Localization of Large ADP-Ribosylation Factor-Guanine Nucleotide Exchange Factors to Different Golgi Compartments: Evidence for Distinct Functions in Protein Traffic

Abstract

Activation of several ADP-ribosylation factors (ARFs) by guanine nucleotide exchange factors (GEFs) regulates recruitment of coat proteins (COPs) on the Golgi complex and is generally assumed to be the target of brefeldin A (BFA). The large ARF-GEFs Golgi-specific BFA resistance factor 1 (GBF1) and BFA-inhibited GEFs (BIGs) localize to this organelle but catalyze exchange preferentially on class II and class I ARFs, respectively. We now demonstrate using quantitative confocal microscopy that these GEFs show a very limited overlap with each other (15 and 23%). In contrast, GBF1 colocalizes with the cis-marker p115 (86%), whereas BIGs overlap extensively with TGN38 (83%). Consistent with these distributions, GBF1, but not BIG1, partially relocalized to peripheral sites after incubation at 15 degrees C. The new GBF1 structures represent peripheral vesicular tubular clusters (VTCs) because 88% of structures analyzed stained for both GBF1 and p115. Furthermore, as expected of VTCs, they rapidly reclustered to the Golgi complex in a microtubule-dependent manner upon warm-up. These observations suggest that GBF1 and BIGs activate distinct subclasses of ARFs in specific locations to regulate different types of reactions. In agreement with this possibility, COPI overlapped to a greater extent with GBF1 (64%) than BIG1 (31%), whereas clathrin showed limited overlap with BIG1, and virtually none with GBF1.

Dynamics of GBF1, a Brefeldin A-Sensitive Arf1 Exchange Factor at the Golgi

“…Fractionation experiments also have shown GBF1 to be distributed in both membrane and cytosolic pools (Claude et al, 1999). GBF1 has been described previously to localize to endoplasmic reticulum (ER) exit sites, VTC/intermediate compartment structures, and early Golgi compartments (Claude et al, 1999;Kawamoto et al, 2002;Zhao et al, 2002;. YFP-GBF1, like endogenous GBF1, colocalizes with p58 at the Golgi and at peripheral sites ( Figure 1).…”

“…GBF1 is a high-molecular-weight Arf GEF that at steady state is localized to early Golgi compartments in mammalian cells Zhao et al, 2002;. We constructed a yellow fluorescent protein (YFP)-tagged version of GBF1 to assess whether GBF1 associates stably or transiently with Golgi membranes.…”

Early embryonic lethality in gene trap mice with disruption of the Arfgef2 gene

“…All ARFGEFs identified so far possess a Sec7 domain composed of 200 amino acids as a minimum unit for the catalysis of replacement of (Ramaen et al 2007;Ishizaki et al 2008). So far in mammals only one member of the Gea/GBF group, the GBF1 has been described, that primarily functions in the trafficking between the cisGolgi and ER-Golgi intermediate compartment (Claude et al 1999;Kawamoto et al 2002;Zhao et al 2002;Garcia-Mata et al 2003). From the Sec7/BIG group BIG1 and BIG2 proteins were purified from bovine brain together with macromolecular complexes (>670 kDa) (Morinaga et al 1996).…”

Mutations in a Highly Conserved Region of the Arf1p ActivatorGEA2Block Anterograde Golgi Transport but Not COPI Recruitment to Membranes

“…The Sec7 domain is sufficient to catalyze exchange of GTP for GDP on class I Arf proteins such as Arf1 and Arf3 in humans, and Arf1p and Arf2p in Saccharomyces cerevisiae. Two subfamilies of Arf GEFs localize to the Golgi complex in both yeast and mammalian cells: GBF1 and its yeast homologues Gea1p and Gea2p localize at steady state to the early Golgi, and Sec7p and its mammalian homologues BIG1 and BIG2 localize at steady state to the late Golgi (Zhao et al, 2002;Shin and Nakayama, 2004). In yeast, Gea1p and Gea2p are functionally redundant in that one or the other can be deleted with no detectable effect on growth rate or rates of transport to the cell surface and vacuole, whereas a gea1⌬ gea2⌬ double mutant is inviable (Peyroche et al, 1996.…”