p200 ARF-GEP1: A Golgi-localized guanine nucleotide exchange protein whoseSec7 domain is targeted by the drug brefeldin A

The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism. Here, we investigated the cellular localization and BFA sensitiv ity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleot ide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structu res that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGICS3/58. Analysis of several truncated fo rms mapped the Golgi-localization signal to the N-terminal third of p200. B FA treatment of transiently or stably transfected cells resulted in the red istribution of Golgi markers and in loss of cell viability, thereby indicat ing that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of gu anosine 5'-[gamma-thio] triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete w ith ARF for interaction with p200 but, rather, acted as an uncompetitive in hibitor that only targeted the p200-ARF complex with an inhibition constant of 7 mu M. On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disrupt ion of Golgi morphology, p200 mapped to chromosome 8q13, 3.56 centirays fro m WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.