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© The Rockefeller University Press, 0021-9525/1999//71 $5.00
The Journal of Cell Biology, Volume 146, Number 1, , 1999 71-84

Original Article

Gbf1

: A Novel Golgi-Associated Bfa-Resistant Guanine Nucleotide Exchange Factor That Displays Specificity for Adp-Ribosylation Factor 5



Alejandro Claudea, Bao-Ping Zhaoa, Craig E. Kuziemskya, Sophie Dahanb, Scott J. Bergerc, Jian-Ping Yanc, Adrian D. Armoldc, Eric M. Sullivanc, and Paul Melançona

a Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7
b Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 2B2
c Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309

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.

Key Words: Golgi complex • Brefeldin A • Sec7 • ADP-ribosylation factor • protein traffic

© 1999 The Rockefeller University Press

1.used in this paper: ARF, ADP-ribosylation factor; ARF-GEF, ARF-specific guanine nucleotide exchange activity; BFA, Brefeldin A; DPBS, Dulbecco's PBS; EBNA, Epstein-Barr nuclear antigen; G6PDH, glucose-6-phosphate dehydrogenase; GBF, Golgi-specific Brefeldin A resistance factor; KLH, keyhole limpet hemocyanin; LB, Luria-Bertani medium; NRK, normal rat kidney; PA, polyacrylamide; PNS, postnuclear supernatant

The current address of S. Dahan is Mayo Clinic, Rochester, MN 55905. The current address of J. Berger is Pacific Northwest National Lab, Richland, WA 99352. The current address of J.-P. Yan is Department of Biochemistry, Colorado State University, Fort Collins, CO 80523. Address correspondence to Paul Melançon, Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7. Tel.: (780) 492-6183. Fax: (780) 492-0450. E-mail: paul.melancon@ualberta.ca


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