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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 and BFA on the distribution of ts-O45-G. ts-O45 VSV-infected  Vero cells, kept for 2 h at 39.5°C,  were microinjected at nonpermissive  temperature with Fab-fragments of  anti-EAGE (a–d) or with anti-110-12  (e and f). After injection control cells  were incubated for further 6 h at  39.5°C (a and b), or for 2.5 h at 39.5°C  with 5 μg/ml BFA (c–f). Cells were  then fixed and labeled for injected  antibodies (b, d, and f) and ts-O45-G  (a, c, and e). Injected anti-EAGE has  no effect on ts-O45-G in the ER in  the control cells kept at 39.5°C (a and  b). BFA treatment, however, induces accumulation of ts-O45-G in  cytosolic patches (arrows in c and e)  which are distinct from the patches  where anti-EAGE accumulate (arrowheads in d). Anti-110-12 maintains its diffuse cytosolic distribution  after BFA treatment (f). Bar, 15 μm.
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Figure 3: Effect of microinjected anti-EAGE and BFA on the distribution of ts-O45-G. ts-O45 VSV-infected Vero cells, kept for 2 h at 39.5°C, were microinjected at nonpermissive temperature with Fab-fragments of anti-EAGE (a–d) or with anti-110-12 (e and f). After injection control cells were incubated for further 6 h at 39.5°C (a and b), or for 2.5 h at 39.5°C with 5 μg/ml BFA (c–f). Cells were then fixed and labeled for injected antibodies (b, d, and f) and ts-O45-G (a, c, and e). Injected anti-EAGE has no effect on ts-O45-G in the ER in the control cells kept at 39.5°C (a and b). BFA treatment, however, induces accumulation of ts-O45-G in cytosolic patches (arrows in c and e) which are distinct from the patches where anti-EAGE accumulate (arrowheads in d). Anti-110-12 maintains its diffuse cytosolic distribution after BFA treatment (f). Bar, 15 μm.

Mentions: In control noninjected cells ts-O45-G accumulates in the ER at 39.5°C and colocalizes with PDI or calnexin (data not shown). Microinjection of anti-EAGE has no effect on the localization of ts-O45-G in the ER at nonpermissive temperature (e.g., Fig. 3, a and b). The lack of BFA-induced acquisition of endo H resistance of ts-O45-G in these cells must therefore be due to inhibition of delivery of Golgi enzymes to the ER. To analyze this transport block morphologically, we colocalized microinjected antibodies and several marker proteins for the ER, IC, and Golgi complex by immunofluorescence microscopy.


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 and BFA on the distribution of ts-O45-G. ts-O45 VSV-infected  Vero cells, kept for 2 h at 39.5°C,  were microinjected at nonpermissive  temperature with Fab-fragments of  anti-EAGE (a–d) or with anti-110-12  (e and f). After injection control cells  were incubated for further 6 h at  39.5°C (a and b), or for 2.5 h at 39.5°C  with 5 μg/ml BFA (c–f). Cells were  then fixed and labeled for injected  antibodies (b, d, and f) and ts-O45-G  (a, c, and e). Injected anti-EAGE has  no effect on ts-O45-G in the ER in  the control cells kept at 39.5°C (a and  b). BFA treatment, however, induces accumulation of ts-O45-G in  cytosolic patches (arrows in c and e)  which are distinct from the patches  where anti-EAGE accumulate (arrowheads in d). Anti-110-12 maintains its diffuse cytosolic distribution  after BFA treatment (f). Bar, 15 μm.
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Related In: Results  -  Collection

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Figure 3: Effect of microinjected anti-EAGE and BFA on the distribution of ts-O45-G. ts-O45 VSV-infected Vero cells, kept for 2 h at 39.5°C, were microinjected at nonpermissive temperature with Fab-fragments of anti-EAGE (a–d) or with anti-110-12 (e and f). After injection control cells were incubated for further 6 h at 39.5°C (a and b), or for 2.5 h at 39.5°C with 5 μg/ml BFA (c–f). Cells were then fixed and labeled for injected antibodies (b, d, and f) and ts-O45-G (a, c, and e). Injected anti-EAGE has no effect on ts-O45-G in the ER in the control cells kept at 39.5°C (a and b). BFA treatment, however, induces accumulation of ts-O45-G in cytosolic patches (arrows in c and e) which are distinct from the patches where anti-EAGE accumulate (arrowheads in d). Anti-110-12 maintains its diffuse cytosolic distribution after BFA treatment (f). Bar, 15 μm.
Mentions: In control noninjected cells ts-O45-G accumulates in the ER at 39.5°C and colocalizes with PDI or calnexin (data not shown). Microinjection of anti-EAGE has no effect on the localization of ts-O45-G in the ER at nonpermissive temperature (e.g., Fig. 3, a and b). The lack of BFA-induced acquisition of endo H resistance of ts-O45-G in these cells must therefore be due to inhibition of delivery of Golgi enzymes to the ER. To analyze this transport block morphologically, we colocalized microinjected antibodies and several marker proteins for the ER, IC, and Golgi complex by immunofluorescence microscopy.

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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