GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5.
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 beta-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.
Claude
A
A
Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Zhao
B P
BP
Kuziemsky
C E
CE
Dahan
S
S
Berger
S J
SJ
Yan
J P
JP
Armold
A D
AD
Sullivan
E M
EM
Melançon
P
P
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D020756:Coatomer Protein; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D004351:Drug Resistance / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D020756:Coatomer Protein; D005656:Fungal Proteins; C120445:GBF1 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D011506:Proteins; C066755:Sec7 guanine nucleotide exchange factors; D020126:Brefeldin A; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; C486478:Arf5 protein, rat; D008274:Magnesium