ADP-ribosylation factor machinery mediates endocytosis in plant cells

Abstract

Endocytosis is crucial for various cellular functions and development of multicellular organisms. In mammals and yeast, ADP-ribosylation factor (ARF) GTPases, key components of vesicle formation, and their regulators ARF-guanine nucleotide exchange factors (GEFs) and ARF-GTPase-activating protein (GAPs) mediate endocytosis. A similar role has not been established in plants, mainly because of the lack of the canonical ARF and ARF-GEF components that are involved in endocytosis in other eukaryotes. In this study, we revealed a regulatory mechanism of endocytosis in plants based on ARF GTPase activity. We identified that ARF-GEF GNOM and ARF-GAP VASCULAR NETWORK DEFECTIVE 3 (VAN3), both of which are involved in polar auxin transport-dependent morphogenesis, localize at the plasma membranes as well as in intracellular structures. Variable angle epifluorescence microscopy revealed that GNOM and VAN3 localize to partially overlapping discrete foci at the plasma membranes that are regularly associated with the endocytic vesicle coat clathrin. Genetic studies revealed that GNOM and VAN3 activities are required for endocytosis and internalization of plasma membrane proteins, including PIN-FORMED auxin transporters. These findings identified ARF GTPase-based regulatory mechanisms for endocytosis in plants. GNOM and VAN3 previously were proposed to function solely at the recycling endosomes and trans-Golgi networks, respectively. Therefore our findings uncovered an additional cellular function of these prominent developmental regulators.vesicle trafficking | plant development | polarity

The dynamics of plant plasma membrane proteins: PINs and beyond

“…DRP1A also interacts with the ARF-GAP VAN3, which, together with GNOM, localizes to clathrin foci at the plasma membrane, thus linking ARF activity to CME . Consistently, mutants in components of the ARF machinery, including GNOM, GNL1, VAN3 and ARF1, are impaired in endocytosis and internalization of plasma membrane proteins, including PINs (Xu and Scheres, 2005;Teh and Moore, 2007;Naramoto et al, 2010;Irani et al, 2012). The apparently widespread function of ARF GTPase-based signaling in both intracellular compartments and CME explains the strong developmental alterations associated with these mutants (Geldner et al, 2003(Geldner et al, , 2004Xu and Scheres, 2005;Richter et al, 2007;Teh and Moore, 2007;Naramoto et al, 2010).…”

“…VAN3 localizes to the TGN/EE and appears to modulate intracellular PIN distribution in response to BFA Sieburth et al, 2006). Furthermore, VAN3 and GNOM have been implicated in the regulation of cargo endocytic sorting and transcytosis (Naramoto et al, 2010) (see below), highlighting the versatility of the small GTP-binding protein molecular toolbox found in higher plants (Fig. 3).…”

“…In addition, BFA treatment of plants expressing a mutant pin allele mimicking constitutive phosphorylation revealed reduced BFA sensitivity of the PIN1 variant, indicating that phosphorylated PINs do not represent preferred targets for GNOM ARF-GEFmediated protein sorting (Kleine-Vehn et al, 2009). In light of the various sorting decisions mediated by the GNOM-and PIDdependent pathways (Geldner et al, 2003;Kleine-Vehn et al, 2008bNaramoto et al, 2010;Ding et al, 2011;Rakusova et al, 2011), variations in PIN phosphorylation could thus play a key role during distinct developmental responses by modulating the efficiency with which PINs enter either GNOM-dependent or -independent trafficking routes. This predicts the existence of mechanisms that sense and transmit variations in the phosphorylation status of PINs into variations in their intracellular sorting.…”

Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development

“…To visualize PIN1-GFP endocytosis, we inhibited endocytic recycling using the fungal toxin brefeldin A (BFA), which causes the heterogeneous aggregation of internalized PIN1 in intracellular compartments (known as 'BFA bodies') (Pan et al, 2009;Naramoto et al, 2010). As expected, the aggregation of PIN1-GFP in BFA bodies was clearly observed in the wild type when treated with BFA (Fig.…”

Salicylic acid interferes with clathrin-mediated endocytic protein trafficking

“…We visualized the auxin transporters PIN1 and PIN2 or the aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN2 (PIP2) that constitutively undergo cycles of endocytosis and recycling back to the PM (17). This recycling (18) and, to a lesser extent, endocytosis (19) are inhibited by the trafficking inhibitor brefeldin A (BFA). The imbalance in recycling and endocytosis caused by the BFA treatment results in intracellular accumulation of internalized PM proteins, which end up in BFA-induced aggregations of endosomes, called BFA bodies (20).…”