Melançon, Paul - University of Alberta - Department of Cell Biology

研究领域

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 determine if class I and class II Arfs regulate different processes. 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]. Our working hypothesis is that the multiplicity of Arfs and their regulatory GEFs, play a critical role in providing selectivity to the process of coat recruitment, and ultimately cargo sorting. We have already 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]. Further evidence of specialization for Arfs and their GEFs comes from the recent demonstration that class II Arfs, Arf4 in particular, can associate with membranes in the GDP-bound and do so selectively on membranes of the ERGIC [Chun et al. 2008]. We are currently using a combination of live cell microscopy, as well as biochemical and genetic methods to examine the regulation and recruitment of Arfs and their GEFs onto unique membranes. 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. Specific projects include examining (1) the relative function of BIG1 and BIG2 in cargo sorting to and from the TGN, (2) the recruitment and function of Arf4 at ERGIC and of Arf3 at the TGN, (3) the relative function and partners of each domains of GBF1, (4) the role of Arfs and lipids in regulating the localization and function of BRAG1 isoforms at the plasma membrane and endosomes.

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Boulay, P.L, Cotton. M, Melançon, P. and Claing, A. (2008) ARF1 controls the activation of the PI3K pathway to regulate EGF dependent growth and migration of breast cancer cells, J Biol. Chem., 283(52): 36425-36434. Chun, J., Shapovalova, Z., Dejgaard, S.Y., Presley, J.F., and Melançon, P. (2008) Characterization of class I and II Arfs in live cells: GDP-bound class II Arfs associate with the ER-Golgi intermediate compartment independently of GBF1. Molec. Biol Cell 19(8): 3488-500. 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. Dunphy, J.L., Moravec, R., Ly, K., Lasell, T.K., Melançon, P., and Casanova, J.E. (2006) The Arf6 GEF GEP100/BRAG2 regulates cell adhesion by controlling endocytosis of b1 integrins. Current Biology 16: 315-320. 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, PNAS 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.