Regulation of protein traffic to and through the Golgi complex
My laboratory investigates the regulation of protein traffic within the secretory pathway of animal cells. Extensive bi-directional traffic between these organelles involves the specific recruitment of coat proteins for “cargo sorting” and “membrane deformation”. Small GTPases of the Arf family and their regulatory guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) play a critical role in the recruitment of these protein coats. Although originally identified as required cofactors for the ADP-ribosylation of Gs by cholera toxin, these proteins have been shown to regulate intracellular vesicle transport throughout the eukaryotic secretory pathway. Six Arfs have been identified in mammals, and except for Arf6, which acts preferentially at the plasma membrane, specific activities for the Golgi-associated Arfs 1-5 have yet to be established. We previously demonstrated that Arfs are required for the formation of coated vesicles on Golgi membranes [Taylor et al. 1992, 1994] and these studies are being extended in order to examine the mechanism of Arf activation at the Golgi.
Fig. 1. GBF1 and BIG1 localize to cis- and trans-compartments of the Golgi complex, respectively
. NRK cells were processed for immuno-fluorescence using antibodies raised against the indicated antigens.Panel A The X-Y slice shown in the centre demonstrates that GBF1 and BIG1 localize to distinct sub-compartments. Panel B Localization of Arf-GEFs was compared to that of markers of the cis- (p115) and trans- (TGN38) Golgi. [from Zhao et al, 2002].
Arf activity is itself regulated by guanine nucleotide exchange factors, or GEFs, that catalyze exchange of GDP for GTP and thus promote association of Arfs with Golgi membranes. The drug Brefeldin A (BFA) blocks this recruitment, disrupts traffic between organelles and provided us with an entry point to dissect the process. Those studies identified a Golgi-specific BFA resistance factor, or GBF1, whose overexpression allows growth in the presence of BFA [Claude et al, 1999]. This 206 kDa protein contains a Sec 7 domain which displays Arf-specific GEF activity. Subsequent work established that GBF1 activity is sensitive to BFA in vivo and is largely responsible for the effects of this drug on protein traffic [Zhao et al. 2006]. We have also characterized members of two other related Arf-GEF sub-families, the BFA inhibited Arf-GEFs or BIGs, and the BFA-resistant Arf-GEFs or BRAGs. These studies established that Arf-GEFs such as GBF1 and BIGs associate with specific cellular compartments [see Figure 1 above; Zhao et al. 2002, 2006].
Fig. 2. BIGs depletion blocks sorting to the TGN but does not prevent assembly of a polarized Golgi stack. Panels show confocal slices of Hela cells treated with control (left panels) or siRNAs that target BIG1 and BIG2 (right panels). Knockdown of BIGs (panel B) completely blocked the monensin-induced redistribution of galactosyl transferase (green) from the Golgi stack (mannosidase II, red) readily observed in control cells (panel A). Surprisingly, knockdown of BIGs (center panels) and consequent loss of the TGN had no impact on assembly on a polarized Golgi stack (panels C & D) in which a cis-Golgi marker (p115, red) remains well-resolved from galactosyl transferase (green).
We demonstrated that GBF1 and BIGs regulate the recruitment of distinct coat proteins [Manolea et al, 2008]. GBF1 localizes to cis-Golgi compartments where it is required for recruitment of the COP1 coat and assembly of the Golgi complex. In contrast, the trans-Golgi localized BIGs appear essential for recruitment of clathrin adaptors and maintenance of the TGN; surprisingly BIGs depletion using RNA oligos revealed they are not required for assembly of a polarized Golgi stack (see Figure 2 above; [Manolea et al., 2008]).
We are currently using a combination of live cell microscopy, as well as biochemical methods to examine the regulation and recruitment of Arf-GEFs onto unique membranes. We first established using several methods that the substrate Arf-GDP promotes the recruitment of GBF1 onto membranes. We then developed a cell-free assay that reconstitutes this Arf-GDP sensitive recruitment. Our ultimate goal is to demonstrate that Arf-GEFs interact with unique sets of components on the membrane, possibly involving higher order transient complexes with effectors. Towards this goal we have performed a proximity biotinylation experiment with GBF1 tagged with BirA*. Current efforts are focused on characterizing targets identified by this approach.
Quilty, D., Gray, F., Summerfeldt, N., Cassel, D. and Melançon, P. (2014) Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1, J. Cell Science, 127: 354-364.
Manolea, F., Claude, A., Chun, J., Rosas, J. and Melançon, P. (2008). Distinct functions for Arf nucleotide exchange factors at the Golgi complex: GBF1 and BIGs are required for assembly and maintenance of the Golgi stack and TGN, respectively. Molec. Biol Cell, 19: 19: 523-535.
Zhao, X., Claude, A., Chun, J., Shields, D.J., and Melançon, P. (2006) GBF1, a cis-Golgi and ERGIC localized ARF-GEF implicated in ER-to-Golgi protein traffic. J. Cell Science. 119: 3743-3753.
Zhao, X, Lasell, T.R.K. and Melançon, P. (2002) Localization of large ARF-GEFs to different Golgi compartments: Evidence for distinct functions in protein traffic. Molec. Biol Cell, 13: 119-23.
Claude, A., Zhao, B.P., Kuziemsky, C.E., Dahan, S., Berger, S.J., Yan, J.P., Armold, A. Sullivan, E.M., and Melançon, P. (1999) GBF1: A novel Golgi-associated BFA resistant guanine nucleotide exchange factor for ADP-ribosylation factors, J. Cell Biol. 146, 71-84.
Mansour, S.J., Skaug, J., Zhao, X.-H., Giordano, J., Scherer, S.W., Melançon, P. (1999) p200 ARF-GEP1: A Golgi- localized Guanine Nucleotide Exchange Factor whose Sec7 Domain is Targeted by the Drug Brefeldin A, Proc. Nat. Acad. Sci. 96, 7968-7973.
Yan, J.P., Colon, M.E., Beebe, L.A., and Melançon, P. (1994) Isolation and characterization of CHO cell lines with compartment-specific resistance to Brefeldin A., J. Cell Biol. 126: 65-75.
Taylor, T.C., Kanstein, M., Weidman, P, and Melançon, P. (1994) Cytosolic Arfs are required for vesicle formation but not for cell-free intra-Golgi transport: Evidence for coated vesicle-independent transport. Molec. Biol. Cell, 5, 237-252.
Taylor, T.C., Kahn, R., and Melançon, P. (1992) Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport. Cell 70: 69-80.