- Email: [email protected]
Dissecting the plant exocyst
Available online at www.sciencedirect.com
ScienceDirect Dissecting the plant exocyst Bushra Saeed1, Carla Brillada1 and Marco Trujillo The exocyst is an evolutionary conserved complex that mediates tethering of post-Golgi vesicles derived from the conventional secretory pathway to the plasma membrane (PM), before SNARE-mediated fusion. Through its tethering function, connecting secretory vesicles to the PM, it mediates spatiotemporal regulation of exocytosis. As an integral element of the secretory machinery, the exocyst has been implicated in a large variety of processes. However, emerging evidence suggests that it may also cater for unconventional secretory pathways, as well as autophagy. The exocyst entertains a multitude of interactions with proteins and membrane phospholipids, reflecting its highly dynamic nature and the complex regulatory processes that hardwire it with cellular signalling networks. However, our molecular understanding of this essential complex remains fragmentary. Here we review recent work focusing on the molecular features that have revealed both commonalities with yeast and animals, as well as unique characteristics of the plant exocyst.
compartments of the Golgi apparatus, ending at the PM, as well as other target membranes. Transport of cargoes between the different stations of the secretory endomembrane system is mediated by vesicular traffic, which entails five major stages starting with the (i) budding of a vesicle and its (ii) transport towards a target membrane. The vesicle is initially (iii) tethered to the target membrane, and subsequently, the association is tightened during (iv) docking, to finally allow (v) fusion.
Address Albert-Ludwigs-University Freiburg, Faculty of Biology, Institute of Biology II, 79104 Freiburg, Germany
Exocyst interaction with the PM
Corresponding author: Trujillo, Marco ([email protected]) 1 Equal contribution. Current Opinion in Plant Biology 2019, 52:69–76 This review comes from a themed issue on Cell biology Edited by Eva Benkova and Yasin Dagdas
https://doi.org/10.1016/j.pbi.2019.08.004
The exocyst complex provides the first attachment of the vesicle to the PM, and by serving as a convergence point for GTPases, cellular signalling and the cytoskeleton, it orchestrates cargo delivery. It is an octameric tethering factor, first identified in the budding yeast Saccharomyces cerevisiae that consists of the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 [5] (Figure 1).
Early work from Segui-Simarro et al. [6] using electron tomography, identified ‘Y’ or ‘L’-shaped structures linking vesicles that were reminiscent of the mammalian exocyst complex [7]. Insensitivity to Brefeldin A [8,9] suggests that the exocyst complex is recruited to the secretory vesicle after budding from the trans-Golgi network (TGN), independently of vesicular traffic. In yeast, Sec15 binds to the vesicle in cooperation with the GTP-bound GTPase Sec4 [10]. Although, in Arabidopsis Sec15 is required for exocyst function [11], it remains unknown whether it carries out the same function.
1369-5266/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Secretion is responsible for the transport of de novo synthesized material, and it culminates with the exocytic event at the PM. Transported cargoes include integral PM proteins such as receptor kinases or CELLULOSE SYNTHASE COMPLEXES (CSCs), but additionally encompasses material released out to the apoplast such as, proteins, signalling peptides, small RNAs, polysaccharides required for cell wall growth and defence compounds [1–3,4]. The entry port of the secretory pathway is the endoplasmic reticulum (ER). Cargo traverses the different www.sciencedirect.com
Points of contact to the PM are created by the Sec3 and Exo70 subunits (Figure 1). The mammalian and yeast Sec3 and Exo70 subunits are able to directly bind phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phospholipid that decorates the inner leaflet of the PM [10]. Sec3 of budding yeast interacts with PI(4,5)P2 through a cryptic pleckstrin homology (PH) fold in its N-terminus, whereas Exo70 interacts with PI(4,5)P2 through a patch of basic residues at its C-terminus [10]. In plants, the first indication of exocyst PM binding was provided by the small molecule Endosidin2 (ES2). Exo70A1 association to the PM was inhibited by ES2 treatment, resulting in its increased transport into the vacuole and reduced PM-localized PIN2 [12]. ES2 directly binds to Exo70A1, as well as the human Exo70, suggesting that it docks to a conserved structural feature required for PM binding [12]. Current Opinion in Plant Biology 2019, 52:69–76
70 Cell biology
Figure 1
PM
KEULE
PI(4,5)P2
PI?
SNAP33 (B1)
ICR1 RIC7 (B1) MtVapyrin (I) RIN4 (B1) TN2 (B1) PUB22-24 (B2) PUB18 & 19 (B1) ES2 (A1)
3
70
5
10
6
84
8
15
SCD1/2 For1F (Pp) actin
COG2
vesicle microtubules
Current Opinion in Plant Biology
Schematic of the plant exocyst complex and its known interactions. Schematic of the exocyst as a holocomplex composed of eight subunits. Arrows indicate interactions in which the exocyst engages (out), or is a potential effector or substrate (in) of other proteins or molecules. Sec3 and Exo70 directly bind to phospholipids (shown in red) to the plasma membrane via their positively charged residues (+++). Interacting Exo70 paralogue are indicated (e.g. Exo70B1 is B1). Specific phospholipids to which plant Exo70 subunits bind are unknown. The PH domain of Sec3 is probably connected via a flexible linker [16]. For1F is a Sec10 fusion to a formin in Physcomitrella patens (Pp). Subunit arrangement guided by the yeast exocyst structure revealed by cryo-EM [16]. Colour code and subunit arrangement (indicated by number: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) in two subcomplexes (SC1 and SC2) [16,28]. Proteins are from Arabidopsis thaliana, unless specified. Abbreviations: Phosphoinositides (PI), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), plasma membrane (PM), Medicago truncatula (Mt), +++ indicate basic residues.
The Arabidopsis Sec3 directly binds to PI(4,5)P2 through basic residues partially conserved in yeast and humans [13,14]. Further, overexpression of the PIP5 kinase resulted in increased association of Nicotiana benthamiana Sec3a to the PM [15]. However, the PH-domain was dispensable to target Sec3 to the PM, and the complementation of the sec3a-1 mutant [13]. One possible explanation is that the truncated Sec3 is integrated into the exocyst, which can still associate to the PM through the Exo70 subunit. It also suggests that in analogy to yeast, the C-terminal portion of Sec3, may be required for complex assembly and stability [16].
to the cytokinesis-specific syntaxin KNOLLE. Sec6 interaction is conserved in Physcomitrella patens, and its silencing delays the appearance of KEULE to the early cell plate [20]. KEULE was additionally shown to dynamically compete with SNAP33 for SYP121 (PEN1) binding, a close homologue of KNOLLE [21]. It is therefore conceivable that the exocyst alleviates this competition by sequestering KEULE via Sec6 upon arrival to the PM, to initiate SNARE-complex formation and membrane fusion. Of note, analyses of Exo70B1 function during immunity, suggest that also SNAP33 acts downstream of an exocyst–Exo70B1 complex [22].
Beyond its function in tethering, the exocyst may also be implicated in the regulation of later stages of exocytosis mediated by SNARE complexes. In budding yeast, Sec6 interacts mutually exclusively with the Q-SNARE Sec9 [17] and Sec1/Munc18, a regulator of membrane fusion [18]. In analogy, the Arabidopsis Sec6 physically interacts with the Sec1 orthologue KEULE [19], which itself binds
Functional specialization of exocyst subtypes
Current Opinion in Plant Biology 2019, 52:69–76
It was not until recently that cargoes were identified which required exocyst function for polarized [9,23,24], as well as general secretion involved in growth [4]. However, the irradiation of Exo70 genes in plants has led to the hypothesis that exocyst subtypes, ‘equipped’ with specific Exo70 subunits, may have www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 71
adopted specialized functions (Figure 2a). A study showed that exo70A1 mutants display secretory defects specific to CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN 1 (CASP1) [23], while lateral polarity of BOR1 and PDR6, was unaffected. In an elegant approach, Kulich et al. [25] addressed Exo70 specificity by assaying complementation of the cell wall defect in trichomes-specific to exo70H4. They tested 18 different Arabidopsis EXO70 paralogues under control
of the EXO70H4 promoter, and showed that functional complementation was exquisitely specific to Exo70H4. [25]. Another example is the evolution of EXO70 homologues exclusively present in plants forming symbiosis with arbuscular mycorrhiza fungi. These include the Medicago truncatula Exo70I, which is required for the development of the arbuscule and spatially restricted to hyphal tip areas of the periarbuscular membrane, suggesting a specialized function [26].
Figure 2
(a) PM
Golgi
Golgi (c)
(b)
PM
Receptor kinase A
Integral trans-membrane protein A
Soluble secreted molecule A
PM
Soluble secreted molecule C
Exocyst subtype X (Exo70X) Exocyst subtype Y (Exo70Y)
Receptor kinase B
Integral trans-membrane protein B
Soluble secreted molecule B
Small RNA Exocyst subtype Z (Exo70Z) Current Opinion in Plant Biology
Secretory pathways potentially involving the exocyst complex. (a) Conventional secretory pathway. Exocyst tethers secretory vesicles derived from the Golgi containing various cargoes such as integral membrane proteins, soluble secreted proteins, and potentially signalling peptides and carbohydrates. The Exo70 subunit (highlighted in red and purple) and Sec3 (red) are able to directly associate to the PM by binding phospholipids. Additional proteins participating in membrane association, that indirectly complex with exocyst subunits, are not included. Exocyst subtypes characterized by distinct Exo70 subunits, may participate in the delivery of specific cargo proteins, including different types of integral membrane proteins. (b) Unconventional secretion via multivesicular bodies (MVB) or late endosomes. MVBs are large bodies containing smaller intraluminal vesicles that have been reported to fuse to the PM. The exocyst may participate in their tethering to the PM. (c) Unconventional secretion via the exocyst-positive organelle (EXPO). The EXPO is characterized by a double membrane that fuses to the PM and releases a vesicle to the apoplast. The organelle of origin for EXPO remains unknown. (b–c) Both pathways remain poorly described, but may be involved in the delivery of extracellular vesicles shown to contain different types of proteins and small RNAs. (a–c) Docking stage of exocytosis mediated by SNARE proteins is not included.
www.sciencedirect.com
Current Opinion in Plant Biology 2019, 52:69–76
72 Cell biology
Complex dynamics A detailed study of exocyst dynamics at the PM showed that its subunits are localized to mobile foci, differing from endocytic sites [24]. However, whether the exocyst acts as a holocomplex [27,28], or assembles dynamically [29], has remained highly controversial. The Cryo-EM structure indicates that the eight subunits assemble hierarchically through helical bundles into the exocyst complex, and are organized in two subcomplexes (SCs) composed of SC1 (Sec8, Sec6, Sec5, and Sec3), and SC2 (Sec15, Sec10, Exo84, and Exo70) [16,28] (Figure 1). An elegant study in mammalian cells using knock-in tagged subunits, revealed that the two subcomplexes are highly dynamic and assemble at the PM independently of each other [30]. SC1 arrives before SC2, and Sec3, which is associated only loosely with the complex, departs before the rest of the complex [30]. During cytokinesis, Sec6 of Physcomitrella was visible at the phragmoplast midzone before detectable membrane and preceding Sec3a and Sec5b [20]. It is unlikely that this reflects the dynamics of exocyst subcomplexes, as these subunits are predicted to reside in SC1. It does suggest however that subunits assemble dynamically into a holocomplex or have complex-independent functions.
Points of contact with the cytoskeleton New studies have begun to reveal an intertwined relation between the exocyst with actin and microtubule cytoskeletal systems. A link to the actin cytoskeleton is provided by the formin For1F from P. patens. For1F is a fusion of a Sec10 exocyst subunit with a class I formin, which act as nucleators of actin filaments [31]. Sec6 dynamically colocalized with For1F, but increased density of cortical For1F in the absence of actin, suggests that targeting to the cell cortex is actin independent. Instead, actin may play a role in post vesicle delivery removal of For1F, and potentially, of the complex [31]. This would also provide an explanation for the observed clustering of the Arabidopsis Exo84b only after long term actin disruption [24]. By contrast, the interaction between the coiled-coil containing proteins VESICLE TETHERING 1 (VETH1) and VETH2 with CONSERVED OLIGOMERIC GOLGI 2 (COG2), promoted the association between cortical microtubules and Exo70A1 in xylem cells [32]. COG2 was able to interact with Sec10 in vivo, and thus, potentially build a Sec10–COG2–VETH1/2 connection with microtubules [33]. Accordingly, Sec8 arrival at the PM preceded secondary cell wall growth in xylem cells, and exo84b mutants displayed abnormal development of tracheary elements [33].
Exocyst regulation The intricate dynamics of the exocyst complex are indicative of a multi-layered regulation required to integrate its manifold intra and inter-molecular interactions, to Current Opinion in Plant Biology 2019, 52:69–76
provide a spatiotemporal orchestration of the exocytic event. Several exocyst subunits are known GTPase effectors [10]. In plants however, GTPase-mediated exocyst regulation appears to be achieved indirectly via adaptor proteins [34]. In one example, Exo70B1 interacts with the RHO OF PLANTS 2 (ROP2) effector RAC-INTERACTIVE BINDING MOTIF-CONTAINING PROTEIN 7 (RIC7) [35]. In the presence of a constitutive active ROP2, Exo70B1 and RIC7 relocalized to the PM of Vicia faba stomata. More recently, Sec15b and RabE1 were shown to interact with STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) and SCD2 [36]. Although the constellation of the interaction is not clear, SCDs may also act as adaptors connecting Sec15b and RabE1. Given that in yeast and animals Sec15 is a GTPase effector through which the exocyst is bound to the vesicle [10], it is possible that in plants Sec15b–SCD1 carry out this function via RabE1. Intriguingly, both SCDs bind to the inactive mutant of RabE1, leading authors to propose that SCDs may function in its activation. Additional exocyst-interacting proteins show parallels to adaptor proteins, for example, RPM1 INTERACTING PROTEIN 4 (RIN4), a PM-anchored protein involved in the immune response. RIN4 coexpression experiments, revealed the specific recruitment of Exo70B1 to the PM, but not that of its nearest homologue Exo70B2 [37]. Cleavage by the bacterial effector AvrRpt2 releases Exo70B1 from the PM, potentially blocking its function during immune responses [37,38,39]. Exocyst subunits are also regulated by post-translational modifications. Exo70B1 and Exo70B2 are actively regulated by ubiquitination and subsequent degradation, mediated by the related U-box type E3 ligases PUB18 and PUB22, respectively [39,40,41,42]. PUB22 and related E3s, interact with E2s dedicated to generate Lys63-linked ubiquitin chains, and this interaction was induced by immunostimulation in the case of PUB22 [43]. Lys-63 ubiquitin chains are key to regulate various steps of vesicle trafficking and transport to the vacuole [44,45]. The recruitment of exocyst subunits to the vacuole [25,46,47], either via the endocytic pathway or potentially by autophagy (see below), suggests that ubiquitination possibly contributes to this process. However, the reason for the induced degradation of Exo70 subunits still remains unclear. On the one hand, it may help to dampen the secretory activity, but alternatively, it could reflect a change in the levels of exocyst subtypes that mediate secretion of distinct cargoes (Figure 2a). Similar to PUB22, our unpublished data uncovered the phosphorylation of Exo70B2 at its C-terminus, which is predicted to bind phospholipids [47]. Mimicking phosphorylation reduced Exo70B2 levels at sites of active secretion, revealing a direct crosstalk between the secretory machinery and cellular signalling. www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 73
Different roles of the exocyst in vesicle tethering Several studies have underlined the essential nature of the exocyst [48], which is most likely indispensable for conventional secretion. Its relevance is nicely illustrated by the polarized secretion at sites of contact between the stigmatic papillae and a compatible pollen grains to promote pollen reception, which required all eight exocyst subunits [49]. Thus providing evidence for the requirement of the entire complex during pollination [11]. In line with this, disruption of Exo70A1 in Arabidopsis, results in a loss of secretory vesicles at the stigmatic papillar PM [50]. By contrast, unconventional protein secretion mediates the transport of proteins that lack an amino-terminal signal peptide, and bypass the conventional pathway. There are at least two possible routes in which the exocyst may be involved. The first involves the fusion of multivesicular bodies (MVBs/late endosomes) to the PM. During the acceptance of compatible pollen, MVBs fuse to the PM in Brassica napus [50] (Figure 2b), a process reminiscent to responses against penetrating fungal pathogens [51]. Accordingly, silencing of Exo70A1 resulted in a reduced incidence of MVBs at the PM [50]. A second route, involves the so called exocyst positive organelle (EXPO). It was first identified through the characterization of the Arabidopsis exocyst subunit Exo70E2, which does not colocalize with standard organelle markers [46,52]. EM analyses captured Exo70E2 labelled double membrane vesicles fusing with the PM and releasing a single membrane vesicle into the apoplast [52] (Figure 2c). These observations open the exciting possibility that the exocyst participates in the transport of extracellular vesicles (EVs). EVs contain proteins, as well as small RNAs, and their release to the apoplast is induced during infection [1,2,53]. Another exciting development in the field is the involvement of the exocyst in autophagy. Silencing of exocyst components in human cells was shown to have autophagy-inducing or inhibiting effects, which relied on subunit ability to recruit distinct autophagy signalling components [54]. The first link to autophagy in plants was provided by the observation that Exo70B1 is transported into the vacuole, where it partially colocalized with ATG8 [55]. In addition, ATG8 transport was reduced in exo70B1 hypocotyls of etiolated seedlings after tunicamycin treatment. However, Exo70B1 may be guarded by the LEUCINE-RICH REPEAT-CONTAINING 2 (TN2), which together with CALCIUM-DEPENDENT PROTEIN KINASE 5 (CPK5), induce a spontaneous cell death phenotype in www.sciencedirect.com
the null mutant [22,38,55,56]. Because the cell death phenotype, and potentially other phenotypes, which are reminiscent of autophagy-deficient mutants, are exo70B1independent, its function may also be downstream of autophagy activation. A subsequent study showed that Exo70E2 relocalized to autophagosomes only after the induction of autophagy with BTH treatment [46]. Interestingly, Exo70 homologues display an overrepresentation of predicted ATG8-interacting motifs (AIMs), compared to other subunits in Arabidopsis [57,58]. Our unpublished work shows that Exo70B2 displays a similar behavior as Exo70E2, and moreover, is able to physically interact with ATG8 [47]. Thus, Exo7B2 may directly link the secretory machinery and autophagy. Of note, during self-incompatible pollinations, secretory vesicles and MVBs, are absent from the PM of stigmatic papillae, and autophagy appeared to be induced to redirect vesicles and MVBs to the vacuole [50].
Conclusions and open questions The exocyst represents the last step in the secretory pathway before a vesicle is committed to exocytosis. Its diverse functions and interactions may therefore, be involved in gaging secretion, that together with the endocytic/vacuolar degradation pathways, control membrane protein levels and maintenance of cellular homeostasis. New studies have underlined the existence of specialized exocyst subtypes carrying specific Exo70 homologues that tether vesicles with distinct cargoes. This fascinating possibility opens however many new questions, for example: How are vesicles loaded with specific cargoes? And, how do exocyst subtypes, equipped with alternate Exo70 subunits, recognize the corresponding vesicles? Several lines of evidence support a link between the exocyst and autophagy, as well as unconventional secretion. However, our knowledge here remains limited, and future studies should reveal whether exocyst subunits are substrates or play a leading part. It is nevertheless tempting to speculate that the exocyst is able to switch between activities depending on the subtype, its cargo and/or cellular status. The ultimate goal will be to discover the molecular changes induced by crosstalk with interacting partners including GTPases and their scaffolds, signalling components, cytoskeleton, and so on, that modulate exocyst activity and secretory output.
Conflict of interest statement Nothing declared.
Acknowledgements The work in the authors’ laboratory is supported by grants from the German Research Foundation (DFG: TR 1073/6-1, TR 1073/4-1) and German Current Opinion in Plant Biology 2019, 52:69–76
74 Cell biology
Academic Exchange Service (DAAD:PPP-HK 57447859, Germany) to M.T. and SERB-Overseas Postdoctoral Fellowship (SB/OS/PDF-286/2016-17, India) of the Government of India to B.S. The authors apologize to colleagues whose work could not be included due to length restrictions of this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
3.
Cai Q, Qiao L, Wang M, He B, Lin FM, Palmquist J, Huang SD, Jin H: Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science 2018, 360:1126-1129. Baldrich P, Rutter BD, Zand Karimi H, Podicheti R, Meyers BC, Innes RW: Plant extracellular vesicles contain diverse small RNA species and are enriched in 10 to 17 nucleotide “tiny” RNAs. Plant Cell 2019, 31:315-324. Stegmann M, Monaghan J, Smakowska-Luzan E, Rovenich H, Lehner A, Holton N, Belkhadir Y, Zipfel C: The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling. Science 2017, 355:287-289.
4.
Zhu X, Li S, Pan S, Xin X, Gu Y: CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane. Proc Natl Acad Sci U S A 2018, 115:E3578-E3587. Exocyst function is required for the secretion of non-polar localized proteins such as CSI, thus a central protein of general plant growth. 5.
TerBush DR, Maurice T, Roth D, Novick P: The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 1996, 15:6483-6494.
6.
Segui-Simarro JM, Austin JR 2nd, White EA, Staehelin LA: Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by highpressure freezing. Plant Cell 2004, 16:836-856.
7.
Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH: Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 1998, 20:1111-1122.
8.
Drdova EJ, Synek L, Pecenkova T, Hala M, Kulich I, Fowler JE, Murphy AS, Zarsky V: The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J 2013, 73:709-719.
Mao H, Nakamura M, Viotti C, Grebe M: A framework for lateral membrane trafficking and polar tethering of the PEN3 ATPbinding cassette transporter. Plant Physiol 2016, 172:22452260. Exocyst function is required for the secretion of non-polar localized proteins such as CSI, thus a central protein of general plant growth.
9.
10. Wu B, Guo W: The exocyst at a glance. J Cell Sci 2015, 128:29572964. 11. Safavian D, Zayed Y, Indriolo E, Chapman L, Ahmed A, Goring DR: RNA silencing of exocyst genes in the stigma impairs the acceptance of compatible pollen in Arabidopsis. Plant Physiol 2015, 169:2526-2538. The acceptance of compatible pollen is the first process shown to require all exocyst subunits. 12. Zhang C, Brown MQ, van de Ven W, Zhang ZM, Wu B, Young MC, Synek L, Borchardt D, Harrison R, Pan S et al.: Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc Natl Acad Sci U S A 2016, 113:E41-50. ES2 disrupted Exo70A1 association to the PM and the transport of PIN2 to the PM, indicating that PM association of Exo70A1 is required for its function. ES2 directly binds to Exo70A1 in a region predicted to mediate PM association. 13. Bloch D, Pleskot R, Pejchar P, Potocky M, Trpkosova P, Cwiklik L, Vukasinovic N, Sternberg H, Yalovsky S, Zarsky V: Exocyst SEC3 Current Opinion in Plant Biology 2019, 52:69–76
and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiol 2016, 172:980-1002. Sec3 was shown to bind PI(4,5)P2 and dynamically bind to the PM at the tip of growing pollen tubes. Relocalization of Sec3 predicted changes in growth direction in agreement of its function in secretion required for cell growth. 14. Pleskot R, Cwiklik L, Jungwirth P, Zarsky V, Potocky M: Membrane targeting of the yeast exocyst complex. Biochim Biophys Acta 2015, 1848:1481-1489. 15. Sekeres J, Pejchar P, Santrucek J, Vukasinovic N, Zarsky V, Potocky M: Analysis of exocyst subunit EXO70 family reveals distinct membrane polar domains in tobacco pollen tubes. Plant Physiol 2017, 173:1659-1675. This study supports the existence of distinct PM domains occupied by different Exo70 paralogues, suggesting also that exocyst-subtype specificities are dictated by Exo70 phospholipid binding traits. 16. Mei K, Li Y, Wang S, Shao G, Wang J, Ding Y, Luo G, Yue P, Liu JJ, Wang X et al.: Cryo-EM structure of the exocyst complex. Nat Struct Mol Biol 2018. 17. Sivaram MV, Saporita JA, Furgason ML, Boettcher AJ, Munson M: Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p. Biochemistry 2005, 44:6302-6311. 18. Morgera F, Sallah MR, Dubuke ML, Gandhi P, Brewer DN, Carr CM, Munson M: Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol Biol Cell 2012, 23:337-346. 19. Wu J, Tan X, Wu C, Cao K, Li Y, Bao Y: Regulation of cytokinesis by exocyst subunit SEC6 and KEULE in Arabidopsis thaliana. Mol Plant 2013, 6:1863-1876. 20. Tang H, de Keijzer J, Overdijk E, Sweep E, Steentjes M, Vermeer JE, Janson ME, Ketelaar T: Exocyst subunit Sec6 is positioned by microtubule overlaps in the moss phragmoplast prior to cell plate membrane arrival. J Cell Sci 2019. 21. Karnik R, Grefen C, Bayne R, Honsbein A, Kohler T, Kioumourtzoglou D, Williams M, Bryant NJ, Blatt MR: Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic. Plant Cell 2013, 25:1368-1382. 22. Zhao T, Rui L, Li J, Nishimura MT, Vogel JP, Liu N, Liu S, Zhao Y, Dangl JL, Tang D: A truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant. PLoS Genet 2015, 11:e1004945. Exo70B1 may be a guarded protein and its absence in the null mutant potentially activates TN2. 23. Kalmbach L, Hematy K, De Bellis D, Barberon M, Fujita S, Ursache R, Daraspe J, Geldner N: Transient cell-specific EXO70A1 activity in the CASP domain and Casparian strip localization. Nat Plants 2017, 3:17058. Authors demonstrate that Exo70A1 is required for the delivery of CASP1, but not other tested PM proteins, supporting a functional specialization of exocyst subtypes. 24. Fendrych M, Synek L, Pecenkova T, Drdova EJ, Sekeres J, de Rycke R, Nowack MK, Zarsky V: Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol Biol Cell 2013, 24:510-520. 25. Kulich I, Vojtikova Z, Sabol P, Ortmannova J, Nedela V, Tihlarikova E, Zarsky V: Exocyst subunit EXO70H4 has a specific role in callose synthase secretion and silica accumulation. Plant Physiol 2018, 176:2040-2051. Exo70H4, but not other Exo70 paralogues, is able to functionally complement secretory defects in the exo70H1 mutant, further supporting a functional specialization of exocyst subtypes. 26. Zhang X, Pumplin N, Ivanov S, Harrison MJ: EXO70I is required for development of a sub-domain of the periarbuscular membrane during arbuscular mycorrhizal symbiosis. Curr Biol 2015, 25:2189-2195. 27. Picco A, Irastorza-Azcarate I, Specht T, Boke D, Pazos I, RivierCordey AS, Devos DP, Kaksonen M, Gallego O: The in vivo architecture of the exocyst provides structural basis for exocytosis. Cell 2017, 168:400-412 e418. www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 75
28. Heider MR, Gu M, Duffy CM, Mirza AM, Marcotte LL, Walls AC, Farrall N, Hakhverdyan Z, Field MC, Rout MP et al.: Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat Struct Mol Biol 2016, 23:59-66. 29. Boyd C, Hughes T, Pypaert M, Novick P: Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J Cell Biol 2004, 167:889-901. 30. Ahmed SM, Nishida-Fukuda H, Li Y, McDonald WH, Gradinaru CC, Macara IG: Exocyst dynamics during vesicle tethering and fusion. Nat Commun 2018, 9:5140. This work illuminates the exocyst dynamics at the PM of human cells in unparalleled detail showing the dynamic assembly and disassembly of the exocyst complex during vesicle tethering and membrane fusion. 31. van Gisbergen PAC, Wu SZ, Chang M, Pattavina KA, Bartlett ME, Bezanilla M: An ancient Sec10-formin fusion provides insights into actin-mediated regulation of exocytosis. J Cell Biol 2018, 217:945-957. Interaction with the SNARE-interacting KEULE protein is conserved in the Physcomitrella patens. Authors show that Sec6 arrives at the phragmoplast before other exocyst subunits suggesting that exocyst subunits reside outside the holocomplex also in plants. 32. Oda Y, Iida Y, Nagashima Y, Sugiyama Y, Fukuda H: Novel coiled coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein. Plant Cell Physiol 2015, 56:277-286. VETH1 and VETH2 may contribute to the recruitment of the exocyst to microtubules, revealing a possible link of the exocyst to microtubules. 33. Vukasinovic N, Oda Y, Pejchar P, Synek L, Pecenkova T, Rawat A, Sekeres J, Potocky M, Zarsky V: Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis. New Phytol 2017, 213:1052-1067. Authors provide evidence that the exocyst is recruited to microtubules by interacting with COG2. COG2 was previously shown to complex with VETH1/2 which associates to microtubules [32]. 34. Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S: A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 2007, 17:947-952. 35. Hong D, Jeon BW, Kim SY, Hwang JU, Lee Y: The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. New Phytol 2016, 209:624-635. The ROP2 effector RIC7 is shown to interact with Exo70B1 and to recruit and mediate its recruitment to the PM. This work underlines the apparently indirect regulation by GTPases via adaptor proteins [34,36]. 36. Mayers JR, Hu T, Wang C, Cardenas JJ, Tan Y, Pan J, Bednarek SY: SCD1 and SCD2 form a complex that functions with the exocyst and RabE1 in exocytosis and cytokinesis. Plant Cell 2017, 29:2610-2625. SCD1/2 associate to RabE1, as well as Sec13b, suggesting that they potentially act as adaptors (see also Refs. [34,35]). In budding yeast, Sec15 is an effector of the Sec4 GTPase through which the exocyst is attached to the vesicle. Hence, author may have laid the foundation to elucidate this exocyst vesicle binding in plants. 37. Sabol P, Kulich I, Zarsky V: RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane. J Exp Bot 2017, 68:32533265. The immunity related RIN4, may act as an adaptor protein regulating Exo70B1 recruitment to the PM. RIN4 has been shown to be guarded by different NLR (see also Ref. [22]). 38. Stegmann M, Anderson RG, Westphal L, Rosahl S, McDowell JM, Trujillo M: The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death. Plant Signal Behav 2013, 8:e27421. 39. Stegmann M, Anderson RG, Ichimura K, Pecenkova T, Reuter P, Zarsky V, McDowell JM, Shirasu K, Trujillo M: The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell 2012, 24:4703-4716. 40. Furlan G, Nakagami H, Eschen-Lippold L, Jiang X, Majovsky P, Kowarschik K, Hoehenwarter W, Lee J, Trujillo M: Changes in PUB22 ubiquitination modes triggered by MITOGENwww.sciencedirect.com
ACTIVATED PROTEIN KINASE3 dampen the immune response. Plant Cell 2017, 29:726-745. 41. Seo DH, Ahn MY, Park KY, Kim EY, Kim WT: The N-terminal UND motif of the Arabidopsis U-Box E3 ligase PUB18 is critical for the negative regulation of ABA-mediated stomatal movement and determines its ubiquitination specificity for exocyst subunit Exo70B1. Plant Cell 2016, 28:2952-2973. PUB18 regulates Exo70B1 levels via ubiquitination and degradation. In contrast to PUB22 [35] which targets Exo70B2 via its armadillo repeat, PUB18 interacts with Exo70B1 via the U-box N-ter domain (UND). 42. Trujillo M: News from the PUB: plant U-box type E3 ubiquitin ligases. J Exp Bot 2018, 69:371-384. 43. Turek I, Tischer N, Lassig R, Trujillo M: Multi-tiered pairing selectivity between E2 ubiquitin-conjugating enzymes and E3 ligases. J Biol Chem 2018, 293:16324-16336. 44. Dubeaux G, Vert G: Zooming into plant ubiquitin-mediated endocytosis. Curr Opin Plant Biol 2017, 40:56-62. 45. Isono E, Kalinowska K: ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants. Curr Opin Plant Biol 2017, 40:49-55. 46. Lin Y, Ding Y, Wang J, Shen J, Kung CH, Zhuang X, Cui Y, Yin Z, Xia Y, Lin H et al.: Exocyst-positive organelles and autophagosomes are distinct organelles in plants. Plant Physiol 2015, 169:1917-1932. The exocyst subunit Exo70E2 resides in the so-called EXPO, but is relocated to autophagosomes and recruited into the vacuole upon autophagy induction. 47. Teh O-K, Lee C-W, Ditengou FA, Klecker T, Furlan G, Zietz M, Hause G, Eschen-Lippold L, Hoehenwarter W, Lee J et al.: Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function. bioRxiv 2018. 48. Zarsky V, Kulich I, Fendrych M, Pecenkova T: Exocyst complexes multiple functions in plant cells secretory pathways. Curr Opin Plant Biol 2013, 16:726-733. 49. Doucet J, Lee HK, Goring DR: Pollen acceptance or rejection: a tale of two pathways. Trends Plant Sci 2016, 21:1058-1067. 50. Safavian D, Goring DR: Secretory activity is rapidly induced in stigmatic papillae by compatible pollen, but inhibited for selfincompatible pollen in the Brassicaceae. PLoS One 2013, 8: e84286. This work shows the differences between secretory responses during the pollen reception in Brassica napus and Arabidopsis thaliana. Self-incompatible pollen does not elicit similar reactions. Importantly, this work shows that Exo70A1 is required for vesicle delivery during pollen reception. 51. Dormann P, Kim H, Ott T, Schulze-Lefert P, Trujillo M, Wewer V, Huckelhoven R: Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions. New Phytol 2014, 204:815-822. 52. Wang J, Ding Y, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L: EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell 2010, 22:4009-4030. 53. Rutter BD, Innes RW: Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol 2017, 173:728-741. 54. Bodemann BO, Orvedahl A, Cheng T, Ram RR, Ou YH, Formstecher E, Maiti M, Hazelett CC, Wauson EM, Balakireva M et al.: RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 2011, 144:253-267. 55. Kulich I, Pecenkova T, Sekeres J, Smetana O, Fendrych M, Foissner I, Hoftberger M, Zarsky V: Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 2013, 14:1155-1165. Authors uncover the first link between exocyst and autophagy by showing that Exo70B1 is transported into the vacuole where it colocalizes with the autophagy marker ATG8. Current Opinion in Plant Biology 2019, 52:69–76
76 Cell biology
56. Liu N, Hake K, Wang W, Zhao T, Romeis T, Tang D: CALCIUMDEPENDENT PROTEIN KINASE5 associates with the truncated NLR protein TIR-NBS2 to contribute to exo70B1mediated immunity. Plant Cell 2017, 29:746-759. 57. Tzfadia O, Galili G: The Arabidopsis exocyst subcomplex subunits involved in a golgi-independent transport into the
Current Opinion in Plant Biology 2019, 52:69–76
vacuole possess consensus autophagy-associated atg8 interacting motifs. Plant Signal Behav 2013, 8 http://dx.doi.org/ 10.4161/psb 26732. 58. Cvrckova F, Zarsky V: Old AIMs of the exocyst: evidence for an ancestral association of exocyst subunits with autophagyassociated Atg8 proteins. Plant Signal Behav 2013, 8:e27099.
www.sciencedirect.com
ScienceDirect Dissecting the plant exocyst Bushra Saeed1, Carla Brillada1 and Marco Trujillo The exocyst is an evolutionary conserved complex that mediates tethering of post-Golgi vesicles derived from the conventional secretory pathway to the plasma membrane (PM), before SNARE-mediated fusion. Through its tethering function, connecting secretory vesicles to the PM, it mediates spatiotemporal regulation of exocytosis. As an integral element of the secretory machinery, the exocyst has been implicated in a large variety of processes. However, emerging evidence suggests that it may also cater for unconventional secretory pathways, as well as autophagy. The exocyst entertains a multitude of interactions with proteins and membrane phospholipids, reflecting its highly dynamic nature and the complex regulatory processes that hardwire it with cellular signalling networks. However, our molecular understanding of this essential complex remains fragmentary. Here we review recent work focusing on the molecular features that have revealed both commonalities with yeast and animals, as well as unique characteristics of the plant exocyst.
compartments of the Golgi apparatus, ending at the PM, as well as other target membranes. Transport of cargoes between the different stations of the secretory endomembrane system is mediated by vesicular traffic, which entails five major stages starting with the (i) budding of a vesicle and its (ii) transport towards a target membrane. The vesicle is initially (iii) tethered to the target membrane, and subsequently, the association is tightened during (iv) docking, to finally allow (v) fusion.
Address Albert-Ludwigs-University Freiburg, Faculty of Biology, Institute of Biology II, 79104 Freiburg, Germany
Exocyst interaction with the PM
Corresponding author: Trujillo, Marco ([email protected]) 1 Equal contribution. Current Opinion in Plant Biology 2019, 52:69–76 This review comes from a themed issue on Cell biology Edited by Eva Benkova and Yasin Dagdas
https://doi.org/10.1016/j.pbi.2019.08.004
The exocyst complex provides the first attachment of the vesicle to the PM, and by serving as a convergence point for GTPases, cellular signalling and the cytoskeleton, it orchestrates cargo delivery. It is an octameric tethering factor, first identified in the budding yeast Saccharomyces cerevisiae that consists of the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 [5] (Figure 1).
Early work from Segui-Simarro et al. [6] using electron tomography, identified ‘Y’ or ‘L’-shaped structures linking vesicles that were reminiscent of the mammalian exocyst complex [7]. Insensitivity to Brefeldin A [8,9] suggests that the exocyst complex is recruited to the secretory vesicle after budding from the trans-Golgi network (TGN), independently of vesicular traffic. In yeast, Sec15 binds to the vesicle in cooperation with the GTP-bound GTPase Sec4 [10]. Although, in Arabidopsis Sec15 is required for exocyst function [11], it remains unknown whether it carries out the same function.
1369-5266/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Secretion is responsible for the transport of de novo synthesized material, and it culminates with the exocytic event at the PM. Transported cargoes include integral PM proteins such as receptor kinases or CELLULOSE SYNTHASE COMPLEXES (CSCs), but additionally encompasses material released out to the apoplast such as, proteins, signalling peptides, small RNAs, polysaccharides required for cell wall growth and defence compounds [1–3,4]. The entry port of the secretory pathway is the endoplasmic reticulum (ER). Cargo traverses the different www.sciencedirect.com
Points of contact to the PM are created by the Sec3 and Exo70 subunits (Figure 1). The mammalian and yeast Sec3 and Exo70 subunits are able to directly bind phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a phospholipid that decorates the inner leaflet of the PM [10]. Sec3 of budding yeast interacts with PI(4,5)P2 through a cryptic pleckstrin homology (PH) fold in its N-terminus, whereas Exo70 interacts with PI(4,5)P2 through a patch of basic residues at its C-terminus [10]. In plants, the first indication of exocyst PM binding was provided by the small molecule Endosidin2 (ES2). Exo70A1 association to the PM was inhibited by ES2 treatment, resulting in its increased transport into the vacuole and reduced PM-localized PIN2 [12]. ES2 directly binds to Exo70A1, as well as the human Exo70, suggesting that it docks to a conserved structural feature required for PM binding [12]. Current Opinion in Plant Biology 2019, 52:69–76
70 Cell biology
Figure 1
PM
KEULE
PI(4,5)P2
PI?
SNAP33 (B1)
ICR1 RIC7 (B1) MtVapyrin (I) RIN4 (B1) TN2 (B1) PUB22-24 (B2) PUB18 & 19 (B1) ES2 (A1)
3
70
5
10
6
84
8
15
SCD1/2 For1F (Pp) actin
COG2
vesicle microtubules
Current Opinion in Plant Biology
Schematic of the plant exocyst complex and its known interactions. Schematic of the exocyst as a holocomplex composed of eight subunits. Arrows indicate interactions in which the exocyst engages (out), or is a potential effector or substrate (in) of other proteins or molecules. Sec3 and Exo70 directly bind to phospholipids (shown in red) to the plasma membrane via their positively charged residues (+++). Interacting Exo70 paralogue are indicated (e.g. Exo70B1 is B1). Specific phospholipids to which plant Exo70 subunits bind are unknown. The PH domain of Sec3 is probably connected via a flexible linker [16]. For1F is a Sec10 fusion to a formin in Physcomitrella patens (Pp). Subunit arrangement guided by the yeast exocyst structure revealed by cryo-EM [16]. Colour code and subunit arrangement (indicated by number: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) in two subcomplexes (SC1 and SC2) [16,28]. Proteins are from Arabidopsis thaliana, unless specified. Abbreviations: Phosphoinositides (PI), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), plasma membrane (PM), Medicago truncatula (Mt), +++ indicate basic residues.
The Arabidopsis Sec3 directly binds to PI(4,5)P2 through basic residues partially conserved in yeast and humans [13,14]. Further, overexpression of the PIP5 kinase resulted in increased association of Nicotiana benthamiana Sec3a to the PM [15]. However, the PH-domain was dispensable to target Sec3 to the PM, and the complementation of the sec3a-1 mutant [13]. One possible explanation is that the truncated Sec3 is integrated into the exocyst, which can still associate to the PM through the Exo70 subunit. It also suggests that in analogy to yeast, the C-terminal portion of Sec3, may be required for complex assembly and stability [16].
to the cytokinesis-specific syntaxin KNOLLE. Sec6 interaction is conserved in Physcomitrella patens, and its silencing delays the appearance of KEULE to the early cell plate [20]. KEULE was additionally shown to dynamically compete with SNAP33 for SYP121 (PEN1) binding, a close homologue of KNOLLE [21]. It is therefore conceivable that the exocyst alleviates this competition by sequestering KEULE via Sec6 upon arrival to the PM, to initiate SNARE-complex formation and membrane fusion. Of note, analyses of Exo70B1 function during immunity, suggest that also SNAP33 acts downstream of an exocyst–Exo70B1 complex [22].
Beyond its function in tethering, the exocyst may also be implicated in the regulation of later stages of exocytosis mediated by SNARE complexes. In budding yeast, Sec6 interacts mutually exclusively with the Q-SNARE Sec9 [17] and Sec1/Munc18, a regulator of membrane fusion [18]. In analogy, the Arabidopsis Sec6 physically interacts with the Sec1 orthologue KEULE [19], which itself binds
Functional specialization of exocyst subtypes
Current Opinion in Plant Biology 2019, 52:69–76
It was not until recently that cargoes were identified which required exocyst function for polarized [9,23,24], as well as general secretion involved in growth [4]. However, the irradiation of Exo70 genes in plants has led to the hypothesis that exocyst subtypes, ‘equipped’ with specific Exo70 subunits, may have www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 71
adopted specialized functions (Figure 2a). A study showed that exo70A1 mutants display secretory defects specific to CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN 1 (CASP1) [23], while lateral polarity of BOR1 and PDR6, was unaffected. In an elegant approach, Kulich et al. [25] addressed Exo70 specificity by assaying complementation of the cell wall defect in trichomes-specific to exo70H4. They tested 18 different Arabidopsis EXO70 paralogues under control
of the EXO70H4 promoter, and showed that functional complementation was exquisitely specific to Exo70H4. [25]. Another example is the evolution of EXO70 homologues exclusively present in plants forming symbiosis with arbuscular mycorrhiza fungi. These include the Medicago truncatula Exo70I, which is required for the development of the arbuscule and spatially restricted to hyphal tip areas of the periarbuscular membrane, suggesting a specialized function [26].
Figure 2
(a) PM
Golgi
Golgi (c)
(b)
PM
Receptor kinase A
Integral trans-membrane protein A
Soluble secreted molecule A
PM
Soluble secreted molecule C
Exocyst subtype X (Exo70X) Exocyst subtype Y (Exo70Y)
Receptor kinase B
Integral trans-membrane protein B
Soluble secreted molecule B
Small RNA Exocyst subtype Z (Exo70Z) Current Opinion in Plant Biology
Secretory pathways potentially involving the exocyst complex. (a) Conventional secretory pathway. Exocyst tethers secretory vesicles derived from the Golgi containing various cargoes such as integral membrane proteins, soluble secreted proteins, and potentially signalling peptides and carbohydrates. The Exo70 subunit (highlighted in red and purple) and Sec3 (red) are able to directly associate to the PM by binding phospholipids. Additional proteins participating in membrane association, that indirectly complex with exocyst subunits, are not included. Exocyst subtypes characterized by distinct Exo70 subunits, may participate in the delivery of specific cargo proteins, including different types of integral membrane proteins. (b) Unconventional secretion via multivesicular bodies (MVB) or late endosomes. MVBs are large bodies containing smaller intraluminal vesicles that have been reported to fuse to the PM. The exocyst may participate in their tethering to the PM. (c) Unconventional secretion via the exocyst-positive organelle (EXPO). The EXPO is characterized by a double membrane that fuses to the PM and releases a vesicle to the apoplast. The organelle of origin for EXPO remains unknown. (b–c) Both pathways remain poorly described, but may be involved in the delivery of extracellular vesicles shown to contain different types of proteins and small RNAs. (a–c) Docking stage of exocytosis mediated by SNARE proteins is not included.
www.sciencedirect.com
Current Opinion in Plant Biology 2019, 52:69–76
72 Cell biology
Complex dynamics A detailed study of exocyst dynamics at the PM showed that its subunits are localized to mobile foci, differing from endocytic sites [24]. However, whether the exocyst acts as a holocomplex [27,28], or assembles dynamically [29], has remained highly controversial. The Cryo-EM structure indicates that the eight subunits assemble hierarchically through helical bundles into the exocyst complex, and are organized in two subcomplexes (SCs) composed of SC1 (Sec8, Sec6, Sec5, and Sec3), and SC2 (Sec15, Sec10, Exo84, and Exo70) [16,28] (Figure 1). An elegant study in mammalian cells using knock-in tagged subunits, revealed that the two subcomplexes are highly dynamic and assemble at the PM independently of each other [30]. SC1 arrives before SC2, and Sec3, which is associated only loosely with the complex, departs before the rest of the complex [30]. During cytokinesis, Sec6 of Physcomitrella was visible at the phragmoplast midzone before detectable membrane and preceding Sec3a and Sec5b [20]. It is unlikely that this reflects the dynamics of exocyst subcomplexes, as these subunits are predicted to reside in SC1. It does suggest however that subunits assemble dynamically into a holocomplex or have complex-independent functions.
Points of contact with the cytoskeleton New studies have begun to reveal an intertwined relation between the exocyst with actin and microtubule cytoskeletal systems. A link to the actin cytoskeleton is provided by the formin For1F from P. patens. For1F is a fusion of a Sec10 exocyst subunit with a class I formin, which act as nucleators of actin filaments [31]. Sec6 dynamically colocalized with For1F, but increased density of cortical For1F in the absence of actin, suggests that targeting to the cell cortex is actin independent. Instead, actin may play a role in post vesicle delivery removal of For1F, and potentially, of the complex [31]. This would also provide an explanation for the observed clustering of the Arabidopsis Exo84b only after long term actin disruption [24]. By contrast, the interaction between the coiled-coil containing proteins VESICLE TETHERING 1 (VETH1) and VETH2 with CONSERVED OLIGOMERIC GOLGI 2 (COG2), promoted the association between cortical microtubules and Exo70A1 in xylem cells [32]. COG2 was able to interact with Sec10 in vivo, and thus, potentially build a Sec10–COG2–VETH1/2 connection with microtubules [33]. Accordingly, Sec8 arrival at the PM preceded secondary cell wall growth in xylem cells, and exo84b mutants displayed abnormal development of tracheary elements [33].
Exocyst regulation The intricate dynamics of the exocyst complex are indicative of a multi-layered regulation required to integrate its manifold intra and inter-molecular interactions, to Current Opinion in Plant Biology 2019, 52:69–76
provide a spatiotemporal orchestration of the exocytic event. Several exocyst subunits are known GTPase effectors [10]. In plants however, GTPase-mediated exocyst regulation appears to be achieved indirectly via adaptor proteins [34]. In one example, Exo70B1 interacts with the RHO OF PLANTS 2 (ROP2) effector RAC-INTERACTIVE BINDING MOTIF-CONTAINING PROTEIN 7 (RIC7) [35]. In the presence of a constitutive active ROP2, Exo70B1 and RIC7 relocalized to the PM of Vicia faba stomata. More recently, Sec15b and RabE1 were shown to interact with STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) and SCD2 [36]. Although the constellation of the interaction is not clear, SCDs may also act as adaptors connecting Sec15b and RabE1. Given that in yeast and animals Sec15 is a GTPase effector through which the exocyst is bound to the vesicle [10], it is possible that in plants Sec15b–SCD1 carry out this function via RabE1. Intriguingly, both SCDs bind to the inactive mutant of RabE1, leading authors to propose that SCDs may function in its activation. Additional exocyst-interacting proteins show parallels to adaptor proteins, for example, RPM1 INTERACTING PROTEIN 4 (RIN4), a PM-anchored protein involved in the immune response. RIN4 coexpression experiments, revealed the specific recruitment of Exo70B1 to the PM, but not that of its nearest homologue Exo70B2 [37]. Cleavage by the bacterial effector AvrRpt2 releases Exo70B1 from the PM, potentially blocking its function during immune responses [37,38,39]. Exocyst subunits are also regulated by post-translational modifications. Exo70B1 and Exo70B2 are actively regulated by ubiquitination and subsequent degradation, mediated by the related U-box type E3 ligases PUB18 and PUB22, respectively [39,40,41,42]. PUB22 and related E3s, interact with E2s dedicated to generate Lys63-linked ubiquitin chains, and this interaction was induced by immunostimulation in the case of PUB22 [43]. Lys-63 ubiquitin chains are key to regulate various steps of vesicle trafficking and transport to the vacuole [44,45]. The recruitment of exocyst subunits to the vacuole [25,46,47], either via the endocytic pathway or potentially by autophagy (see below), suggests that ubiquitination possibly contributes to this process. However, the reason for the induced degradation of Exo70 subunits still remains unclear. On the one hand, it may help to dampen the secretory activity, but alternatively, it could reflect a change in the levels of exocyst subtypes that mediate secretion of distinct cargoes (Figure 2a). Similar to PUB22, our unpublished data uncovered the phosphorylation of Exo70B2 at its C-terminus, which is predicted to bind phospholipids [47]. Mimicking phosphorylation reduced Exo70B2 levels at sites of active secretion, revealing a direct crosstalk between the secretory machinery and cellular signalling. www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 73
Different roles of the exocyst in vesicle tethering Several studies have underlined the essential nature of the exocyst [48], which is most likely indispensable for conventional secretion. Its relevance is nicely illustrated by the polarized secretion at sites of contact between the stigmatic papillae and a compatible pollen grains to promote pollen reception, which required all eight exocyst subunits [49]. Thus providing evidence for the requirement of the entire complex during pollination [11]. In line with this, disruption of Exo70A1 in Arabidopsis, results in a loss of secretory vesicles at the stigmatic papillar PM [50]. By contrast, unconventional protein secretion mediates the transport of proteins that lack an amino-terminal signal peptide, and bypass the conventional pathway. There are at least two possible routes in which the exocyst may be involved. The first involves the fusion of multivesicular bodies (MVBs/late endosomes) to the PM. During the acceptance of compatible pollen, MVBs fuse to the PM in Brassica napus [50] (Figure 2b), a process reminiscent to responses against penetrating fungal pathogens [51]. Accordingly, silencing of Exo70A1 resulted in a reduced incidence of MVBs at the PM [50]. A second route, involves the so called exocyst positive organelle (EXPO). It was first identified through the characterization of the Arabidopsis exocyst subunit Exo70E2, which does not colocalize with standard organelle markers [46,52]. EM analyses captured Exo70E2 labelled double membrane vesicles fusing with the PM and releasing a single membrane vesicle into the apoplast [52] (Figure 2c). These observations open the exciting possibility that the exocyst participates in the transport of extracellular vesicles (EVs). EVs contain proteins, as well as small RNAs, and their release to the apoplast is induced during infection [1,2,53]. Another exciting development in the field is the involvement of the exocyst in autophagy. Silencing of exocyst components in human cells was shown to have autophagy-inducing or inhibiting effects, which relied on subunit ability to recruit distinct autophagy signalling components [54]. The first link to autophagy in plants was provided by the observation that Exo70B1 is transported into the vacuole, where it partially colocalized with ATG8 [55]. In addition, ATG8 transport was reduced in exo70B1 hypocotyls of etiolated seedlings after tunicamycin treatment. However, Exo70B1 may be guarded by the LEUCINE-RICH REPEAT-CONTAINING 2 (TN2), which together with CALCIUM-DEPENDENT PROTEIN KINASE 5 (CPK5), induce a spontaneous cell death phenotype in www.sciencedirect.com
the null mutant [22,38,55,56]. Because the cell death phenotype, and potentially other phenotypes, which are reminiscent of autophagy-deficient mutants, are exo70B1independent, its function may also be downstream of autophagy activation. A subsequent study showed that Exo70E2 relocalized to autophagosomes only after the induction of autophagy with BTH treatment [46]. Interestingly, Exo70 homologues display an overrepresentation of predicted ATG8-interacting motifs (AIMs), compared to other subunits in Arabidopsis [57,58]. Our unpublished work shows that Exo70B2 displays a similar behavior as Exo70E2, and moreover, is able to physically interact with ATG8 [47]. Thus, Exo7B2 may directly link the secretory machinery and autophagy. Of note, during self-incompatible pollinations, secretory vesicles and MVBs, are absent from the PM of stigmatic papillae, and autophagy appeared to be induced to redirect vesicles and MVBs to the vacuole [50].
Conclusions and open questions The exocyst represents the last step in the secretory pathway before a vesicle is committed to exocytosis. Its diverse functions and interactions may therefore, be involved in gaging secretion, that together with the endocytic/vacuolar degradation pathways, control membrane protein levels and maintenance of cellular homeostasis. New studies have underlined the existence of specialized exocyst subtypes carrying specific Exo70 homologues that tether vesicles with distinct cargoes. This fascinating possibility opens however many new questions, for example: How are vesicles loaded with specific cargoes? And, how do exocyst subtypes, equipped with alternate Exo70 subunits, recognize the corresponding vesicles? Several lines of evidence support a link between the exocyst and autophagy, as well as unconventional secretion. However, our knowledge here remains limited, and future studies should reveal whether exocyst subunits are substrates or play a leading part. It is nevertheless tempting to speculate that the exocyst is able to switch between activities depending on the subtype, its cargo and/or cellular status. The ultimate goal will be to discover the molecular changes induced by crosstalk with interacting partners including GTPases and their scaffolds, signalling components, cytoskeleton, and so on, that modulate exocyst activity and secretory output.
Conflict of interest statement Nothing declared.
Acknowledgements The work in the authors’ laboratory is supported by grants from the German Research Foundation (DFG: TR 1073/6-1, TR 1073/4-1) and German Current Opinion in Plant Biology 2019, 52:69–76
74 Cell biology
Academic Exchange Service (DAAD:PPP-HK 57447859, Germany) to M.T. and SERB-Overseas Postdoctoral Fellowship (SB/OS/PDF-286/2016-17, India) of the Government of India to B.S. The authors apologize to colleagues whose work could not be included due to length restrictions of this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
3.
Cai Q, Qiao L, Wang M, He B, Lin FM, Palmquist J, Huang SD, Jin H: Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science 2018, 360:1126-1129. Baldrich P, Rutter BD, Zand Karimi H, Podicheti R, Meyers BC, Innes RW: Plant extracellular vesicles contain diverse small RNA species and are enriched in 10 to 17 nucleotide “tiny” RNAs. Plant Cell 2019, 31:315-324. Stegmann M, Monaghan J, Smakowska-Luzan E, Rovenich H, Lehner A, Holton N, Belkhadir Y, Zipfel C: The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling. Science 2017, 355:287-289.
4.
Zhu X, Li S, Pan S, Xin X, Gu Y: CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane. Proc Natl Acad Sci U S A 2018, 115:E3578-E3587. Exocyst function is required for the secretion of non-polar localized proteins such as CSI, thus a central protein of general plant growth. 5.
TerBush DR, Maurice T, Roth D, Novick P: The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 1996, 15:6483-6494.
6.
Segui-Simarro JM, Austin JR 2nd, White EA, Staehelin LA: Electron tomographic analysis of somatic cell plate formation in meristematic cells of Arabidopsis preserved by highpressure freezing. Plant Cell 2004, 16:836-856.
7.
Hsu SC, Hazuka CD, Roth R, Foletti DL, Heuser J, Scheller RH: Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments. Neuron 1998, 20:1111-1122.
8.
Drdova EJ, Synek L, Pecenkova T, Hala M, Kulich I, Fowler JE, Murphy AS, Zarsky V: The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J 2013, 73:709-719.
Mao H, Nakamura M, Viotti C, Grebe M: A framework for lateral membrane trafficking and polar tethering of the PEN3 ATPbinding cassette transporter. Plant Physiol 2016, 172:22452260. Exocyst function is required for the secretion of non-polar localized proteins such as CSI, thus a central protein of general plant growth.
9.
10. Wu B, Guo W: The exocyst at a glance. J Cell Sci 2015, 128:29572964. 11. Safavian D, Zayed Y, Indriolo E, Chapman L, Ahmed A, Goring DR: RNA silencing of exocyst genes in the stigma impairs the acceptance of compatible pollen in Arabidopsis. Plant Physiol 2015, 169:2526-2538. The acceptance of compatible pollen is the first process shown to require all exocyst subunits. 12. Zhang C, Brown MQ, van de Ven W, Zhang ZM, Wu B, Young MC, Synek L, Borchardt D, Harrison R, Pan S et al.: Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc Natl Acad Sci U S A 2016, 113:E41-50. ES2 disrupted Exo70A1 association to the PM and the transport of PIN2 to the PM, indicating that PM association of Exo70A1 is required for its function. ES2 directly binds to Exo70A1 in a region predicted to mediate PM association. 13. Bloch D, Pleskot R, Pejchar P, Potocky M, Trpkosova P, Cwiklik L, Vukasinovic N, Sternberg H, Yalovsky S, Zarsky V: Exocyst SEC3 Current Opinion in Plant Biology 2019, 52:69–76
and phosphoinositides define sites of exocytosis in pollen tube initiation and growth. Plant Physiol 2016, 172:980-1002. Sec3 was shown to bind PI(4,5)P2 and dynamically bind to the PM at the tip of growing pollen tubes. Relocalization of Sec3 predicted changes in growth direction in agreement of its function in secretion required for cell growth. 14. Pleskot R, Cwiklik L, Jungwirth P, Zarsky V, Potocky M: Membrane targeting of the yeast exocyst complex. Biochim Biophys Acta 2015, 1848:1481-1489. 15. Sekeres J, Pejchar P, Santrucek J, Vukasinovic N, Zarsky V, Potocky M: Analysis of exocyst subunit EXO70 family reveals distinct membrane polar domains in tobacco pollen tubes. Plant Physiol 2017, 173:1659-1675. This study supports the existence of distinct PM domains occupied by different Exo70 paralogues, suggesting also that exocyst-subtype specificities are dictated by Exo70 phospholipid binding traits. 16. Mei K, Li Y, Wang S, Shao G, Wang J, Ding Y, Luo G, Yue P, Liu JJ, Wang X et al.: Cryo-EM structure of the exocyst complex. Nat Struct Mol Biol 2018. 17. Sivaram MV, Saporita JA, Furgason ML, Boettcher AJ, Munson M: Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p. Biochemistry 2005, 44:6302-6311. 18. Morgera F, Sallah MR, Dubuke ML, Gandhi P, Brewer DN, Carr CM, Munson M: Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1. Mol Biol Cell 2012, 23:337-346. 19. Wu J, Tan X, Wu C, Cao K, Li Y, Bao Y: Regulation of cytokinesis by exocyst subunit SEC6 and KEULE in Arabidopsis thaliana. Mol Plant 2013, 6:1863-1876. 20. Tang H, de Keijzer J, Overdijk E, Sweep E, Steentjes M, Vermeer JE, Janson ME, Ketelaar T: Exocyst subunit Sec6 is positioned by microtubule overlaps in the moss phragmoplast prior to cell plate membrane arrival. J Cell Sci 2019. 21. Karnik R, Grefen C, Bayne R, Honsbein A, Kohler T, Kioumourtzoglou D, Williams M, Bryant NJ, Blatt MR: Arabidopsis Sec1/Munc18 protein SEC11 is a competitive and dynamic modulator of SNARE binding and SYP121-dependent vesicle traffic. Plant Cell 2013, 25:1368-1382. 22. Zhao T, Rui L, Li J, Nishimura MT, Vogel JP, Liu N, Liu S, Zhao Y, Dangl JL, Tang D: A truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant. PLoS Genet 2015, 11:e1004945. Exo70B1 may be a guarded protein and its absence in the null mutant potentially activates TN2. 23. Kalmbach L, Hematy K, De Bellis D, Barberon M, Fujita S, Ursache R, Daraspe J, Geldner N: Transient cell-specific EXO70A1 activity in the CASP domain and Casparian strip localization. Nat Plants 2017, 3:17058. Authors demonstrate that Exo70A1 is required for the delivery of CASP1, but not other tested PM proteins, supporting a functional specialization of exocyst subtypes. 24. Fendrych M, Synek L, Pecenkova T, Drdova EJ, Sekeres J, de Rycke R, Nowack MK, Zarsky V: Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol Biol Cell 2013, 24:510-520. 25. Kulich I, Vojtikova Z, Sabol P, Ortmannova J, Nedela V, Tihlarikova E, Zarsky V: Exocyst subunit EXO70H4 has a specific role in callose synthase secretion and silica accumulation. Plant Physiol 2018, 176:2040-2051. Exo70H4, but not other Exo70 paralogues, is able to functionally complement secretory defects in the exo70H1 mutant, further supporting a functional specialization of exocyst subtypes. 26. Zhang X, Pumplin N, Ivanov S, Harrison MJ: EXO70I is required for development of a sub-domain of the periarbuscular membrane during arbuscular mycorrhizal symbiosis. Curr Biol 2015, 25:2189-2195. 27. Picco A, Irastorza-Azcarate I, Specht T, Boke D, Pazos I, RivierCordey AS, Devos DP, Kaksonen M, Gallego O: The in vivo architecture of the exocyst provides structural basis for exocytosis. Cell 2017, 168:400-412 e418. www.sciencedirect.com
The plant Exocyst complex, beyond secretion Saeed, Brillada and Trujillo 75
28. Heider MR, Gu M, Duffy CM, Mirza AM, Marcotte LL, Walls AC, Farrall N, Hakhverdyan Z, Field MC, Rout MP et al.: Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex. Nat Struct Mol Biol 2016, 23:59-66. 29. Boyd C, Hughes T, Pypaert M, Novick P: Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J Cell Biol 2004, 167:889-901. 30. Ahmed SM, Nishida-Fukuda H, Li Y, McDonald WH, Gradinaru CC, Macara IG: Exocyst dynamics during vesicle tethering and fusion. Nat Commun 2018, 9:5140. This work illuminates the exocyst dynamics at the PM of human cells in unparalleled detail showing the dynamic assembly and disassembly of the exocyst complex during vesicle tethering and membrane fusion. 31. van Gisbergen PAC, Wu SZ, Chang M, Pattavina KA, Bartlett ME, Bezanilla M: An ancient Sec10-formin fusion provides insights into actin-mediated regulation of exocytosis. J Cell Biol 2018, 217:945-957. Interaction with the SNARE-interacting KEULE protein is conserved in the Physcomitrella patens. Authors show that Sec6 arrives at the phragmoplast before other exocyst subunits suggesting that exocyst subunits reside outside the holocomplex also in plants. 32. Oda Y, Iida Y, Nagashima Y, Sugiyama Y, Fukuda H: Novel coiled coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein. Plant Cell Physiol 2015, 56:277-286. VETH1 and VETH2 may contribute to the recruitment of the exocyst to microtubules, revealing a possible link of the exocyst to microtubules. 33. Vukasinovic N, Oda Y, Pejchar P, Synek L, Pecenkova T, Rawat A, Sekeres J, Potocky M, Zarsky V: Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis. New Phytol 2017, 213:1052-1067. Authors provide evidence that the exocyst is recruited to microtubules by interacting with COG2. COG2 was previously shown to complex with VETH1/2 which associates to microtubules [32]. 34. Lavy M, Bloch D, Hazak O, Gutman I, Poraty L, Sorek N, Sternberg H, Yalovsky S: A novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking. Curr Biol 2007, 17:947-952. 35. Hong D, Jeon BW, Kim SY, Hwang JU, Lee Y: The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. New Phytol 2016, 209:624-635. The ROP2 effector RIC7 is shown to interact with Exo70B1 and to recruit and mediate its recruitment to the PM. This work underlines the apparently indirect regulation by GTPases via adaptor proteins [34,36]. 36. Mayers JR, Hu T, Wang C, Cardenas JJ, Tan Y, Pan J, Bednarek SY: SCD1 and SCD2 form a complex that functions with the exocyst and RabE1 in exocytosis and cytokinesis. Plant Cell 2017, 29:2610-2625. SCD1/2 associate to RabE1, as well as Sec13b, suggesting that they potentially act as adaptors (see also Refs. [34,35]). In budding yeast, Sec15 is an effector of the Sec4 GTPase through which the exocyst is attached to the vesicle. Hence, author may have laid the foundation to elucidate this exocyst vesicle binding in plants. 37. Sabol P, Kulich I, Zarsky V: RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane. J Exp Bot 2017, 68:32533265. The immunity related RIN4, may act as an adaptor protein regulating Exo70B1 recruitment to the PM. RIN4 has been shown to be guarded by different NLR (see also Ref. [22]). 38. Stegmann M, Anderson RG, Westphal L, Rosahl S, McDowell JM, Trujillo M: The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death. Plant Signal Behav 2013, 8:e27421. 39. Stegmann M, Anderson RG, Ichimura K, Pecenkova T, Reuter P, Zarsky V, McDowell JM, Shirasu K, Trujillo M: The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell 2012, 24:4703-4716. 40. Furlan G, Nakagami H, Eschen-Lippold L, Jiang X, Majovsky P, Kowarschik K, Hoehenwarter W, Lee J, Trujillo M: Changes in PUB22 ubiquitination modes triggered by MITOGENwww.sciencedirect.com
ACTIVATED PROTEIN KINASE3 dampen the immune response. Plant Cell 2017, 29:726-745. 41. Seo DH, Ahn MY, Park KY, Kim EY, Kim WT: The N-terminal UND motif of the Arabidopsis U-Box E3 ligase PUB18 is critical for the negative regulation of ABA-mediated stomatal movement and determines its ubiquitination specificity for exocyst subunit Exo70B1. Plant Cell 2016, 28:2952-2973. PUB18 regulates Exo70B1 levels via ubiquitination and degradation. In contrast to PUB22 [35] which targets Exo70B2 via its armadillo repeat, PUB18 interacts with Exo70B1 via the U-box N-ter domain (UND). 42. Trujillo M: News from the PUB: plant U-box type E3 ubiquitin ligases. J Exp Bot 2018, 69:371-384. 43. Turek I, Tischer N, Lassig R, Trujillo M: Multi-tiered pairing selectivity between E2 ubiquitin-conjugating enzymes and E3 ligases. J Biol Chem 2018, 293:16324-16336. 44. Dubeaux G, Vert G: Zooming into plant ubiquitin-mediated endocytosis. Curr Opin Plant Biol 2017, 40:56-62. 45. Isono E, Kalinowska K: ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants. Curr Opin Plant Biol 2017, 40:49-55. 46. Lin Y, Ding Y, Wang J, Shen J, Kung CH, Zhuang X, Cui Y, Yin Z, Xia Y, Lin H et al.: Exocyst-positive organelles and autophagosomes are distinct organelles in plants. Plant Physiol 2015, 169:1917-1932. The exocyst subunit Exo70E2 resides in the so-called EXPO, but is relocated to autophagosomes and recruited into the vacuole upon autophagy induction. 47. Teh O-K, Lee C-W, Ditengou FA, Klecker T, Furlan G, Zietz M, Hause G, Eschen-Lippold L, Hoehenwarter W, Lee J et al.: Phosphorylation of the exocyst subunit Exo70B2 contributes to the regulation of its function. bioRxiv 2018. 48. Zarsky V, Kulich I, Fendrych M, Pecenkova T: Exocyst complexes multiple functions in plant cells secretory pathways. Curr Opin Plant Biol 2013, 16:726-733. 49. Doucet J, Lee HK, Goring DR: Pollen acceptance or rejection: a tale of two pathways. Trends Plant Sci 2016, 21:1058-1067. 50. Safavian D, Goring DR: Secretory activity is rapidly induced in stigmatic papillae by compatible pollen, but inhibited for selfincompatible pollen in the Brassicaceae. PLoS One 2013, 8: e84286. This work shows the differences between secretory responses during the pollen reception in Brassica napus and Arabidopsis thaliana. Self-incompatible pollen does not elicit similar reactions. Importantly, this work shows that Exo70A1 is required for vesicle delivery during pollen reception. 51. Dormann P, Kim H, Ott T, Schulze-Lefert P, Trujillo M, Wewer V, Huckelhoven R: Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions. New Phytol 2014, 204:815-822. 52. Wang J, Ding Y, Hillmer S, Miao Y, Lo SW, Wang X, Robinson DG, Jiang L: EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells. Plant Cell 2010, 22:4009-4030. 53. Rutter BD, Innes RW: Extracellular vesicles isolated from the leaf apoplast carry stress-response proteins. Plant Physiol 2017, 173:728-741. 54. Bodemann BO, Orvedahl A, Cheng T, Ram RR, Ou YH, Formstecher E, Maiti M, Hazelett CC, Wauson EM, Balakireva M et al.: RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 2011, 144:253-267. 55. Kulich I, Pecenkova T, Sekeres J, Smetana O, Fendrych M, Foissner I, Hoftberger M, Zarsky V: Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic 2013, 14:1155-1165. Authors uncover the first link between exocyst and autophagy by showing that Exo70B1 is transported into the vacuole where it colocalizes with the autophagy marker ATG8. Current Opinion in Plant Biology 2019, 52:69–76
76 Cell biology
56. Liu N, Hake K, Wang W, Zhao T, Romeis T, Tang D: CALCIUMDEPENDENT PROTEIN KINASE5 associates with the truncated NLR protein TIR-NBS2 to contribute to exo70B1mediated immunity. Plant Cell 2017, 29:746-759. 57. Tzfadia O, Galili G: The Arabidopsis exocyst subcomplex subunits involved in a golgi-independent transport into the
Current Opinion in Plant Biology 2019, 52:69–76
vacuole possess consensus autophagy-associated atg8 interacting motifs. Plant Signal Behav 2013, 8 http://dx.doi.org/ 10.4161/psb 26732. 58. Cvrckova F, Zarsky V: Old AIMs of the exocyst: evidence for an ancestral association of exocyst subunits with autophagyassociated Atg8 proteins. Plant Signal Behav 2013, 8:e27099.
www.sciencedirect.com