Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit

Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved July 19, 2021 (received for review March 26, 2021)
September 1, 2021
118 (36) e2105287118

Significance

Targeted secretion of the plasma membrane (PM) and cell wall material drives plant cell polarity and morphogenesis. Vesicle-tethering complex exocyst regulates this vital process. We uncovered that one of the exocyst subunits, EXO70A1, is responsible for recruiting the plant exocyst to the PM. EXO70A1 binds PM via interactions with several phospholipids that specifically contribute to the PM–lipid signature in plant cells. We demonstrated that in plants, phosphatidylinositol 4-phosphate and phosphatidic acid orchestrate the exocyst recruitment to the PM, and their action is mutually substitutable. Moreover, our results suggest that the PM net negative charge is a principal driving force at the membrane–protein interface. Our study provides a blueprint for future studies analyzing protein–membrane interactions in plant cells.

Abstract

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.
The plasma membrane (PM) of eukaryotic cells is spatially segregated into distinct domains with diverse functions, composition, and scales, a feature essential for many vital processes, including cell polarity regulation, signaling, and interactions with microorganisms (1, 2). Localized exocytosis is a fundamental process contributing to the establishment and maintenance of cellular polarity. An arsenal of small GTPases orchestrates the exocytosis through multiple effectors. Octameric protein complex exocyst is the small GTPase effector that facilitates the fusion of secretory vesicles with the PM (3, 4). The exocyst consists of eight subunits, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84, that are evolutionarily conserved across eukaryotes (5).
The plant exocyst complex is crucial for targeted secretion in cellular processes including the tip growth of root hairs and pollen tubes (69); hypocotyl elongation (10); cell wall maturation in xylem, endodermis, and trichomes (1113); pectin secretion in seed coats (14); recycling of PIN auxin transporters (15); and plant–microbe interactions (16, 17). The exocyst is also important for cell plate initiation and maturation during plant cytokinesis (1820). At the outer lateral PM of plant epidermal cells, the exocyst controls the secretion of polarly localized cargo proteins (21). Notably, exocyst accumulates at the outer lateral PM in dynamic foci that are distinct from sites of endocytosis (22).
While the Exo70 subunit is encoded by a single gene in yeast and animals, many EXO70 isoforms exist in angiosperm plants. Such multiplication enables the existence of diverse, functionally specific exocyst complexes even within one cell (23, 24). Particular EXO70 isoforms are involved in highly localized domain-specific secretion at the PM as documented in pollen tubes and trichomes (8, 13, 25). Some EXO70 isoforms even acquired diverged functions in autophagy regulation (26) or as negative regulators of tip growth (27, 28).
In Arabidopsis, housekeeping secretory processes in most sporophytic tissues involve the EXO70 isoform EXO70A1 (29, 30). On the other hand, these processes employ the closely related EXO70A2 isoform in the male gametophyte (9). Mutant plants lacking EXO70A1, unlike other studied EXO70 mutants, are severely morphologically affected and show secretory defects similarly to several mutants in other exocyst subunits (14, 15, 29, 31, 32). Moreover, among the multiple plant EXO70 isoforms, EXO70A1 is sequentially and structurally the most similar to the yeast and animal Exo70 (24, 29, 33). Hence, we focused on the EXO70A1 function in the plant exocyst architecture and PM recruitment in this study.
Molecular mechanisms of the exocyst function reside in mediating the first contact of secretory vesicles with the PM and facilitating the subsequent fusogenic SNARE complex formation, leading to a vesicle–PM fusion (34, 35). An emerging model of the exocyst complex based on partially solved structures of several exocyst subunits, protein interaction mapping, fluorescent microscopy, and cryogenic electron microscopy indicates that interlaced rod-like exocyst subunits align longitudinally at the core of the complex with distant parts being flexible and available for concomitant molecular interactions bridging vesicles and the PM (36). In yeast, the Sec15p subunit binds secretory vesicles via interaction with Rab GTPase Sec4p (37), while Sec3p and Exo70p subunits interact with the PM and serve as landmarks for the exocyst recruitment (3840). Both Sec3p and Exo70p bind the PM-specific phosphatidylinositol 4,5-bisphosphate (PIP2) (3941) and protein interactors such as Rho GTPases (42, 43). In animal cells, the exocyst membrane recruitment depends on the direct interaction of Exo70 with PIP2 (44). In yeast and animals, PIP2-dependent recruitment represents a general mechanism governing the localization of peripheral membrane proteins to the PM (45). In contrast, other anionic phospholipids, namely phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylinositol 4-phosphate (PI4P), seem to be more important than PIP2 in constituting the PM phospholipid signature in plants (4648).
In this study, we analyzed the overall Arabidopsis exocyst architecture and described the subunit connectivity map. Although the general architecture of the exocyst is evolutionary conserved, the PM recruitment mechanism represents a unique feature of the plant complex. By combining genetics, live-cell imaging, biochemistry, protein structure modeling, and molecular dynamics simulations, we demonstrated that the EXO70A1 subunit plays an essential role in PM–lipid signature recognition and dominates in the plant exocyst–PM recruitment to the PM.

Results

Comparative Proteomic Analysis of the Exocyst Complex Architecture In Planta Reveals the Presence of Two Conserved Modules.

To get insight into the architecture of the Arabidopsis exocyst complex, we performed a series of coimmunoprecipitations using GFP-tagged exocyst subunits as baits followed by mass spectrometry identification and label-free quantitative analysis of the isolated interacting proteins. Each bait coimmunoprecipitated with at least two other exocyst subunits (Fig. 1A). The analysis of the normalized spectral abundance factor (NSAF) suggests a tight association of exocyst subunits within two distinct modules; one composed of SEC3a, SEC5a, SEC6, and SEC8 (3–5–6–8 module) and another of SEC10, SEC15b, EXO70A1, and EXO84b (10–15–70–84 module). While the presence of an exocyst subunit in a mass spectrometry output does not imply a direct pairwise interaction with the particular bait, the high NSAF scores for subunit pairs suggest the proximity of those subunits within the complex. Interestingly, SEC3a and EXO70A1 were able to pull down subunits from both exocyst modules, suggesting their putative function as a bridge connecting the two modules. Furthermore, our bioinformatic analysis uncovered that the CorEx motifs known to be responsible for the major binary interactions within the yeast exocyst (49) are present in all tested eukaryotic lineages (SI Appendix, Fig. S1).
Fig. 1.
The plant exocyst complex consists of two loosely connected modules. (A) Coimmunoprecipitation of the Arabidopsis exocyst complex. Each GFP-tagged exocyst subunit was used as a bait to isolate interacting proteins that were identified by LC-MS/MS. The numbers indicate NSAF scores that suggest the proximity of each subunit pair within the complex. Scores for bait self-identification are depicted diagonally (gray fields). The brightness of the background indicates the relative interaction propensity based on the NSAF quartile ranking of all identified proteins within individual samples. Magenta indicates subunits of the 3–5–6–8 module, while green subunits of the 10–15–70–84 module. (B) A schematic spoke model of the Arabidopsis exocyst complex. Dashed lines represent interactions found via coimmunoprecipitation (Co-IP) in this study, and solid lines represent previously published Y2H interactions (10, 18). Thick and thin lines depict reciprocal or one-way interactions found in individual bait–prey pairs, respectively.
In summary, our current data based on the coimmunoprecipitation experiments, together with previously published yeast two-hybrid (Y2H) interactions (10, 18), show that the modular composition of the exocyst complex together with most of the binary subunit interactions are conserved between opisthokonts and plants (4951) (Fig. 1B). Given the evolutionary distance between these kingdoms, this finding suggests that the two-module organization is a fundamental feature of the exocyst architecture and function.

EXO70A1 Is Essential for the Plant Exocyst Recruitment to the PM.

The exocyst accumulates at the outer lateral PM domain in root epidermal cells (Fig. 2A). However, the molecular determinants of the plant exocyst recruitment to the PM are largely unknown. While both Exo70 and Sec3 subunits are essential for the direct exocyst–PM interaction in yeast and animal cells (3941, 44), our previous work showed that SEC3a membrane binding is dispensable for this process in Arabidopsis (6). This stimulated us to explore the role of EXO70 in the exocyst recruitment to the PM.
Fig. 2.
EXO70A1 is required for the recruitment of exocyst to the PM. (A) GFP-tagged exocyst subunits preferentially localize to outer lateral PM domains in WT root epidermal cells. (B) In exo70a1 mutant cells, GFP-tagged exocyst subunits from both modules are detached from the PM and accumulate in intracellular structures. The cartoon indicates the orientation of epidermal cells in AC. Regions of measurement are schematically displayed in red (PM) and white (cytoplasm) rectangles. (C) In exo84b mutant cells, GFP:EXO70A1 is partially localized to the PM in discrete patches, whereas other GFP-tagged exocyst subunits are detached from the outer lateral PM and accumulate in the cytoplasm. GFP:SEC10b is also localized to the baso-apical PM. (D) Analysis of the PM to cytoplasm (PM/Cyt) fluorescence ratio at the outer lateral PM in WT, exo70a1, and exo84b root epidermal cells. Green bars indicate median; distinct letters denote statistically different groups at a 0.01 significance level. For each line, at least 30 cells from six independent plants were quantified. (Scale Bar, 10 µm.) All images are in scale. (E) GFP:EXO70A1 localization in root epidermal cells of WT and sec3a plants complemented with pollen-specific pLAT52::SEC3aΔN. (Scale Bars, 10 µm.) An analysis of the PM to cytoplasm fluorescence ratio at the outer lateral PM showed nonsignificant difference (NS) using Student’s t test (P = 0.157). Green bars indicate the median. For each line, at least 30 cells from five independent plants were quantified.
We first systematically inspected the PM localization of exocyst subunits (SEC3a, SEC6, SEC8, SEC10b, SEC15b, and EXO84b), representing both structural modules of the exocyst complex, in root epidermal cells lacking EXO70A1. Spinning-disk confocal microscopy revealed that all tested subunits, including SEC3a, lost their PM localization in the exo70a1 background (Fig. 2 B and D). Importantly, the mislocalization of SEC3a implies that this subunit has only an accessory role in the recruitment of plant exocyst. The exocyst complex lacking EXO70A1 seems to be still assembled but is relocalized from the PM to abnormal intracellular structures (Fig. 2B). Two available combinations of tagged exocyst subunits (EXO84b:GFP and SEC10b:mRFP and SEC3a:GFP and EXO84b-mRFP), representing both modules, colocalized in the abnormal structures (SI Appendix, Fig. S2A). In agreement, we also identified SEC3a and SEC6 as interactors of GFP:SEC8 using coimmunoprecipitation followed by mass spectrometry in exo70a1 mutant plants (SI Appendix, Fig. S2B). All these intracellular structures were stained with the styryl FM4-64 dye, thus demonstrating their membrane nature (SI Appendix, Fig. S3A). Interestingly, the structures varied in their size, depending on the tagged exocyst subunit used for the visualization (SI Appendix, Fig. S3B). Moreover, the structures were positive for several endosomal-specific RAB GTPases, suggesting they are coalesced secretory and recycling compartments with endosomal and trans-Golgi identity (SI Appendix, Fig. S3C). They also contained KEULE, a representative of known interactors of the plant exocyst (52) (SI Appendix, Fig. S3D).
To test whether EXO70A1 can bind the PM autonomously in vivo, we introduced GFP-tagged EXO70A1 and other exocyst subunits from both exocyst modules into the exo84b background. It is known that the loss of the EXO84b subunit results in living plantlets (in contrast to gametophytically lethal sec6 and sec8), however, with strong pleiotropic defects and dwarf growth (18). While the localization of other exocyst subunits was completely cytoplasmic with no traces at the outer lateral PM in exo84b mutant cells, GFP:EXO70A1 partially retained its PM localization in discrete patches (Fig. 2 C and D). Interestingly, SEC10b:GFP was still present at the baso-apical cell side (Fig. 2C).
We also tested whether the polar localization of EXO70A1 is dependent on SEC3, which can serve as an exocyst landmark in yeast and mammalian cells (38, 40, 41). Since the loss of SEC3a, the major SEC3 subunit in plants, is gametophytically lethal (6), we transformed GFP:EXO70A1 into sec3a mutant plants expressing SEC3a under the control of the pollen-specific LAT52 promoter to selectively overcome this lethality. Moreover, the expressed SEC3a subunit was lacking its N-terminal domain responsible for the interaction with PM phospholipids in pollen tubes. We found that the EXO70A1 localization to the outer lateral domain of root epidermis in sec3a/pLAT52::SEC3aΔN cells was indistinguishable from wild-type (WT) cells (Fig. 2E), demonstrating that the exocyst recruitment to the PM in the sporophyte is not dependent on SEC3-mediated membrane binding in plants.
Taken together, our data demonstrate that EXO70A1 is essential for the native localization of the exocyst complex to the plant PM and can bind the PM autonomously. Interestingly, the exocyst assembly is likely dependent on the EXO84b subunit rather than on EXO70A1.

Full-Length EXO70A1 Is Required for Exocyst Recruitment to the PM and Functional Complementation of the exo70a1 Mutant.

We further investigated molecular details of the EXO70A1 recruitment to the PM. Based on the structures of nonplant EXO70 (5355) and the partial Arabidopsis EXO70A1 structure (33), we identified four regions corresponding to previously established domains (A, B, C, and D) in Arabidopsis EXO70A1. We then prepared a series of GFP-tagged EXO70A1-truncated variants lacking one or two domains (denoted as -BCD, ABC-, and –CD) and introduced them into the heterozygous exo70a1 mutant plants (Fig. 3A and Dataset S1). We then selected exo70a1 homozygous mutant seedlings and analyzed the subcellular localization of the truncated EXO70A1 variants using spinning-disk confocal microscopy at the outer lateral PM domain of root epidermal cells.
Fig. 3.
Full-length EXO70A1 is required for its efficient localization at the PM and functional complementation of the exo70a1 knockout. (A) Schematic representations of full-length and truncated GFP-tagged EXO70A1 variants. (B) Subcellular localization of GFP-tagged EXO70A1 variants in medial optical sections through root epidermal cells. (Scale Bars, 10 µm.) (C) Quantification of the PM to cytoplasm fluorescence ratio at the outer lateral PM in root epidermal cells. Green bars indicate median; letters denote statistically different groups at a 0.01 significance level. For each line, at least 30 cells from five independent plants were quantified. (D) Colocalization of the GFP-tagged EXO70A1 variants with mOrange-tagged DRP1C (dynamin) in optical sections parallel to the PM in root epidermal cells. (Scale Bars, 10 µm.) (E) Quantification of foci density at the outer lateral PM in root epidermal cells. Bars indicate median (EXO70A1 variants in green; DRP1C in magenta); letters denote statistically different groups at a 0.01 significance level. For each line, 10 to 22 cells from five independent plants were quantified. (F) Growth phenotype of plants expressing GFP-tagged EXO70A1 variants in the exo70a1 background. WT and nontransformed exo70a1 plants are displayed for reference. (Scale Bars, 1 cm.) Insets show the corresponding 5-d-old seedlings. (G) Interactions of EXO70A1 variants with SEC3a and the N-terminal part of EXO84b in the Y2H assay. AD, activating domain; BD, DNA-binding domain.
In contrast to full-length EXO70A1, none of the truncated variants localized to the outer lateral PM of epidermal cells (Fig. 3 B and C). The localization of ABC- and –CD variants was cytoplasmic without any traces at the PM, and the –CD variant often accumulated in cytoplasmic aggregates. The -BCD variant also localized mostly to the cytoplasm but was weakly detectable at baso-apical PM domains (Fig. 3B).
Next, we compared the localization pattern of all EXO70A1 variants in the PM focal plane, where the exocyst subunits appear as dynamic foci representing putative exocytosis sites (22). To unambiguously focus on the PM, we performed the imaging in plants coexpressing a PM endocytic marker DRP1C:mOrange (56). While full-length GFP:EXO70A1 was found in the typical foci at the PM, all truncated variants were completely detached from the PM (Fig. 3 D and E). Consistent with the microscopic observations, none of the truncated GFP:EXO70A1 variants could complement the exo70a1 mutant phenotype during the seedling and adult plant development in contrast to the full-length GFP:EXO70A1 variant (Fig. 3F).
To dissect which parts of EXO70A1 are responsible for the direct interactions with exocyst subunits, we performed a Y2H assay testing pairwise interactions between truncated variants of EXO70A1 with SEC3a and N-terminal parts of EXO84b (EXO84b-N) that were previously shown to interact with full-length EXO70A1 in Y2H assays (10, 18). While the ABC- variant strongly interacted with both SEC3a and EXO84b-N, the AB– variant interacted only weakly with EXO84b-N. The -BCD and –CD variants were insufficient to bind either SEC3a or EXO84b-N (Fig. 3G and SI Appendix, Fig. S4).
In summary, we deduce that the B, C, and D domains are essential for the EXO70A1 interaction with the PM. Although the A domain might be potentially dispensable, it dramatically improves the efficiency of this interaction. On the other hand, the A and B domains with the contribution of the C domain are important for intersubunit interactions within the exocyst complex.

Key Phospholipid Components Uniquely Contribute to the EXO70A1 Recruitment to the Plant PM.

Yeast and animal Exo70 subunits were previously demonstrated to be recruited to the PM via direct binding PIP2, a principal phospholipid determinant of the opisthokont PM identity (39, 44). Therefore, we investigated the role of PIP2 in the EXO70A1 recruitment to the PM in Arabidopsis root epidermal cells. We utilized the pip5k1 pip5k2 double mutant that lacks two predominant sporophytic isoforms of the 11-member phosphatidylinositol-4-phosphate 5-kinase family in Arabidopsis and shows a dramatically reduced PIP2 level (57). Surprisingly, the GFP:EXO70A1 localization at the PM in pip5k1 pip5k2 homozygotes was indistinguishable from control plants (Fig. 4 A and B).
Fig. 4.
PM localization of EXO70A1 depends on phosphatidylinositol 4-phosphate and diacylglycerol kinase–produced pool of phosphatidic acid. (A) GFP:EXO70A1 recruitment to the outer lateral PM domain in root epidermal cells in WT and pip5k1 pip5k2 mutant cells. (Scale Bar, 10 µm.) (B) The PM association index of GFP:EXO70A1 in WT and pip5k1 pip5k2 mutant cells. Each dataset consists of at least 40 evaluated cells from six independent plants. Green bars represent the median; no significant difference was found at a 0.01 significance level. (C) GFP:EXO70A1 recruitment to the outer lateral PM in root epidermal cells upon inhibition of PA-producing diacylglycerol kinases by R59022 and PI4P-producing PI4 kinases by PAO inhibitors (1 h treatment). (Scale Bar, 10 µm.) (D) The PM association index of GFP:EXO70A1 upon inhibition by R59022 and PAO. Each dataset consists of at least 64 cells from eight independent plants. Green bars represent the median; letters indicate statistically different groups at a 0.01 significance level.
Next, we tested a putative role of plant PM signature phospholipids in the EXO70A1 localization to the outer lateral PM domain in root epidermal cells. We applied R59022 and PAO, established pharmacological inhibitors of the PA-producing diacylglycerol kinase (DGK) and phosphatidylinositol 4-kinase, respectively (SI Appendix, Fig. S5 A and B) (46, 48, 58). The GFP:EXO70A1 recruitment to the outer lateral PM was significantly reduced upon the R59022 treatment, drastically decreased upon the PAO treatment, and nearly lost when a combination of both inhibitors was applied (Fig. 4 C and D). Kinetics of the EXO70A1 detachment from the PM was in agreement with the action of R59022 and PAO on genetically encoded markers for PA and PI4P, respectively (SI Appendix, Fig. S6). To test whether PI4P is essential or the net negative PM charge is sufficient for the EXO70A1 PM recruitment, we combined the inhibition of PI4P synthesis by PAO with the external addition of lyso-PA, similarly to ref. 46. We observed a clear recovery of EXO70A1 recruitment to the lateral PM (SI Appendix, Fig. S5C). In contrast, the depletion of sterols by treatment with fenpropimorph, a potent sterol biosynthesis inhibitor (59), had a negligible effect on the EXO70A1 localization to the outer lateral PM domain, further corroborating the dominant role of anionic phospholipids in EXO70A1 recruitment (SI Appendix, Fig. S5D).
Collectively, our results uncovered a synergic contribution of PI4P and PA to the EXO70A1 PM recruitment in vivo in Arabidopsis root epidermal cells and questioned the prominent role of PIP2 in this process.

Conserved C-Terminal Lysine Residues Are Crucial for the Initial EXO70A1 Interaction with Anionic Phospholipids.

To resolve the EXO70A1 interaction mechanism with the plant PM signature phospholipids at the molecular level, we performed coarse-grained molecular dynamics (CGMD) simulations. This computational approach was shown to be highly accurate in predicting the membrane-bound state of peripheral membrane proteins (60). Using the Robetta web server, we generated a comparative model of the full-length EXO70A1 structure, which we used in subsequent simulations. The simulated system contained one molecule of EXO70A1, ions, water molecules, and a complex phospholipid bilayer with the composition of charged phospholipids corresponding to the plant PM (57, 6163).
We carried out five independent CGMD simulations with different starting velocities resulting in a total of 5 μs simulation time. In all simulations, we observed a close association of EXO70A1 with the complex phospholipid bilayer (Fig. 5 A and B). In all cases, the initial association of EXO70A1 with the membrane was mediated by its C-terminal part. Later in the simulation, the whole protein interacted with phospholipids and remained stably bound for the remaining simulation time (Fig. 5 A and B). The same binding behavior was previously described for yeast Exo70p, pointing to the evolutionarily conserved mode of membrane interaction among the EXO70 proteins (41). An analysis of contacts between protein amino acid residues and phosphate groups of the phospholipid bilayer revealed that the highest number of interacting residues was located at the C-terminal part, a region corresponding to the C and D domains of EXO70A1 (Fig. 5 C and D). By comparing the identified membrane-interacting residues of EXO70A1 with a multiple sequence alignment of EXO70A family isoforms from a wide selection of Streptophyta, we found five evolutionary conserved residues which play a dominant role in the membrane association, namely lysines K393, K462, K549, K607, and K611 (Fig. 5 C and D and SI Appendix, Fig. S7). A detailed analysis of the contacts between protein residues and particular anionic phospholipids showed that the EXO70A1 interaction with PI4P and PIP2 is mediated predominantly by the amino acid residues K607 and K611 of the D domain (SI Appendix, Fig. S8). In contrast, the residues interacting with the PA and PS, namely K393 and K462, are distributed beyond the D domain to the C domain and, in the case of PS, also by K137 located at the end of the A domain (SI Appendix, Fig. S8).
Fig. 5.
Molecular dynamics simulations reveal the key residues indispensable for the EXO70A1 interaction with anionic phospholipids. (A) Representative snapshots of the molecular dynamics simulations of EXO70A1 with a complex negatively charged phospholipid bilayer. Three different time points are shown, the starting conditions (0 ns), the initial encounter of EXO70A1 with the phospholipid bilayer mediated by the protein C terminus (390 ns), and the stably membrane-bound protein (1,000 ns). EXO70A1 is colored in white, acyl chains are gray, headgroup atoms are purple, sodium atoms are orange and water molecules are transparent cyan. (B) Progress of five independent molecular dynamics simulations shown as the minimal distance between EXO70A1 and the phospholipid bilayer. Different replicas are depicted in different colors. (C) Time course of EXO70A1 residues in contact with the phosphate groups of the complex phospholipid bilayer. The contacts were defined as the number of phosphate groups within 0.8 nm of protein atoms. The in-scale schematic domain organization of EXO70A1 is shown above the plot. (D) The mean number of EXO70A1–phosphate contacts per amino acid residue of the EXO70A1 molecule from five independent runs. (E) The mean number of EXO70A1–phosphate contacts mapped onto the protein structure. The solvent-excluded surface is shown in a transparent representation. (F) Two-dimensional density of different negatively charged phospholipid molecules in the leaflet adjacent to EXO70A1 calculated over the last 500 ns of the simulation shows preferential clustering of PIP2, PI4P, and PA.
To further characterize the EXO70A1 interaction with the complex membrane, we analyzed a lateral distribution of different phospholipid molecules in the phospholipid leaflet adjacent to the protein. We observed that EXO70A1 caused pronounced clustering of PI4P, PIP2, and PA but not PS (Fig. 5E). Such a differential binding and clustering of several negatively charged phospholipids suggest a complementary contribution of different plant PM signature phospholipids to the EXO70A1–membrane interaction. These computational results are consistent with our in vivo observations in Arabidopsis root epidermal cells. Notably, the observed clustering of PIP2 by EXO70A1 provides a molecular explanation of the recently observed phenomenon showing that EXO70A1 is required for the PIP2 accumulation at prospective Casparian strips (11).

EXO70A1 Recognizes a Specific Subset of Plant PM Signature Phospholipids In Vitro.

To prove the proposed mode of EXO70A1 binding to the PM, we tested the EXO70A1 capability to directly bind major negatively charged PM phospholipids in vitro. Our attempts to obtain recombinant full-length GST:EXO70A1 were not successful. However, we managed to purify near full-length GST:EXO70A1 (residues 70 to 638) from Escherichia coli lysates, corresponding to structurally characterized mouse, yeast, and plant Exo70s (33, 5355) (SI Appendix, Fig. S9A).
We performed protein–lipid overlay assays to get a general overview of the lipid-binding capability of EXO70A1. Consistently with our computational data, we found promiscuous EXO70A1 binding to all negatively charged phospholipids (Fig. 6A). We then addressed the contribution of individual signature phospholipids present in the plant PM under more physiological conditions. To this end, we employed cosedimentation lipid-binding assays using large unilamellar vesicles (LUV) with phospholipid composition that recapitulates the CGMD simulations. Under these conditions, GST:EXO70A1 showed a strong interaction with PA, PI4P, and PIP2, as 80 to 90% of the input protein was bound to LUVs containing any of these phospholipids, without significant binding preference (Fig. 6B and SI Appendix, Fig. S9B). Conversely, the presence of PS in LUVs did not significantly increase GST:EXO70A1 in the phospholipid-bound fraction above the level observed for the electroneutral PC/PE control. GST alone used as a negative control bound no phospholipids in the same experimental setup (Fig. 6C).
Fig. 6.
EXO70A1 binds negatively charged phospholipids in vitro. (A and D) Protein–lipid overlay assays with purified recombinant GST:EXO70A1 or GST:EXO70A1-5xK/E. Bound proteins were visualized using the anti-GST–horseradish peroxidase antibody. TAG, triglyceride; DAG, diacylglycerol; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; CL, cardiolipin; PI, phosphatidylinositol; PI4P, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; Chol, cholesterol; SM, sphingomyelin; Sulf, 3-sulfogalactosylceramide. (B, C, and E) Cosedimentation of LUVs with purified recombinant GST:EXO70A1, GST:EXO70A1-5xK/E or GST. LUVs contained the particular phospholipids at indicated fractions. After sedimentation, proteins in supernatants (S) and pellets (P) were subjected to SDS-PAGE and visualized by Coomassie Blue staining (gel images on top). Means ± SEM of membrane-bound fractions are shown; individual data points from three to six independent experiments are indicated by circles; letters denote statistically different groups at a 0.05 significance level. PM signature phospholipids strongly interacting with EXO70A1 are shown in magenta. The entire gels are displayed in SI Appendix, Fig. S9B.
Next, we tested the importance of the five evolutionarily conserved lysines (K393, K462, K549, K607, and K611) that were predicted to interact with anionic phospholipids in CGMD simulations (Fig. 5). Using site-directed mutagenesis, we substituted the five key lysines for glutamates and tested the ability of this mutated near full-length variant (GST:EXO70A1-5xK/E) to interact with phospholipids. In contrast to WT GST:EXO70A1, the point-mutated GST:EXO70A1-5xK/E variant lost its ability to bind anionic phospholipids in both protein–lipid overlay assays and cosedimentation lipid-binding assays (Fig. 6 D and E and SI Appendix, Fig. S9B).
In summary, our data show that plant EXO70A1 adapted its membrane-binding properties to a specific subset of plant PM signature phospholipids. These multiplex EXO70A1–phospholipid interactions are crucial for the recruitment of the exocyst complex to the PM.

Evolutionarily Conserved EXO70A1 Lysine Residues Are Critical for the Exocyst Function In Planta.

To test the impact of lysine substitutions on the EXO70A1 membrane recruitment in vivo, we introduced GFP-tagged EXO70A1-5xK/E into heterozygous exo70a1 mutant plants. We then analyzed the subcellular localization of GFP:EXO70A1-5xK/E in exo70a1 homozygous seedlings (Fig. 7A). The mutant variant lost the typical EXO70A1 localization pattern at the outer lateral PM, leaving only weak nonpolarized distribution at the PM (Fig. 7B). The PM association index was significantly lower compared to the WT variant (Fig. 7C). Expression of GFP:EXO70A1-5xK/E in exo70a1 homozygotes restored the mutant phenotype on the seedling level but could not fully complement the aberrant exo70a1 morphology of adult plants (SI Appendix, Fig. 8D). These plants could not typically reach the WT height, were more branched than WT, and were mostly sterile (SI Appendix, Fig. S10).
Fig. 7.
Mutations of the key lysine residues in EXO70A1 impair the capability to bind the PM. (A) Schematic representations of GFP-tagged EXO70A1 with introduced point mutations (in red). (B) Subcellular localizations of point-mutated (5xK/E) GFP:EXO70A1 in Arabidopsis root epidermal cells. (Left) Medial optical section through epidermal and cortical cells. (Right) Optical section parallel to the PM. GFP:EXO70A1-5xK/E lost its typical enrichment at the outer lateral PM. For WT GFP:EXO70A1, that was used as control, see Fig. 3 B and C. (Scale Bars, 10 µm.) (C) Quantification of the PM to cytoplasm fluorescence ratio at the outer lateral PM in root epidermal cells. The green bar indicates median; n > 30. The PM association index is significantly different from that of WT at a 0.01 significance level (Fig. 3C). (D) GFP:EXO70A1-5xK/E could only partially complement phenotypic deviations of exo70a1 mutant plants (variability of the phenotype in SI Appendix, Fig. S10). For exo70a1 complemented with WT EXO70A1 used as a control, see Fig. 3F. (Scale Bar, 1 cm.) (Inset) Corresponding 5-d-old seedling. (E) Schematic depiction of the exocyst recruitment to the outer lateral PM via coincidence detection of multiple phospholipids and proteins. EXO70A1 superimposed on the experimentally solved exocyst complex (Protein Data Bank code 5YFP) is shown together with major phospholipid partners and putative polarization protein interactor. The relative number of contacts between EXO70A1 lysine residues and phospholipids obtained from the CGMD simulations is displayed in the purple scale.
To test the functional significance of the evolutionarily conserved lysine residues and their function in other cell types, we prepared an analogical point-mutated variant (5xK/E) of tobacco YFP:NtEXO70A1a and analyzed its localization in tobacco pollen tubes. In contrast to WT YFP:NtEXO70A1a, which localized to PM just behind the pollen tube apex (8), the YFP:NtEXO70A1a-5xK/E variant was completely cytoplasmic (SI Appendix, Fig. S11). To further investigate the involvement of PIP2 and PA on the NtEXO70A1a membrane recruitment, we tested the localization of WT and the point-mutated variant in pollen tubes overexpressing major PIP2- and PA-producing enzymes, PIP5K5 or PLDδ3, respectively (64, 65). While the elevated levels of PIP2 or PA significantly enhanced the recruitment of WT YFP:NtEXO70A1a to the PM, they did not lead to the recruitment of the YFP:NtEXO70A1a-5xK/E variant (SI Appendix, Fig. S11).
Collectively, our data show that the key evolutionarily conserved lysine residues play an essential role in the EXO70A1 function as the major landmark for the exocyst recruitment to the PM (Fig. 7E). The proposed mechanism is thus likely to be conserved among plants and utilized by distinct cell types.

Discussion

The exocyst complex was discovered more than two decades ago (66), and its essential function in polarized exocytosis was demonstrated in various organisms, yet the mechanistic details of the exocyst architecture and membrane recruitment are still not fully understood. Here, we focused on the plant exocyst and demonstrated its several evolutionarily unique features in respect to subunit connectivity and membrane interaction.
Our coimmunoprecipitation and Y2H experiments show that the plant exocyst complex is composed of two conserved modules, similarly to yeast and mammalian cells (50, 51, 67). Given the evolutionary distance between plant, yeast, and mammalian cells, we hypothesize that the two-module architecture is an ancestral feature already present in the last eukaryotic common ancestor. Our data suggest that the overall connectivity of the plant exocyst resembles the situation in mammalian cells, where the 3–5–6–8 module is tightly bound together and only loosely associated with the 10–15–70–84 module (51, 68). However, unlike in mammalian cells, EXO70 and SEC3 subunits directly interact in plant cells, representing a plant-specific bridge between the two modules. Moreover, the colocalization of the two modules in the abnormal intracellular structures in exo70a1 mutant cells suggests the existence of additional intersubunit interactions connecting the two modules, for example, SEC3–SEC10 (Fig. 1B). These structures significantly differed in their size (SI Appendix, Fig. S3B). Subunits from the 3–5-6–8 module labeled small structures, while SEC10b and EXO84b from the 10–15-70–84 module displayed a much broader size distribution. However, the absence of larger structures visualized by GFP:SEC15b suggests that protein–protein interactions of individual subunits, rather than whole modules, determine the size of the membrane structures.
In yeast and animal cells, Exo70 and Sec3 have partially redundant functions in the exocyst–PM recruitment via direct interactions with phospholipids (39, 44). In mammalian cells, the SEC3-containing exocyst module can reach the PM independently of the EXO70-containing module (51). In plants, however, the mutated variants of SEC3a incapable to bind PIP2 were still able to reach the PM in Arabidopsis pollen tubes, pointing to the central role of the EXO70 subunit in phospholipid-mediated exocyst recruitment. Furthermore, the SEC3b isoform cannot substitute for the SEC3a function in the gametophyte as well as sporophyte because of its negligible expression level (6). In concert with this notion, our data show that EXO70A1 is crucial for the delivery of other exocyst subunits, including SEC3a, to the PM in Arabidopsis epidermal root cells. In agreement, the recruitment of exocyst was shown to be EXO70A1 dependent at prospective Casparian strips (11).
We documented that plant EXO70A1 directly binds multiple anionic phospholipids (PA, PI4P, and PIP2) in contrast to yeast and mammalian cells in which only PIP2 is responsible for the Exo70 recruitment (39, 44). Given the almost complete loss of EXO70A1 from the PM upon the pharmacological PA and PI4P depletion and persistent PM recruitment of EXO70A1 in pip5k1 pip5k2 double mutants in root epidermal cells, we conclude that PIP2 contribution to the EXO70A1 PM recruitment is rather auxiliary. However, we cannot completely rule out the contribution of PIP2 to the EXO70A1 PM recruitment because of either residual PIP2 levels in pip5k1 pip5k2 mutants or a specific electrostatic signature in other cell types. For instance, PIP2 seemingly plays an important role in the EXO70A1 recruitment to PM microdomains of prospective Casparian strip deposition (11) or PM subdomains in trichomes (25). Furthermore, the direct EXO70A1 binding to phosphoinositides including PIP2 was independently shown in a lipid–protein overlay (69). We recently described that the pollen-specific EXO70A2 isoform, closely related to EXO70A1, fully complements the exo70a1 mutant phenotype, while EXO70A1 is unable to complement the loss of EXO70A2 in pollen (9). In pollen tubes, PIP2 localizes to the secretory hotspot at the PM and likely dominates PA and PI4P in the regulation of pollen exocytic trafficking (6, 8, 64, 70). Different PM–lipid signatures in sporophytic and gametophytic cells might correspond to distinct phospholipid preferences of particular EXO70A isoforms.
Our discovery of the direct EXO70A1 interaction with PA is a novel and unique feature of the plant exocyst complex. In animal and yeast cells, PA is present in negligible levels in the PM, and its function as a PM signature phospholipid outside plants is not clear (71). In the plant research, however, recent papers documented a vital contribution of PA to the plant PM composition and function. For example, the lipidomics of tobacco leaf and BY2 suspension PM fractions demonstrated 5 to 10 times higher content of PA than PIP2 (62), and the PA level in plant tissues further rises during biotic and abiotic stresses (72). The importance of the plant PA signaling is also reflected by the expansion of genes encoding PA-producing enzymes in plant evolution (73). Furthermore, several candidate PA interactors, including components of signaling pathways, endocytic machinery, and cytoskeletal regulators, were identified in proteomic screens (7478). Interestingly, the actin-capping protein, which is regulated by PIP2 in animals, interacts preferentially with PA in plants due to its lineage-specific features that drive the interaction (79, 80). Collectively, our results and published data support the hypothesis that PI4P and PA have overtaken the role of PIP2 during plant evolution (47, 48). Moreover, our data indicate that the action of PA and PI4P in the protein–membrane recruitment might be mutually substitutable, pointing to the PM net negative charge as a driving force at the membrane–protein interface.
In growing pollen tubes (6, 8), developing endodermis (11), and mature trichomes (25), polarized localization of the exocyst was correlated with genetically encoded fluorescent phospholipid probes. However, in root epidermal cells, neither PIP2, PI4P, nor PA was enriched at the outer lateral PM (46, 81). This suggests that although specific phospholipids are critical for the exocyst membrane recruitment, they are not sufficient for the polarized exocyst localization to the outer lateral PM domain in root epidermal cells. Thus, we hypothesize that coincidence detection of anionic phospholipids and specific protein partners by EXO70 is required for the polar exocyst localization in plants (Fig. 7E) as shown for other peripheral membrane proteins (82). Accordingly, the EXO70B1 recruitment to the PM is dependent on the RIN4 protein, a regulator of plant defense (83). We previously showed that in a single cell, different EXO70 pairs localize to nonoverlapping PM domains enriched with distinct phospholipids (EXO70A1/H4 in Arabidopsis trichomes; EXO70A1a/B1 in tobacco pollen tubes) (8, 25). Therefore, we speculate that the multiple plant EXO70 isoforms might be targeted to distinct membrane domains by the recognition of specific lipid–protein signatures (84).
In summary, our study shows that the overall architecture of the exocyst vesicle-tethering complex is broadly conserved among yeast, animals, and plants. However, the recruitment of the plant exocyst to the PM is substantially different due to the evolutionarily diverged PM composition (85). We conclude that the members of the EXO70A family are critical subunits responsible for the exocyst recruitment to the plant PM, while SEC3 plays only an auxiliary role. Whereas the N-terminal part of Arabidopsis EXO70A1 is responsible for intersubunit interactions within the exocyst complex, the C-terminal part is essential for the exocyst binding to the PM. The EXO70A1–PM binding is orchestrated by interactions of evolutionarily conserved lysine residues with selected plant PM signature phospholipids. The multivalent interaction of EXO70A1 with PA and PI4P represents a unique plant-specific feature of the exocyst complex. By our multidisciplinary approach, combining plant genetics, advanced live-cell imaging, protein–lipid interaction assays, and molecular dynamics simulations, we provide a blueprint for future studies analyzing membrane–protein interfaces in plant cells.

Materials and Methods

Plant Material and Growth Conditions.

Arabidopsis thaliana seeds were surface sterilized (70% ethanol for 3 min, 10% commercial bleach for 10 min, and rinsed three times in sterile distilled water) and stratified for 3 d at 4 °C. Seeds were then germinated on vertical 1/2× MS agar plates (half-strength Murashige and Skoog salts [Duchefa Biochemie] supplemented with 1% sucrose, vitamin mixture, and 1.6% plant agar [Duchefa Biochemie]) at 21 °C and 16 h of light per day. Seedlings that were 7 d-old were transferred into turf pellets (Jiffy Products International) and grown at 22 °C and 16 h of light per day in growth rooms.
Published mutant and transgenic lines (GFP/RFP) are listed in SI Appendix, Supplementary Materials and Methods.
For all crossings and transformations into the exo70a1 or exo84b, plants heterozygous for these mutations were used due to drastically reduced fertility of respective homozygotes. For crossings into pip5k1 pip5k2, plants heterozygous for pip5k1 and homozygous for pip5k2 were used. Homozygous plants were then selected for experiments from segregating populations based on their typical phenotypes on vertical agar plates (29, 57).
To evaluate the complementation capacity of truncated and point-mutated EXO70A1 variants, we analyzed their functionality in the second generation of transformed exo70a1 heterozygotes. GFP-positive seedlings were visually selected and transferred to soil to observe their terminal phenotype 6 wk after germination.

Coimmunoprecipitation and Mass Spectrometry Analysis.

For the analysis of in vivo interactions within the exocyst complex by coimmunoprecipitation, we used 1 g 10 d old Arabidopsis seedlings for each isolation. Protein interactors of GFP-tagged exocyst subunits and of free GFP (used as a negative control) were isolated using the µMACS GFP-Tagged Protein Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Instead of the provided wash buffers, we used the Sec6/8 buffer (10). Bound proteins were eluted with 100 µL the preheated elution buffer, flash frozen in liquid nitrogen, and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Detailed description of LC-MS/MS is provided in SI Appendix, Supplementary Materials and Methods.

Confocal Microscopy, Pharmacological Treatments, and Quantification in Arabidopsis Root Cells.

The subcellular localization of exocyst subunits and EXO70A1 variants was examined in the late meristematic to early elongation zone of 5-d-old seedlings using a spinning-disk confocal microscope (Yokogawa CSU-X1 on the Nikon Ti-E platform, Agilent MLC400 laser box, Andor Zyla sCMOS camera, NIS Elements 4.1 software). A Plan Apochromat 60×/1.2 water immersion objective was used for medial optical sections, while a Plan Apochromat 100×/1.4 oil objective was used for detailed analysis of the PM localization (in the plane on the PM). The GFP and mOrange fluorescence was excited by 488 and 561 nm laser lines, respectively, and recorded at 1-s exposure times, 4× averaging using 525/30 and 607/36 emission filters (Semrock Brightline). Seedlings were observed in chambered coverslips (LabTek II) in 1/2× MS liquid medium covered with a block of 1/2× MS agar medium.
Staining with 5 µM FM4-64 was performed for 30 min, and seedlings were immediately imaged.
For the inhibition of diacylglycerol kinases and/or phosphatidylinositol 4-kinases, seedlings were treated with 12.5 µM R59022 (Sigma-Aldrich; 25 mM stock in DMSO) and/or 30 µM PAO (Sigma-Aldrich; 30 mM stock in DMSO) in liquid 1/2× MS medium for 1 h before imaging. For the lyso-PA addback experiment, seedlings were treated with 54 μM lyso-PA (fresh 13.5 mM stock in 50% ethanol) together with PAO for 1 h before imaging. Control seedlings were treated with equivalent volumes of the corresponding solvent(s). At least 40 cells from six to eight independent plants were evaluated. In time-lapse experiments, eight roots were imaged by 10 min for 80 min in total; samples were mounted in liquid 1/2× MS medium between slides in this case.
For the inhibition of sterol production, seedlings were treated with fenpropimorph (50 μg/mL) on vertical 1/2× MS agar plates for 20 h before imaging. At least 35 cells from eight independent plants were evaluated.
Postacquisition image processing and quantitative analyses were performed in the Fiji software (86), and figures were assembled in Inkscape. The PM association index was calculated as a ratio of the PM/cytoplasm GFP signal based on mean gray values in narrow regions of interest parallel to the PM and next to it inside the cell, avoiding vacuoles or nuclei (Fig. 2 B, Inset). Density of endo- and exocytic foci at the PM was calculated using Find Maxima (value 9) in Fiji after applying Gaussian blur (0.5).
To analyze the size of the abnormal intracellular structures, confocal images of root epidermal cells were binarized using a manual threshold setting to select the structures of interest followed by Particle Analysis in Fiji to acquire the area of every structure. At least 84 structures from eight roots were analyzed for each line.

Y2H Assay.

Cells of Saccharomyces cerevisiae AH109 double transformed with plasmids bearing tested exocyst subunits were selected on -LEU -TRP medium. Single colonies were then scale diluted and grown for 3 d on -ADE -HIS -LEU -TRP selective medium. The assay was repeated three times. SEC3a and EXO84b-N fused to the DNA-binding domain exhibited highly promiscuous interactions (10, 18); therefore, only their fusions with the activating domain were included in the assay.

Homology Modeling and Molecular Dynamics Simulations of AtEXO70A1.

A three-dimensional model of the full-length Arabidopsis EXO70A1 protein structure was predicted using the Robetta web server (87). The structure of EXO70A1 was mapped into the MARTINI coarse-grain (CG) representation using the martinize.py script (88). The ELNEDYN representation with the distance cutoff 0.9 nm and the spring force constant 500 kJ · mol−1 · nm−2 was used to prevent any undesired large conformational changes during CGMD simulations (89). The MARTINI CG model for all phospholipid molecules used in this study was taken from ref. 90. The phospholipid bilayer, in total composed of 1,200 phospholipid molecules, containing POPC:POPE:POPS:POPA:POPI4P:POPI(4,5)P2 (molecular ratio 37:37:10:10:5:1) was prepared using CharmmGUI Martini Maker (91). CGMD simulations were performed in GROMACS version 5 (92). The bond lengths were constrained to equilibrium lengths using the Linear Constraint Solver (LINCS) algorithm (93). Lennard-Jones and electrostatics interactions were cut off at 1.1 nm, with the potentials shifted to zero at the cutoff (88). A relative dielectric constant of 15 was used. The neighbor list was updated every 20 steps using the Verlet neighbor search algorithm. Simulations were run in the isothermal–isobaric (NPT) ensemble. The system was subject to pressure scaling to 1 bar using the Parrinello–Rahman barostat (94) with temperature scaling to 303 K using the velocity-rescaling method (95) with coupling times of 1.0 and 12.0 ps. Simulations were performed using a 20-fs integration time step. Initially, the protein was placed ∼4.0 nm away from the membrane. Subsequently, the standard MARTINI water and Na+ ions were added to ensure the electroneutrality of the system. The whole system was energy minimized using the steepest descent method up to the maximum of 500 steps and equilibrated for 10 ns. Production runs were performed for up to 1 μs. The standard GROMACS tools as well as in-house codes were used for the analysis.

Purification of Recombinant GST-tagged Proteins.

Plasmids (pGEX4T-2) bearing GST:EXO70A1 and GST:EXO70A1-5xK/E, both lacking the N terminus encoding the first 69 amino acids, and GST alone were transformed into E. coli ArcticExpress Codon Plus DE3 cells (Stratagene). Overnight culture (10 mL) was diluted in 1 L of fresh Luria–Bertani medium with gentamicin (25 mg/L) and ampicillin (50 mg/L) and grown at 37 °C to OD 0.8. Bacterial culture was cooled down to room temperature (RT), recombinant protein expression was induced by the addition of powdered lactose (final concentration 1 g/L), and the culture was then cooled down to 15 °C. After 20-h incubation (160 rpm at 15 °C), the bacterial culture was centrifuged in 50-mL falcon tubes (5,000 rpm, 4 °C, and 10 min), and the pellet was frozen at –20 °C overnight. After being thawed on ice, cells were resuspended in 50 mL cold purification buffer (50 mM Tris [pH 7.5], 0.2 M NaCl, 0.1% β-mercaptoethanol, 5% glycerol, and protease inhibitor mixture), disrupted by sonication (3 × 10 min on ice with 10 min breaks at 30% power, Bandelin Sonoplus), and cleared by centrifugation (20,000 × g, 4 °C, 1 h). The supernatant was loaded on a chromatographic column containing 1 mL Glutathione Sepharose (GE Healthcare) and let flow through by gravity. Unbound proteins were washed away with 10 mL purification buffer, and GST-tagged proteins were eluted five times with 1 mL 25 mM glutathione in purification buffer to collect five following fractions. The quality of the eluted protein was evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The whole purification procedure was done at 4 °C.

Protein–Lipid Overlay Assay.

Membrane lipid strips (Echelon Bioscience, P-6002) were blocked with 5 mL blocking solution (3% bovine serum albumin [Sigma, A7030], 0.1% Tween-20 in phosphate-buffered saline [PBS], pH 7.2) at RT for 1 h under gentle agitation. Then, 2 mL blocking solution were removed and purified and GST:EXO70A1 or GST was added (final protein concentration of 1 µg/mL). Strips were incubated at RT for 2 h under gentle agitation and then washed three times for 10 min with PBS containing 0.1% Tween-20. Anti-GST HRP (Cytiva, RPN1236) diluted 1:5,000 in the blocking solution was layered over the strips and incubated at RT for 1 h under gentle agitation. After three washes for 10 min with PBS containing 0.1% Tween-20, strips were incubated with 600 µL Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2236) for 2 min, and lipid-bound proteins were visualized using the ChemiDoc XRS+ Imaging system (Bio-Rad).

Cosedimentation Lipid-binding Assay with LUVs.

Dioleyl PA, PE, PC, and PS were dissolved in chloroform as 4 mM stocks, whereas PI4P and PIP2 in chloroform/methanol/water (20:9:1) as 500 µM stock. Phospholipid mixtures of different composition were then mixed to give 400 nmol total phospholipids per sample, and the solvent was evaporated in a SpeedVac at 35 °C for 45 min. Phospholipids were rehydrated in 500 µL extrusion buffer (250 mM raffinose pentahydrate [Sigma, R0250], 25 mM Tris-HCl [pH 7.5], and 1 mM dithiothreitol), incubated at RT for 1.5 h on a rotator wheel, and sonicated for 30 s at 75 kHz. The MiniExtruder (Avanti) with a filter pore diameter of 200 nm was washed with the extrusion buffer, and LUVs were prepared by pushing the phospholipid suspensions through the extruder 20 times. Each sample was filled up to 1 mL with the binding buffer (750 mM KCl, 150 mM Tris-HCl [pH 7.5], 6 mM dithiothreitol, and 3 mM EDTA [pH 7.5]). After centrifugation (72,000 × g, RT, 30 min), the supernatant was discarded and the pellet resuspended in 1 mL binding buffer. LUVs were centrifuged again, supernatant discarded, and the pellet resuspended in 100 µL of the binding buffer. GST:EXO70A1 (WT or 5xK/E) protein was added (7 µL), and samples were incubated at RT for 45 min on a rotator wheel. After centrifugation (72,000 × g, RT, 30 min), supernatants were removed and precipitated using 400 µL cold acetone or concentrated in SpeedVac to a final volume of 25 µL. Pellets were washed with 100 µL binding buffer and centrifuged again. Pellets were resuspended in 25 µL 6× SDS loading buffer and denatured at 70 °C for 10 min. The supernatants and pellets were subjected to 10% SDS-PAGE and stained with Coomassie Blue for quantification of free (supernatant) and phospholipid-bound (pellet) proteins.

Transient Expression and Confocal Microscopy in Tobacco Pollen Tubes.

Plasmids encoding YFP:NtEXO70A1a, YFP:NtEXO70A1a-5xK/E, or phospholipid-modifying enzymes (AtPIP5K5, NtPLDδ3) were introduced into germinating tobacco pollen grains on solid culture medium by particle bombardment using a helium-driven particle delivery system PDS-1000/He (Bio-Rad) as described previously (96). Gold particles were coated with 1 μg plasmid DNA for NtEXO70A1a variant expression and with 5 μg plasmid DNA for the overexpression of phospholipid-modifying enzymes, namely the Arabidopsis AtPIP5K5:CFP (64) and tobacco mRFP:NtPLDδ3 variants (65). Imaging was performed on 8- to 10-h-old pollen tubes using the spinning-disk confocal microscope as described above (Plan Apochromat 60× WI objective, 488 nm laser line excitation, Semrock Brightline 542/27 emission filter).
For measurements of the total length of the NtEXO70A1a membrane signal under various conditions, we subtracted the background signal and then manually measured the length of the pollen tube membrane covered with the YFP signal in Fiji.

Statistics.

Unless stated otherwise, experimental values were tested for significant differences with the agricolae package in R using either Kruskal–Wallis or Welch Anova with Tukey’s post hoc comparisons with the Benjamini–Hochberg correction. In the case of two data sets comparison the Student’s t test was used (indicated in figure legends).
Plant material, molecular cloning, mass spectrometry analysis, and phylogenetic analysis are described in detail in SI Appendix, Supplementary Materials and Methods.

Data Availability

All study data are included in the article and/or SI Appendix.

Acknowledgments

This work was supported by the Czech Science Foundation (project GA17-27477S to P.P., GA15-24711S to L.S., and GA19-21758S to M.P.) and by the Operational Programme Prague – Competitiveness (project no. CZ.2.16/3.1.00/21519 to the Institute of Experimental Botany). Part of T.P.’s and V.Ž.’s income was covered by the Plant Experimental Biology Center (project no. CZ.02.1.01/0.0/0.0/16_019/0000738). N.S. was supported by the Postdoctoral Fellowship Programme of the Czech Academy of Sciences (L200382051) and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR)/Charles University (OP RDE, call no. 02_18_053). We acknowledge the Imaging Facility of the Institute of Experimental Botany of the Czech Academy of Sciences (IEB AS CR) supported by the MEYS CR (LM2018129 Czech BioImaging) and IEB AS CR. We thank Prof. Ingo Heilmann and Dr. Till Ischebeck for providing the plasmid encoding PIP5K5:CFP and the pip5k1 pip5k2 double mutant, Prof. Sebastian Bednarek and his colleagues for the DRP1C:mOrange-expressing line, Dr. Farhah Assaad for the KEULE:GFP-expressing line, Dr. Matyáš Fendrych for critical reading of the manuscript, Hana Soukupová for plant crossing, and our technician Jana Šťovíčková for her great assistance. We also thank the developers of the Linux operating system and the open‐source software used in this study, particularly Fiji, Inkscape, Gimp, Gnumeric, and Gromacs.

Supporting Information

Appendix (PDF)
Dataset_S01 (PDF)

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Information & Authors

Information

Published in

The cover image for PNAS Vol.118; No.36
Proceedings of the National Academy of Sciences
Vol. 118 | No. 36
September 7, 2021
PubMed: 34470819

Classifications

Data Availability

All study data are included in the article and/or SI Appendix.

Submission history

Published online: September 1, 2021
Published in issue: September 7, 2021

Keywords

  1. cell polarity
  2. exocyst
  3. EXO70A1
  4. phospholipids
  5. plasma membrane

Acknowledgments

This work was supported by the Czech Science Foundation (project GA17-27477S to P.P., GA15-24711S to L.S., and GA19-21758S to M.P.) and by the Operational Programme Prague – Competitiveness (project no. CZ.2.16/3.1.00/21519 to the Institute of Experimental Botany). Part of T.P.’s and V.Ž.’s income was covered by the Plant Experimental Biology Center (project no. CZ.02.1.01/0.0/0.0/16_019/0000738). N.S. was supported by the Postdoctoral Fellowship Programme of the Czech Academy of Sciences (L200382051) and the Ministry of Education, Youth and Sports of the Czech Republic (MEYS CR)/Charles University (OP RDE, call no. 02_18_053). We acknowledge the Imaging Facility of the Institute of Experimental Botany of the Czech Academy of Sciences (IEB AS CR) supported by the MEYS CR (LM2018129 Czech BioImaging) and IEB AS CR. We thank Prof. Ingo Heilmann and Dr. Till Ischebeck for providing the plasmid encoding PIP5K5:CFP and the pip5k1 pip5k2 double mutant, Prof. Sebastian Bednarek and his colleagues for the DRP1C:mOrange-expressing line, Dr. Farhah Assaad for the KEULE:GFP-expressing line, Dr. Matyáš Fendrych for critical reading of the manuscript, Hana Soukupová for plant crossing, and our technician Jana Šťovíčková for her great assistance. We also thank the developers of the Linux operating system and the open‐source software used in this study, particularly Fiji, Inkscape, Gimp, Gnumeric, and Gromacs.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

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;
Juraj Sekereš1
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;
Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, 128 44 Prague, Czech Republic;
Nemanja Vukašinović
Institute of Experimental Botany, Czech Academy of Sciences, 165 02 Prague, Czech Republic;
Present address: Department of Plant Biotechnology and Bioinformatics, Center for Plant Systems Biology, Ghent University, and VIB Center for Plant Systems Biology, 9052 Ghent, Belgium.
Jitka Ortmannová
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;
Přemysl Pejchar
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;
Department of Experimental Plant Biology, Faculty of Science, Charles University in 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;
Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, 128 44 Prague, Czech Republic;
Institute of Experimental Botany, Czech Academy of Sciences, 165 02 Prague, Czech Republic;
Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology, University of Chemistry and Technology, 166 28 Prague, Czech Republic
Institute of Experimental Botany, Czech Academy of Sciences, 165 02 Prague, Czech Republic;
Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, 128 44 Prague, Czech Republic;
Institute of Experimental Botany, Czech Academy of Sciences, 165 02 Prague, Czech Republic;

Notes

3
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: L.S., R.P., V.Ž., and M.P. designed research; L.S., R.P., J.S., N.S., N.V., J.O., M.K., P.P., K.B., M.G., E.J.-D., V.M., T.P., J.Š., and M.P. performed research; L.S., R.P., N.V., P.P., K.B., E.J.-D., V.M., and T.P. contributed new reagents/analytic tools; L.S., R.P., J.S., and M.P. analyzed data; and L.S., R.P., J.S., and M.P. wrote the paper.
1
L.S., R.P., and J.S. contributed equally to this work.

Competing Interests

The authors declare no competing interest.

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    Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit
    Proceedings of the National Academy of Sciences
    • Vol. 118
    • No. 36

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