Abstract
BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.
Highlights
Membrane traffic between intracellular organelles in eukaryotic cells is mediated primarily by small vesicles that bud from a donor compartment and fuse with a target compartment to deliver cargo molecules
These observations indicate that BFAinduced adaptor protein (AP)-1 dissociation from trans-Golgi network (TGN) membranes and tubulation of TGN membranes are not coupled events and suggest that a brefeldin A (BFA) target other than ADP-ribosylation factor (ARF)-guanine nucleotide exchange factor (GEF) exists in the cell
Brefeldin A (BFA), which blocks guanine nucleotide exchange on ARF, inhibits the assembly of the COPI and AP-1 complexes onto membranes and induce the formation of membrane tubules from the Golgi complex and the TGN that subsequently fuse with the endoplasmic reticulum and with endosomes, respectively (11–14, reviewed in Refs. 6, 15, and 16)
Summary
Membrane traffic between intracellular organelles in eukaryotic cells is mediated primarily by small vesicles that bud from a donor compartment and fuse with a target compartment to deliver cargo molecules. ARF1-GTP promotes the formation of COPI-coated and clathrin/AP-1-coated vesicles at the pre-Golgi intermediates and at the TGN, respectively, by inducing an assembly of the coat complexes onto membranes and possibly by activating lipid mediators Brefeldin A (BFA), which blocks guanine nucleotide exchange on ARF, inhibits the assembly of the COPI and AP-1 complexes onto membranes and induce the formation of membrane tubules from the Golgi complex and the TGN that subsequently fuse with the endoplasmic reticulum and with endosomes, respectively In contrast to the high molecular weight group, all low molecular weight ARF-GEFs are insensitive to BFA and are believed to be involved in endosomal recycling and cytoskeletal reorganization through activating ARF6 primarily In contrast to the high molecular weight group, all low molecular weight ARF-GEFs are insensitive to BFA and are believed to be involved in endosomal recycling and cytoskeletal reorganization through activating ARF6 primarily (reviewed in Refs. 26 and 27)
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