CHARACTERIZATION OF BREFELDIN-A INDUCED VESICULAR STRUCTURES CONTAINING CYCLING PROTEINS OF THE INTERMEDIATE COMPARTMENT CIS-GOLGI NETWORK/

Residence of luminal ER proteins is mediated by a cyclic process which involves binding of escaped proteins to a KDEL receptor in a post-ER compartment and redistribution of the ligand-receptor complex back to the ER. We examined the relocation of the KDEL receptor after treatmen t with the fungal metabolite brefeldin A and compared this with the re trograde transport of the KDEL receptor observed after ligand or recep tor overexpression. Incubation with brefeldin A led to the formation o f vesicular structures containing the KDEL receptor and ERGIC-53, a ma rker for the ER-Golgi intermediate compartment. Immunoelectron microsc opy revealed that these structures are composed of tubulo-vesicular cl usters. The brefeldin A induced vesicular structures were morphologica lly and biochemically distinct from the ER-Golgi hybrid compartment as demonstrated by double immunofluorescence microscopy and subcellular fractionation. Overexpression of the receptor itself or a lysozyme-KDE L construct led to a shift of the KDEL receptor together with ERGIC-53 , an intermediate compartment marker to the ER but not to structures r esembling BFA induced vesicular structures. Moreover, overexpression o f the receptor resulted in the partial redistribution of marker protei ns of the medial Golgi and the trans-Golgi network to ER-like structur es. We conclude that the effects of brefeldin A on the redistribution of the KDEL receptor do not reflect physiological events occurring dur ing increased occupancy of the receptor with ligands. (C) 1997 Federat ion of European Biochemical Societies.