Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

Department of Biochemistry and Microbiology Faculty of Food and Biochemical Technology University of Chemistry and Technology 166 28 Prague Czech Republic

Department of Experimental Plant Biology Faculty of Science Charles University Prague 128 44 Prague Czech Republic

Institute of Biochemistry and Biophysics Polish Academy of Sciences 02 106 Warsaw Poland

Institute of Experimental Botany Czech Academy of Sciences 165 02 Prague Czech Republic

Institute of Experimental Botany Czech Academy of Sciences 165 02 Prague Czech Republic;

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Konrad S. S. A., Ott T., Molecular principles of membrane microdomain targeting in plants. Trends Plant Sci. 20, 351–361 (2015). PubMed

Sekereš J., Pleskot R., Pejchar P., Žárský V., Potocký M., The song of lipids and proteins: Dynamic lipid-protein interfaces in the regulation of plant cell polarity at different scales. J. Exp. Bot. 66, 1587–1598 (2015). PubMed

Heider M. R., Munson M., Exorcising the exocyst complex. Traffic 13, 898–907 (2012). PubMed PMC

Zárský V., Kulich I., Fendrych M., Pečenková T., Exocyst complexes multiple functions in plant cells secretory pathways. Curr. Opin. Plant Biol. 16, 726–733 (2013). PubMed

Koumandou V. L., Dacks J. B., Coulson R. M., Field M. C., Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins. BMC Evol. Biol. 7, 29 (2007). PubMed PMC

Bloch D., et al. ., Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiol. 172, 980–1002 (2016). PubMed PMC

Pecenková T., et al. ., Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae. Ann. Bot. 120, 437–446 (2017). PubMed PMC

Sekereš J., et al. ., Analysis of exocyst subunit EXO70 family reveals distinct membrane polar domains in tobacco pollen tubes. Plant Physiol. 173, 1659–1675 (2017). PubMed PMC

Marković V., et al. ., EXO70A2 is critical for exocyst complex function in pollen development. Plant Physiol. 184, 1823–1839 (2020). PubMed PMC

Hála M., et al. ., An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell 20, 1330–1345 (2008). PubMed PMC

Kalmbach L., et al. ., Transient cell-specific EXO70A1 activity in the CASP domain and Casparian strip localization. Nat. Plants 3, 17058 (2017). PubMed

Vukašinović N., et al. ., Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis. New Phytol. 213, 1052–1067 (2017). PubMed

Kulich I., et al. ., Exocyst subunit EXO70H4 has a specific role in callose synthase secretion and silica accumulation. Plant Physiol. 176, 2040–2051 (2018). PubMed PMC

Kulich I., et al. ., Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin. New Phytol. 188, 615–625 (2010). PubMed

Drdová E. J., et al. ., The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J. 73, 709–719 (2013). PubMed

Pecenková T., et al. ., The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction. J. Exp. Bot. 62, 2107–2116 (2011). PubMed PMC

Zhang X., Pumplin N., Ivanov S., Harrison M. J., EXO70I is required for development of a sub-domain of the periarbuscular membrane during arbuscular mycorrhizal symbiosis. Curr. Biol. 25, 2189–2195 (2015). PubMed

Fendrych M., et al. ., The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell 22, 3053–3065 (2010). PubMed PMC

Rybak K., et al. ., Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes. Dev. Cell 29, 607–620 (2014). PubMed

Mayers J. R., et al. ., SCD1 and SCD2 form a complex that functions with the exocyst and RabE1 in exocytosis and cytokinesis. Plant Cell 29, 2610–2625 (2017). PubMed PMC

Mao H., Nakamura M., Viotti C., Grebe M., A framework for lateral membrane trafficking and polar tethering of the PEN3 ATP-binding cassette transporter. Plant Physiol. 172, 2245–2260 (2016). PubMed PMC

Fendrych M., et al. ., Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol. Biol. Cell 24, 510–520 (2013). PubMed PMC

Cvrčková F., et al. ., Evolution of the land plant exocyst complexes. Front. Plant Sci. 3, 159 (2012). PubMed PMC

Žárský V., Cvrčková F., Potocký M., Hála M., Exocytosis and cell polarity in plants – Exocyst and recycling domains. New Phytol. 183, 255–272 (2009). PubMed

Kubátová Z., et al. ., Arabidopsis trichome contains two plasma membrane domains with different lipid compositions which attract distinct EXO70 subunits. Int. J. Mol. Sci. 20, 3803 (2019). PubMed PMC

Kulich I., et al. ., Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 14, 1155–1165 (2013). PubMed

Synek L., et al. ., EXO70C2 is a key regulatory factor for optimal tip growth of pollen. Plant Physiol. 174, 223–240 (2017). PubMed PMC

Saccomanno A., et al. ., Regulation of exocyst function in pollen tube growth by phosphorylation of exocyst subunit EXO70C2. Front. Plant Sci. 11, 609600 (2021). PubMed PMC

Synek L., et al. ., AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development. Plant J. 48, 54–72 (2006). PubMed PMC

Li S., et al. ., Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell type-specific exocytosis. Plant Physiol. 154, 1819–1830 (2010). PubMed PMC

Cole R. A., Synek L., Zarsky V., Fowler J. E., SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol. 138, 2005–2018 (2005). PubMed PMC

Janková Drdová E., et al. ., Developmental plasticity of Arabidopsis hypocotyl is dependent on exocyst complex function. J. Exp. Bot. 70, 1255–1265 (2019). PubMed PMC

Zhang C., et al. ., Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc. Natl. Acad. Sci. U.S.A. 113, E41–E50 (2016). PubMed PMC

Morgera F., et al. ., Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol. Biol. Cell 23, 337–346 (2012). PubMed PMC

Yue P., et al. ., Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion. Nat. Commun. 8, 14236 (2017). PubMed PMC

Lepore D. M., Martínez-Núñez L., Munson M., Exposing the elusive exocyst structure. Trends Biochem. Sci. 43, 714–725 (2018). PubMed PMC

Guo W., Roth D., Walch-Solimena C., Novick P., The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18, 1071–1080 (1999). PubMed PMC

Finger F. P., Hughes T. E., Novick P., Sec3p is a spatial landmark for polarized secretion in budding yeast. Cell 92, 559–571 (1998). PubMed

He B., Xi F., Zhang X., Zhang J., Guo W., Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. EMBO J. 26, 4053–4065 (2007). PubMed PMC

Zhang X., et al. ., Membrane association and functional regulation of Sec3 by phospholipids and Cdc42. J. Cell Biol. 180, 145–158 (2008). PubMed PMC

Pleskot R., Cwiklik L., Jungwirth P., Žárský V., Potocký M., Membrane targeting of the yeast exocyst complex. Biochim. Biophys. Acta BBA - Biomembr. 1848, 1481–1489 (2015). PubMed

Robinson N. G. G., et al. ., Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol. Cell. Biol. 19, 3580–3587 (1999). PubMed PMC

Zhang X., et al. ., Cdc42 interacts with the exocyst and regulates polarized secretion. J. Biol. Chem. 276, 46745–46750 (2001). PubMed

Liu J., Zuo X., Yue P., Guo W., Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells. Mol. Biol. Cell 18, 4483–4492 (2007). PubMed PMC

Kolay S., Basu U., Raghu P., Control of diverse subcellular processes by a single multi-functional lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Biochem. J. 473, 1681–1692 (2016). PubMed PMC

Platre M. P., et al. ., A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev. Cell 45, 465–480.e11 (2018). PubMed

Pleskot R., Pejchar P., Staiger C. J., Potocký M., When fat is not bad: The regulation of actin dynamics by phospholipid signaling molecules. Front. Plant Sci. 5, 5 (2014). PubMed PMC

Simon M. L. A., et al. ., A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat. Plants 2, 16089 (2016). PubMed PMC

Mei K., et al. ., Cryo-EM structure of the exocyst complex. Nat. Struct. Mol. Biol. 25, 139–146 (2018). PubMed PMC

Heider M. R., et al. ., Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat. Struct. Mol. Biol. 23, 59–66 (2016). PubMed PMC

Ahmed S. M., et al. ., Exocyst dynamics during vesicle tethering and fusion. Nat. Commun. 9, 5140 (2018). PubMed PMC

Wu J., et al. ., Regulation of cytokinesis by exocyst subunit SEC6 and KEULE in Arabidopsis thaliana. Mol. Plant 6, 1863–1876 (2013). PubMed

Moore B. A., Robinson H. H., Xu Z., The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst. J. Mol. Biol. 371, 410–421 (2007). PubMed PMC

Dong G., Hutagalung A. H., Fu C., Novick P., Reinisch K. M., The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol. 12, 1094–1100 (2005). PubMed

Hamburger Z. A., Hamburger A. E., West A. P. Jr, Weis W. I., Crystal structure of the S.cerevisiae exocyst component Exo70p. J. Mol. Biol. 356, 9–21 (2006). PubMed

Konopka C. A., Bednarek S. Y., Comparison of the dynamics and functional redundancy of the Arabidopsis dynamin-related isoforms DRP1A and DRP1C during plant development. Plant Physiol. 147, 1590–1602 (2008). PubMed PMC

Ischebeck T., et al. ., Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis. Plant Cell 25, 4894–4911 (2013). PubMed PMC

Barbosa I. C. R., et al. ., Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses. Development 143, 4687–4700 (2016). PubMed

He J.-X., et al. ., Sterols regulate development and gene expression in Arabidopsis. Plant Physiol. 131, 1258–1269 (2003). PubMed PMC

Yamamoto E., et al. ., Multiple lipid binding sites determine the affinity of PH domains for phosphoinositide-containing membranes. Sci. Adv. 6, eaay5736 (2020). PubMed PMC

Im Y. J., et al. ., Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum. Plant Cell 19, 1603–1616 (2007). PubMed PMC

Furt F., et al. ., Polyphosphoinositides are enriched in plant membrane rafts and form microdomains in the plasma membrane. Plant Physiol. 152, 2173–2187 (2010). PubMed PMC

König S., Hoffmann M., Mosblech A., Heilmann I., Determination of content and fatty acid composition of unlabeled phosphoinositide species by thin-layer chromatography and gas chromatography. Anal. Biochem. 378, 197–201 (2008). PubMed

Ischebeck T., Stenzel I., Heilmann I., Type B phosphatidylinositol-4-phosphate 5-kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion. Plant Cell 20, 3312–3330 (2008). PubMed PMC

Pejchar P., Sekereš J., Novotný O., Žárský V., Potocký M., Functional analysis of phospholipase Dδ family in tobacco pollen tubes. Plant J. 103, 212–226 (2020). PubMed

TerBush D. R., Maurice T., Roth D., Novick P., The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494 (1996). PubMed PMC

Picco A., et al. ., The in vivo architecture of the exocyst provides structural basis for exocytosis. Cell 168, 400–412.e18 (2017). PubMed

Katoh Y., Nozaki S., Hartanto D., Miyano R., Nakayama K., Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J. Cell Sci. 128, 2351–2362 (2015). PubMed

Wu C., et al. ., Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit. J. Integr. Plant Biol. 59, 851–865 (2017). PubMed

Potocký M., et al. ., Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor. New Phytol. 203, 483–494 (2014). PubMed

Zeniou-Meyer M., et al. ., Phospholipase D1 production of phosphatidic acid at the plasma membrane promotes exocytosis of large dense-core granules at a late stage. J. Biol. Chem. 282, 21746–21757 (2007). PubMed

Testerink C., Munnik T., Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J. Exp. Bot. 62, 2349–2361 (2011). PubMed

Pleskot R., et al. ., Turnover of phosphatidic acid through distinct signaling pathways affects multiple aspects of pollen tube growth in tobacco. Front. Plant Sci. 3, 54 (2012). PubMed PMC

Zhang Q., et al. ., Phosphatidic acid regulates microtubule organization by interacting with MAP65-1 in response to salt stress in Arabidopsis. Plant Cell 24, 4555–4576 (2012). PubMed PMC

McLoughlin F., et al. ., Identification of novel candidate phosphatidic acid-binding proteins involved in the salt-stress response of Arabidopsis thaliana roots. Biochem. J. 450, 573–581 (2013). PubMed

Julkowska M. M., et al. ., Identification and functional characterization of the A rabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain: PA-dependent regulation of SnRK2.4. Plant Cell Environ. 38, 614–624 (2015). PubMed

Putta P., et al. ., Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties. Biochim. Biophys. Acta BBA - Biomembr. 1858, 2709–2716 (2016). PubMed

Putta P., Creque E., Piontkivska H., Kooijman E. E., Lipid-protein interactions for ECA1 an N-ANTH domain protein involved in stress signaling in plants. Chem. Phys. Lipids 231, 104919 (2020). PubMed

Huang S., Gao L., Blanchoin L., Staiger C. J., Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid. Mol. Biol. Cell 17, 1946–1958 (2006). PubMed PMC

Pleskot R., Pejchar P., Žárský V., Staiger C. J., Potocký M., Structural insights into the inhibition of actin-capping protein by interactions with phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate. PLoS Comput. Biol. 8, e1002765 (2012). PubMed PMC

Simon M. L. A., et al. ., A multi-colour/multi-affinity marker set to visualize phosphoinositide dynamics in Arabidopsis. Plant J. 77, 322–337 (2014). PubMed PMC

Moravcevic K., Oxley C. L., Lemmon M. A., Conditional peripheral membrane proteins: Facing up to limited specificity. Structure 20, 15–27 (2012). PubMed PMC

Sabol P., Kulich I., Žárský V., RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane. J. Exp. Bot. 68, 3253–3265 (2017). PubMed PMC

Žárský V., Sekereš J., Kubátová Z., Pečenková T., Cvrčková F., Three subfamilies of exocyst EXO70 family subunits in land plants: Early divergence and ongoing functional specialization. J. Exp. Bot. 71, 49–62 (2020). PubMed

Noack L. C., Jaillais Y., Functions of anionic lipids in plants. Annu. Rev. Plant Biol. 71, 71–102 (2020). PubMed

Schindelin J., et al. ., Fiji: An open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012). PubMed PMC

Kim D. E., Chivian D., Baker D., Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res. 32, W526–W531 (2004). PubMed PMC

de Jong D. H., et al. ., Improved parameters for the martini coarse-grained protein force field. J. Chem. Theory Comput. 9, 687–697 (2013). PubMed

Periole X., Cavalli M., Marrink S.-J., Ceruso M. A., Combining an elastic network with a coarse-grained molecular force field: Structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009). PubMed

Ingólfsson H. I., et al. ., Lipid organization of the plasma membrane. J. Am. Chem. Soc. 136, 14554–14559 (2014). PubMed

Hsu P.-C., et al. ., CHARMM-GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides. J. Comput. Chem. 38, 2354–2363 (2017). PubMed PMC

Abraham M. J., et al. ., GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1–2, 19–25 (2015).

Hess B., Bekker H., Berendsen H. J. C., Fraaije J. G. E. M., LINCS: A linear constraint solver for molecular simulations. J. Comput. Chem. 18, 1463–1472 (1997).

Parrinello M., Rahman A., Polymorphic transitions in single crystals: A new molecular dynamics method. J. Appl. Phys. 52, 7182–7190 (1981).

Bussi G., Donadio D., Parrinello M., Canonical sampling through velocity rescaling. J. Chem. Phys. 126, 014101 (2007). PubMed

Kost B., Spielhofer P., Chua N.-H., A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16, 393–401 (1998). PubMed

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