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© The Rockefeller University Press, 0021-9525/1997//319 $5.00
The Journal of Cell Biology, Volume 137, Number 2, , 1997 319-333

Article

Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum



Jochen Scheel, Rainer Pepperkok, Martin Lowe, Gareth Griffiths*, and Thomas E. Kreis

Department of Cell Biology, Sciences III, University of Geneva, Geneva, Switzerland, and * Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Germany

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 β-COP, inhibit BFA-induced redistribution of β-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 β-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 ~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.

Abbreviations used in this paper: BFA, brefeldin A; COP, coat protein; EM, electron microscopy; endo H, endoglycosidase H; IC, intermediate compartment.

R. Pepperkok was a recipient of a European Molecular Biology Organization (EMBO) longterm postdoctoral fellowship and M. Lowe was supported by a Traveling Research Fellowship from the Welcome Trust. T.E. Kreis was supported by grants from the Fonds Nationale Suisse, the Canton de Genève, and the International Human Frontier Science Program.

J. Scheel and R. Pepperkok contributed equally to this work and are first co-authors.


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