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 (PIN) transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, 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 (Sacharomyces cerevisiae) 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.
The Plant Cell
We thank Gerd Jürgens, Sandra Richter, and Sheng Yang He for providing antibodies; Maciek Adamowski, Fernando Aniento, Sebastian Bednarek, Nico Callewaert, Matyás Fendrych, Elena Feraru, and Mugurel I. Feraru for helpful suggestions; Siamsa Doyle for critical reading of the manuscript and helpful comments and suggestions; and Stephanie Smith and Martine De Cock for help in editing and language corrections. We acknowledge the core facility Cellular Imaging of CEITEC supported by the Czech-BioImaging large RI project (LM2015062 funded by MEYS CR) for their support with obtaining scientific data presented in this article. Plant Sciences Core Facility of CEITEC Masaryk University is gratefully acknowledged for obtaining part of the scientific data presented in this article. We acknowledge support from the Fondation pour la Recherche Médicale and from the Institut National du Cancer (J.C.). The research leading to these results was funded by the European Research Council under the European Union's 7th Framework Program (FP7/2007-2013)/ERC grant agreement numbers 282300 and 742985 and the Czech Science Foundation GAČR (GA18-26981S; J.F.); Ministry of Education, Youth, and Sports/MEYS of the Czech Republic under the Project CEITEC 2020 (LQ1601; T.N.); the China Science Council for a predoctoral fellowship (Q.L.); a joint research project within the framework of cooperation between the Research Foundation-Flanders and the Bulgarian Academy of Sciences (VS.025.13N; K.M. and E.R.); Vetenskapsrådet and Vinnova (Verket för Innovationssystem; S.R.), Knut och Alice Wallenbergs Stiftelse via “Shapesystem” Grant 2012.0050 (S.R.), Kempe stiftelserna (P.G.), Tryggers CTS410 (P.G.).
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Kania U, Nodzyński T, Lu Q, et al. The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with sub cellular trafficking in eukaryotes. The Plant Cell. 2018;30(10):2553-2572. doi:10.1105/tpc.18.00127
Kania, U., Nodzyński, T., Lu, Q., Hicks, G. R., Nerinckx, W., Mishev, K., … Friml, J. (2018). The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with sub cellular trafficking in eukaryotes. The Plant Cell. Oxford University Press. https://doi.org/10.1105/tpc.18.00127
Kania, Urszula, Tomasz Nodzyński, Qing Lu, Glenn R Hicks, Wim Nerinckx, Kiril Mishev, Francois Peurois, et al. “The Inhibitor Endosidin 4 Targets SEC7 Domain-Type ARF GTPase Exchange Factors and Interferes with Sub Cellular Trafficking in Eukaryotes.” The Plant Cell. Oxford University Press, 2018. https://doi.org/10.1105/tpc.18.00127.
U. Kania et al., “The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with sub cellular trafficking in eukaryotes,” The Plant Cell, vol. 30, no. 10. Oxford University Press, pp. 2553–2572, 2018.
Kania U, Nodzyński T, Lu Q, Hicks GR, Nerinckx W, Mishev K, Peurois F, Cherfils J, De RRM, Grones P, Robert S, Russinova E, Friml J. 2018. The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with sub cellular trafficking in eukaryotes. The Plant Cell. 30(10), 2553–2572.
Kania, Urszula, et al. “The Inhibitor Endosidin 4 Targets SEC7 Domain-Type ARF GTPase Exchange Factors and Interferes with Sub Cellular Trafficking in Eukaryotes.” The Plant Cell, vol. 30, no. 10, Oxford University Press, 2018, pp. 2553–72, doi:10.1105/tpc.18.00127.