Regulation of trafficking pathways by ARF-GEFs and ARFs

The Arabidopsis genome encodes eight 150-220kDa large ARF-GEFs that are members of a conserved eukaryotic protein family comprising two classes (GGG, BIG): 3 (GNOM, GNL1 and GNL2) are related to the human cis-Golgi ARF-GEF GBF1 whereas 5 (BIG1-5) are related to the human trans-Golgi ARF-GEF BIG1 (Fig. 2b; Anders & Jürgens, 2008). GNOM and GNL1 jointly regulate the retrograde COPI-mediated traffic from the Golgi to the ER, which is the ancient eukaryotic function of the GBF1 class (Fig. 2e,f; Richter et al., 2007). Inhibition of GNOM activity by the fungal toxin brefeldin A (BFA) in the gnl1 mutant blocks the early secretory pathway, resulting in the retention of secretory cargo proteins in the ER. Two additional plant-specific ARF-GEF functions have evolved: GNOM mediates polar recycling of the auxin-efflux transporter PIN1 from endosomes to the basal plasma membrane, which is required for cell polarity and axis formation in embryogenesis (Fig. 2c,d; Geldner et al., 2003), whereas GNL2 plays a specific role in pollen germination and pollen-tube growth (Richter et al., 2012). The ARF-GEFs BIG1 to BIG4 jointly perform an essential function in the late secretory pathway whereas the role of BIG5 is not clear (Richter et al., unpubl.).

Figure 2 ARF-GEFs and vesicle formation (b) The Arabidopsis family of ARF-GEFs related to human GBF1 and BIG1. (c,d) GNOM (green) regulates polar recycling of PIN1 (red) from endosomes to the basal plasma membrane. (c) BFA-induced inhibition of GNOM activity causes accumulation of GNOM and its cargo PIN1 in BFA compartments. (d) No BFA effect in BFA-resistant GNOM plants. (e,f) GNL1 and GNOM regulate retrograde Golgi-ER traffic. BFA treatment prevents Golgi-membrane association of COPI (red) in gnl1 mutant (e) but not in wild-type (BFA-resistant GNL1) (f). Blue, DAPI-stained nuclei.

Our research aims to analyse mechanisms underlying the pathway specificity of ARF-GEF action. To this end, we analyse how large ARF-GEFs associate with specific membrane compartments and attempt to identify their endogenous ARF substrates as well as the associated coat proteins for cargo selection.