Limits...
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.

Show MeSH

Related in: MedlinePlus

Biochemical analysis of transfer of Golgi enzymes to  the ER. Transfer of Golgi enzymes to the ER was determined  biochemically by analyzing acquisition of endo H resistance of tsO45-G arrested in the ER. 500–1000 Vero cells infected with  tsO45-VSV were incubated for 1.5 h at 39.5°C, metabolically labeled for 10 min with 35S-met and microinjected at 39.5°C or 15°C  with Fab-fragments of anti-EAGE (EAGE), divalent anti–110-12  (110-12), or kept as noninjected controls (n.i.). (A) Noninjected  control cells were lysed directly after the pulse with 35S-met and  ts-O45-G analyzed for endo H sensitivity (0 h). 39.5°C: control or  injected cells were lysed and ts-O45-G analyzed for endo H sensitivity after a chase of 6 h at 39.5°C; 15°C: temperature was shifted  for 2 h to 15°C before injection at 15°C, and the cells were kept  for an additional 4 h at 15°C before lysis and analysis of endo H  resistance of ts-O45-G. (B) Control and injected cells were  chased for 0 h or 2.5 h at 39.5°C in the presence of 5 μg/ml BFA  before lysis and analysis of endo H resistance of ts-O45-G. Microinjected anti-EAGE, but not anti-110-12 block BFA induced acquisition of endo H resistance of ts-O45-G.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139784&req=5

Figure 1: Biochemical analysis of transfer of Golgi enzymes to the ER. Transfer of Golgi enzymes to the ER was determined biochemically by analyzing acquisition of endo H resistance of tsO45-G arrested in the ER. 500–1000 Vero cells infected with tsO45-VSV were incubated for 1.5 h at 39.5°C, metabolically labeled for 10 min with 35S-met and microinjected at 39.5°C or 15°C with Fab-fragments of anti-EAGE (EAGE), divalent anti–110-12 (110-12), or kept as noninjected controls (n.i.). (A) Noninjected control cells were lysed directly after the pulse with 35S-met and ts-O45-G analyzed for endo H sensitivity (0 h). 39.5°C: control or injected cells were lysed and ts-O45-G analyzed for endo H sensitivity after a chase of 6 h at 39.5°C; 15°C: temperature was shifted for 2 h to 15°C before injection at 15°C, and the cells were kept for an additional 4 h at 15°C before lysis and analysis of endo H resistance of ts-O45-G. (B) Control and injected cells were chased for 0 h or 2.5 h at 39.5°C in the presence of 5 μg/ml BFA before lysis and analysis of endo H resistance of ts-O45-G. Microinjected anti-EAGE, but not anti-110-12 block BFA induced acquisition of endo H resistance of ts-O45-G.

Mentions: To analyze a possible recycling of Golgi enzymes through the ER/IC, Vero cells were infected with tsO45-VSV and kept at nonpermissive temperature (39.5°C, predominantly ER accumulation), or 15°C (IC accumulation). Under these conditions, ts-O45-G arrested in the ER or IC may serve as a substrate for oligosaccharide-processing Golgi enzymes, and transport of these enzymes into the ER/IC can be studied by biochemical analyses of modifications of the oligosaccharide side chains of the viral glycoprotein (Doms et al., 1989). Even after accumulation of ts-O45-G in the ER or IC for up to 6 h no trace of endo H-resistant glycoprotein could be detected in cells injected with anti-EAGE at the respective temperature (as in noninjected control cells; Fig. 1 A). Moreover, when cells were injected with anti-EAGE at 39.5°C and kept for three additional hours at the nonpermissive temperature and then for 3 h at 15°C with ts-O45-G finally accumulated in the IC (anti-EAGE does not inhibit ER to IC transport) no endo H-resistant fraction of the viral glycoprotein could be detected (not shown). Thus, either the recycling of (even a small fraction of) Golgi enzymes through the ER/IC is inhibited by the injected antibodies, or these enzymes, once located at their proper site of function within the Golgi complex, are retained. To investigate directly the role of coatomer in transport from the Golgi complex to the ER/IC in vivo, we used BFA-induced transfer of Golgi enzymes into the ER combined with microinjection of specific antibodies raised against synthetic peptides of β-COP (anti-EAGE and anti-110-12; Pepperkok et al., 1993) and β′-COP (Lowe and Kreis, 1995).


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)

Biochemical analysis of transfer of Golgi enzymes to  the ER. Transfer of Golgi enzymes to the ER was determined  biochemically by analyzing acquisition of endo H resistance of tsO45-G arrested in the ER. 500–1000 Vero cells infected with  tsO45-VSV were incubated for 1.5 h at 39.5°C, metabolically labeled for 10 min with 35S-met and microinjected at 39.5°C or 15°C  with Fab-fragments of anti-EAGE (EAGE), divalent anti–110-12  (110-12), or kept as noninjected controls (n.i.). (A) Noninjected  control cells were lysed directly after the pulse with 35S-met and  ts-O45-G analyzed for endo H sensitivity (0 h). 39.5°C: control or  injected cells were lysed and ts-O45-G analyzed for endo H sensitivity after a chase of 6 h at 39.5°C; 15°C: temperature was shifted  for 2 h to 15°C before injection at 15°C, and the cells were kept  for an additional 4 h at 15°C before lysis and analysis of endo H  resistance of ts-O45-G. (B) Control and injected cells were  chased for 0 h or 2.5 h at 39.5°C in the presence of 5 μg/ml BFA  before lysis and analysis of endo H resistance of ts-O45-G. Microinjected anti-EAGE, but not anti-110-12 block BFA induced acquisition of endo H resistance of ts-O45-G.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139784&req=5

Figure 1: Biochemical analysis of transfer of Golgi enzymes to the ER. Transfer of Golgi enzymes to the ER was determined biochemically by analyzing acquisition of endo H resistance of tsO45-G arrested in the ER. 500–1000 Vero cells infected with tsO45-VSV were incubated for 1.5 h at 39.5°C, metabolically labeled for 10 min with 35S-met and microinjected at 39.5°C or 15°C with Fab-fragments of anti-EAGE (EAGE), divalent anti–110-12 (110-12), or kept as noninjected controls (n.i.). (A) Noninjected control cells were lysed directly after the pulse with 35S-met and ts-O45-G analyzed for endo H sensitivity (0 h). 39.5°C: control or injected cells were lysed and ts-O45-G analyzed for endo H sensitivity after a chase of 6 h at 39.5°C; 15°C: temperature was shifted for 2 h to 15°C before injection at 15°C, and the cells were kept for an additional 4 h at 15°C before lysis and analysis of endo H resistance of ts-O45-G. (B) Control and injected cells were chased for 0 h or 2.5 h at 39.5°C in the presence of 5 μg/ml BFA before lysis and analysis of endo H resistance of ts-O45-G. Microinjected anti-EAGE, but not anti-110-12 block BFA induced acquisition of endo H resistance of ts-O45-G.
Mentions: To analyze a possible recycling of Golgi enzymes through the ER/IC, Vero cells were infected with tsO45-VSV and kept at nonpermissive temperature (39.5°C, predominantly ER accumulation), or 15°C (IC accumulation). Under these conditions, ts-O45-G arrested in the ER or IC may serve as a substrate for oligosaccharide-processing Golgi enzymes, and transport of these enzymes into the ER/IC can be studied by biochemical analyses of modifications of the oligosaccharide side chains of the viral glycoprotein (Doms et al., 1989). Even after accumulation of ts-O45-G in the ER or IC for up to 6 h no trace of endo H-resistant glycoprotein could be detected in cells injected with anti-EAGE at the respective temperature (as in noninjected control cells; Fig. 1 A). Moreover, when cells were injected with anti-EAGE at 39.5°C and kept for three additional hours at the nonpermissive temperature and then for 3 h at 15°C with ts-O45-G finally accumulated in the IC (anti-EAGE does not inhibit ER to IC transport) no endo H-resistant fraction of the viral glycoprotein could be detected (not shown). Thus, either the recycling of (even a small fraction of) Golgi enzymes through the ER/IC is inhibited by the injected antibodies, or these enzymes, once located at their proper site of function within the Golgi complex, are retained. To investigate directly the role of coatomer in transport from the Golgi complex to the ER/IC in vivo, we used BFA-induced transfer of Golgi enzymes into the ER combined with microinjection of specific antibodies raised against synthetic peptides of β-COP (anti-EAGE and anti-110-12; Pepperkok et al., 1993) and β′-COP (Lowe and Kreis, 1995).

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