Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

Abstract

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.T he EXO70 (exocyst component of 70 kDa) protein is a component of the evolutionarily conserved octameric exocyst complex that tethers post-Golgi vesicles to the plasma membrane before SNARE-mediated membrane fusion (1). As an important component of the exocyst complex that mediates exocytosis, EXO70 regulates, for example, neurite outgrowth, epithelial cell polarity establishment, cell motility, and cell morphogenesis in animal cells (2-6). In plants, EXO70 proteins participate in polarized pollen tube growth, root hair growth, deposition of cell wall material, cell plate initiation and maturation, defense, and autophagy (7-12). In humans, EXO70 mediates the trafficking of the glucose transporter Glut4 to the plasma membrane that is stimulated by insulin and involved in the development of diabetes (13). A specific isoform of human EXO70 is also involved in cancer cell invasion (13-15). Endosidin2 (ES2) was identified from a plantbased chemical screen as an inhibitor of trafficking. We demonstrate that the target of ES2 is the EXO70 subunit of the exocyst and that ES2 is active in plants and mammalian systems. Significantly, no inhibitor of the exocyst complex has been reported, yet such compounds could be important for understanding the basic mechanisms of exocyst-mediated processes, for modifying secretion in biotechnological applications, and for the development of potential new drugs with higher affinity and more potent activity to control exocyst-related diseases. Results ES2 InhibitsTrafficking to the Plasma Membrane. ES2 is a previously identified plant endomembrane trafficking disruptor (Fig. 1A) that inhibits polarized growth of pollen tubes in a dose-dependent manner (Fig. S1 A and B) (16). Arabidopsis seedlings grown on media containing ES2 have shorter roots and fewer and shorter root hairs and are less sensitive to gravity stimulation (Fig. S1 C-G). ES2 disrupted the trafficking of proteins that are actively recycled between the plasma membrane and endosome...

EXO70C2 Is a Key Regulatory Factor for Optimal Tip Growth of Pollen

“…365 Second, we performed a direct comparison to another EXO70 isoform -EXO70A1, 377 which is a sporophytic experimentally-proven exocyst subunit with described PM localization 378 (Drdová et al, 2013;Zhang et al, 2016). Imaging in root hairs showed 379 a PM decoration of developing root hairs by pUBQ10::RFP:EXO70A1, while 380 pEXO70C2::EXO70C2:GFP was again found only in the cytoplasm in the same cell (Figure 7 …”

Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth

“…Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al, 2005;Synek et al, 2006;Hála et al, 2008;Fendrych et al, 2010;Kulich et al, 2010;Pecenková et al, 2011;Safavian and Goring, 2013;Wu et al, 2013;Safavian et al, 2015;Zhang et al, 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al, 2005;Hála et al, 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al, 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al, 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al, 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013;Safavian et al, 2015) have implicated the exocyst in polarized exocytosis in plants. Given their function, it was likely that exocyst subunits could be used as markers for polarized exocytosis.…”

Plant Chemical Genetics: From Phenotype-Based Screens to Synthetic Biology

“…The small molecules ES3, ES5, and ES7 affect cell polarity, vacuolar targeting and recycling, and callose deposition during cell plate maturation, respectively (Drakakaki et al, 2011;Park et al, 2014). Recently, ES2 has been found to inhibit exocytosis in plants and human cells and to target the EXO70 subunit of the exocyst complex (Zhang et al, 2016). ES8 affects secretory pathways, exclusively toward the basal plasma membrane of the cell, thereby affecting PIN-FORMED1 trafficking and auxin distribution (Doyle et al, 2015), whereas ES16 specifically perturbs apically localized proteins through regulation of the small GTPase RabA proteins (Li et al, 2017).…”

“…This method often is used in drug discovery (Ziegler et al, 2013), but is less popular in plant chemical genetics (Dejonghe and Russinova, 2014). One of the few recent examples of affinity-based target identification in plant chemical biology was obtained from the ES2 MoA study, in which a biotin-tagged ES2 analog was used to identify EXO70 as an ES2 target (Zhang et al, 2016). Despite recent progress, direct target identification still remains the most challenging, and painstaking, step in chemical genetics biology projects.…”