Characterization of brefeldin A induced vesicular structures containing cycling proteins of the intermediate compartment/cis-Golgi network

Abstract

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 treatment with the fungal metabolite brefeldin A and compared this with the retrograde transport of the KDEL receptor observed after ligand or receptor overexpression. Incubation with brefeldin A led to the formation of vesicular structures containing the KDEL receptor and ERGIC-53, a marker for the ER-Golgi intermediate compartment. Immunoelectron microscopy revealed that these structures are composed of tubulo-vesicular clusters. The brefeldin A induced vesicular structures were morphologically 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-KDEL construct led to a shift of the KDEL receptor together with ERGIC-53, an intermediate compartment marker to the ER but not to structures resembling BFA induced vesicular structures. Moreover, overexpression of the receptor resulted in the partial redistribution of marker proteins of the medial Golgi and the trans-Golgi network to ER-like structures. We conclude that the effects of brefeldin A on the redistribution of the KDEL receptor do not reflect physiological events occurring during increased occupancy of the receptor with ligands.