Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking

Abstract

COPI recruitment to membranes appears to be essential for the biogenesis of the Golgi and for secretory trafficking. Preventing COPI recruitment by expressing inactive forms of the ADP-ribosylation factor (ARF) or the ARF-activating guanine nucleotide exchange factor GBF1, or by treating cells with brefeldin A (BFA), causes the collapse of the Golgi into the endoplasmic reticulum (ER) and arrests trafficking of soluble and transmembrane proteins at the ER. Here, we assess COPI function in Golgi biogenesis and protein trafficking by preventing COPI recruitment to membranes by removing GBF1. We report that siRNA-mediated depletion of GBF1 causes COPI dispersal but does not lead to collapse of the Golgi. Instead, it causes extensive tubulation of the cis-Golgi. The Golgi-derived tubules target to peripheral ER-Golgi intermediate compartment (ERGIC) sites and create dynamic continuities between the ERGIC and the cis-Golgi compartment. COPI dispersal in GBF1-depleted cells causes dramatic inhibition of the trafficking of transmembrane proteins. Unexpectedly, soluble proteins continue to be secreted from GBF1-depleted cells. Our findings suggest that a secretory pathway capable of trafficking soluble proteins can be maintained in cells in which COPI recruitment is compromised by GBF1 depletion. However, the trafficking of transmembrane proteins through the existing pathway requires GBF1-mediated ARF activation and COPI recruitment.

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…We tested this prediction by examining the Golgi and ERGIC in cells overexpressing GBF1, a Golgi associated Arf exchange factor. [42][43][44][45] The overexpression of GBF1 was similar to the effect of wild-type ArfGAP1. At lower expression levels, GBF1 was juxtanuclear, associated with the Golgi apparatus.…”

“…C-terminally Myc and His-tagged human GBF1 have been described. 44 The following antibodies were used; mouse monoclonal antibody against Myc epitope (9E10, Santa Cruz), rabbit polyclonal antibody against Myc epitope (Santa Cruz), goat polyclonal antibody against GM130 (Santa Cruz), mouse monoclonal antibody against ERGIC53 (G1/93, Alexis Biochemicals), mouse monoclonal antibody against GM130 (BD), rabbit polyclonal antibody against ERGIC53 (Sigma). Polyclonal anti-giantin antibodies were purchased from Covance and rat monoclonal anti-HA antibodies were from Roche Applied Science.…”

“…44 Cells 16 h posttransfection were washed in phosphate-buffered saline (PBS), fixed in 3% paraformaldehyde for 10 min and quenched with 10 mM ammonium chloride. Cells were permeabilized with 0.1% Triton-X-100 in PBS.…”

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

“…The heptameric COPI complex associates with the Golgi membranes, resulting in the budding of COPI-containing vesicles, whose major function is to mediate transport of cellular proteins and cargo from the Golgi to the ER as well as the intra-Golgi transport (20). Depletion of COPI also perturbs the steady-state distribution of the Golgi enzymes (43) and thus inhibits processing of the VSV G protein and its transport to the plasma membrane (44,45). Further, we have found that depletion of COPZ1 subunit had no adverse effect on VSV G protein-mediated entry of pseudotyped HIV (Fig.…”

The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila

“…[9][10][11][12][13] GBF1 is essential for trafficking of cargoes between the ER and the Golgi and GBF1 depletion in cultured cells disrupts trafficking of surface adhesion proteins such as E-selectin ligand I (ESL-1), a broadly expressed protein implicated in leukocyte-endothelial cell interactions.…”