GBF1: A Novel Golgi-Associated BFA-Resistant Guanine Nucleotide Exchange Factor That Displays Specificity for ADP-Ribosylation Factor 5

• Published in: The Journal of cell biology Vol. 146; no. 1; pp. 71 - 84
• Main Authors: Claude, Alejandro, Zhao, Bao-Ping, Kuziemsky, Craig E., Dahan, Sophie, Berger, Scott J., Yan, Jian-Ping, Armold, Adrian D., Sullivan, Eric M., Melançon, Paul
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 12-07-1999; The Rockefeller University Press
• Subjects: ADP-Ribosylation Factors; Amino Acid Sequence; Animals; Biochemistry; Biological Transport - drug effects; Brefeldin A - pharmacology; CDNA libraries; Cell Division - drug effects; Cell lines; Cells; Cellular biology; CHO Cells; Cloning, Molecular; Coatomer Protein; Complementary DNA; Cricetinae; Cytosol - metabolism; Cytosol - ultrastructure; Drug Resistance - genetics; Fungal Proteins - chemistry; Fungal Proteins - metabolism; Gene Expression; Genetics; Golgi apparatus; Golgi Apparatus - drug effects; Golgi Apparatus - metabolism; Golgi Apparatus - ultrastructure; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; Guanine Nucleotide Exchange Factors; Humans; Intracellular Membranes - metabolism; Intracellular Membranes - ultrastructure; Libraries; Magnesium - pharmacology; Male; Membrane Proteins - metabolism; Molecular Sequence Data; Nucleotides; Original; P branes; Plasmids; Proteins; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Rats; Rats, Sprague-Dawley
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.146.1.71

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi-enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+ concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the β-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.