GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic.
The formation and maturation of membrane carriers that transport cargo from the ER to the Golgi complex involves the sequential action of the coat protein complexes COPII and COPI. Recruitment of COPI to nascent carriers requires activation of ADP-ribosylation factors by a BrefeldinA-sensitive guanine nucleotide exchange factor. Using new antisera and a GFP-tagged protein, we demonstrate that the exchange factor GBF1 localized to both Golgi membranes and peripheral puncta, near but separate from ER exit sites. Live cell imaging revealed that GFP-GBF1 associates dynamically with both membranes through rapid exchange with a large cytosolic pool. Treatment with BrefeldinA dramatically altered this rapid exchange, causing accumulation of GBF1 on both Golgi and peripheral puncta before eventual redistribution to the ER in a microtubule-dependent manner. Measurement of diffusion coefficients and subcellular fractionation confirmed this shift in GBF1 from cytosolic to membrane bound. BrefeldinA-induced accumulation of GBF1 coincided with loss of COPI from peripheral puncta. Furthermore, recruitment of GBF1 to cargo-containing peripheral puncta coincided with recruitment of COPI, but not COPII. Strikingly, microinjection of anti-GBF1 antibodies specifically caused dissociation of COPI from membranes. These observations strongly suggest that GBF1 regulates COPI membrane recruitment in the early secretory pathway.
Zhao
Xinhua
X
Department of Cell Biology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Claude
Alejandro
A
Chun
Justin
J
Shields
David J
DJ
Presley
John F
JF
Melançon
Paul
P
D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D020756:Coatomer Protein / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D007700:Kinetics; D008845:Microinjections; D015739:Nocodazole / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*
D020756:Coatomer Protein; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors; D015739:Nocodazole