodies 10 μg IgG was incubated with 30 μg EAGE peptide in PBS for 30 min on ice immediately before addition to the reaction. Membrane bound β−COP was detected by immunoblotting with M3A5 as described in Materials and Methods. Shown is a quantitation of the immunoblots from two duplicate experiments (top) and a corresponding autoradiogram (bottom). Error bars represent the standard mean error.
Mentions: A small fraction of β-COP remains associated in vitro with membranes enriched in Golgi and IC after incubation with rat liver cytosol (Fig. 10). This binding is significantly increased (up to threefold) in the presence of GTPγS, in agreement with previous reports (Donaldson et al., 1991). A 2–3-fold enhanced binding of coatomer to Golgi membranes, similar to the levels obtained with GTPγS, can also be observed in the presence of anti-EAGE (Fig. 10). This increased binding occurs also with monovalent Fab-fragments, can be completely competed by pre-incubation of anti-EAGE with the EAGE peptide, and is specific since it does not occur with control antibodies anti-110-12 (Fig. 10). Thus, anti-EAGE significantly increases the fraction of membrane-bound coatomer. These results and the block of BFA's activity to remove β-COP from membranes in living cells microinjected with anti-EAGE suggest that anti-EAGE interferes with the regulation of dissociation of β-COP from membranes, most likely by locking it together with the other subunits of coatomer, in its membrane-bound form. We assume that this inhibition of membrane dissociation of coatomer by anti-EAGE interferes with membrane traffic between the ER/IC and the Golgi complex.

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