The Inhibitor Endosidin 4 Targets SEC7 Domain-Type ARF GTPase Exchange Factors and Interferes with Subcellular Trafficking in Eukaryotes

Abstract

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in , we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast () mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.

All Roads Lead to Auxin: Post-translational Regulation of Auxin Transport by Multiple Hormonal Pathways

“…Genetic and pharmacological perturbations of endocytosis exhibit dramatic effects on BFA compartmentation of PIN proteins. In particular, this has been reported for the coat protein clathrin, putative clathrin uncoating factors AUXILIN-LIKEs, GNOM, and other BFA-sensitive ARF-GEFs, the ARF-GTPase-activating protein VASCULAR NETWORK DEFECTIVE3, and the small GTPase Rab1b ( Geldner et al., 2001 , Geldner et al., 2003 , Kitakura et al., 2011 , Feraru et al., 2012 , Adamowski et al., 2018 , Kania et al., 2018 , Mishev et al., 2018 , Dejonghe et al., 2019 ). Notably, clathrin-mediated endocytosis, together with de novo protein synthesis, is essential for PIN2 polarity re-establishment post cytokinesis ( Glanc et al., 2018 ).…”

Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection

“…Arabidopsis encodes eight ARF-GEFs, each of which contains a Brefeldin A (BFA)-inhibited Sec7 catalytic domain that activates ARF proteins by exchanging GDP for GTP [40]. These are divided into two subfamilies: the GGG class which includes GNOM, GNOM-LIKE1(GNL1), and GNL2; and the BIG (BFA-inhibited guanine nucleotide exchange proteins) class which includes five members (BIG1-5) [39].…”

“…TGN/EE serves as a key sorting station at the intersection of secretory and endocytic pathways [27]. ARF-GEF proteins play a critical role in vesicle budding and vesicular protein sorting [39,40]. In the present study, the TGN/EE-localized AtMIN7, an Arabidopsis ARF-GEF, is discovered to be critical for the cutin formation, which may play important roles in the sorting, assembling, or delivering cutin precursors or other related important components, such as the proteins and enzymes, involved in cutin formation through the intracellular trafficking pathway.…”

“…ARF proteins switch between the active (GTP-bound) and inactive (GDP-bound) states. Upon activation, ARFs bind to membranes and promote vesicle budding at the TGN, endosomal compartments, and PM [39,40]. Highlighting the importance of AtMIN7 for plant immunity, the HopM1 virulence effector from Pto DC3000 contributes to bacterial virulence by interacting with and triggering proteasome-mediated degradation of AtMIN7 [35,36].…”

Emerging strategies for the identification of protein–metabolite interactions

“…While these techniques have been used in microbial and animal cells for a number of years, SPROX, CETSA/TPP, and DARTS/LiP-SMap have only recently been adapted to plant cells. Published examples in plants include (i) DARTS validation of the interaction between the drug endosidin2 and its protein target, Arabidopsis exocyst complex subunit EXO70 (Zhang et al , 2016), (ii) thermal proteome profiling characterization of the Arabidopsis Mg-ATP interactome (Volkening et al , 2019), and (iii) DARTS validation of the association between the drug endosidin4 and Arabidopsis ADP-ribosylation factor guanine nucleotide exchange factors (ARF-GEFs) (Kania et al , 2018).…”