p200 ARF–GEP1: A Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A

Abstract

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 sensitivity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53͞ 58. Analysis of several truncated forms mapped the Golgilocalization signal to the N-terminal third of p200. BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5-[␥-thio]triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 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 disruption of Golgi morphology. p200 mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…29,36 Unlike in cytohesins, this extended PH domain is not autoinhibitory. 29,46 The crystal structure of a human BRAG2 construct comprising the Sec7 and PH domains in complex with Arf showed that the PH domain forms extensive intramolecular interactions with the Sec7 domain.…”

Chlamydial Intracellular Survival Strategies

“…BFA inhibits activation of Arf1 (a key player in vesicle formation) by targeting its activators, the GEFs GBF1 and BIG1/2 (Claude et al 1999;Mansour et al 1999;Yamaji et al 2000;Kawamoto et al 2002), which are localized to the cis-and trans-Golgi, respectively, and have distinct functions at the Golgi (Manolea et al 2008). Recent studies reveal that Chlamydia uses GBF1 (but not BIG1/2) to acquire sphingomyelin, which is necessary for inclusion membrane growth and stability but not bacterial replication (Elwell et al 2011).…”

Sponge Bcl-2 homologous protein (BHP2-GC) confers distinct stress resistance to human HEK-293 cells

“…After digestion with KpnI and XbaI the fragment was subcloned into the KpnI and XbaI sites of the plasmid pcDNA3 (Invitrogen). Cells were transfected using the FuGENE 6 reagent following the procedure described by Mansour et al 63 using the pcDNA3 containing the GCBHP2 in transcriptional orientation. As a control, mock-transfection was performed with the pcDNA3 without the GCBHP2 insert.…”