Exocyst Research Articles

Exo70 Promotes the Invasion of Pancreatic Cancer Cells via the Regulation of Exosomes.

Pancreatic cancer (PC) is an aggressive and fatal malignant tumor, and exosomes have been reported to be closely related to PC invasion and metastasis. Here we found that Exo70, a key subunit of the exocyst complex, promoted PC metastasis by regulating the secretion of tumor exosomes. Clinical sample studies showed that Exo70 was highly expressed in PC and negatively correlated with patients' survival. Exo70 promoted PC cell lines' invasion and migration. Interestingly, knockdown of Exo70, or using an Exo70 inhibitor (ES2) inhibited the secretion of tumor exosomes and increased the accumulation of cellular vesicles. Furthermore, Exo70 was found to accumulate in the exosomes, which then fused with neighboring PC cells and promoted their invasion. Moreover, Exo70 increased the expression of exosomal PD-L1, leading to the immune escape of PC cells. In vivo, knockdown of Exo70 or treatment with ES2 both decreased the tumor metastasis of PC cells in mice. This study provides new insight into the mechanism of invasion and metastasis in PC and identifies Exo70 as a potential prognostic factor and therapeutic target for PC.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Deletion of Exoc7, but not Exoc3, in male germ cells causes severe spermatogenesis failure with spermatocyte aggregation in mice.

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.

Interplay of EXO70 and MLO proteins modulates trichome cell wall composition and susceptibility to powdery mildew.

Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

Ralb and Exocyst Regulate Endothelial Cell Von Willebrand Factor Release

Background: Identification of novel therapeutic targets for pharmacologic modulation of circulating von Willebrand factor (vWF) would be invaluable in treatment of von Willebrand disease, but also in other pathophysiologic processes where vWF plays a vital role. These include cardiovascular disease, cancer metastasis, angiogenesis, and smooth muscle proliferation. vWF is almost exclusively produced by the endothelial cells and stored in specialized secretory granules, Weibel Palade bodies (WPBs). We have previously shown that loss of the exocyst complex, an essential multi-subunit trafficking protein, which physically tethers secretory granules to the plasma membrane in yeast, increases vWF release. Small GTPases act as regulatable molecular switches. Cycling between GTP-bound ('on') and GDP-bound ('off') states, they control all essential cellular processes. Among these, Ral GTPase has previously been shown to be important in WPB secretion and vWF release. Since Ral specifically regulates exocyst in other cellular functions, we hypothesized that exocyst is also under direct control of Ral in vWF release. Methods: All experiments were performed on human umbilical vein endothelial cells (HUVECs). RalA/B and exocyst depletion were performed via targeted siRNAs. vWF antigen was estimated using ELISA. RalB activation was determined by a commercial pulldown assay specific for the activated form of RalB. Expression of FLAG-tagged RalB was performed by lentiviral transduction. RalB mutants were generated using site-directed mutagenesis of FLAG-RalB (WT). Spinning disc confocal microscope was used for immunofluorescence (IF). Immuno-electron microscopy was performed using immune-gold labeling. FLAG-pulldown and immunoprecipitation (IP) was performed on cell lysates using antibody-labeled magnetic beads. Results: RalB but not RalA depletion, or degradation with the RalB-selective inhibitor dihydroartemisinin, significantly impaired thrombin-induced vWF exocytosis (55±7% decrease). RalB was activated (GTP-bound) and phosphorylated upon thrombin stimulation of HUVECs. Correspondingly, expression of constitutively active RalB-G23V mutant, which stays in a GTP-bound state, significantly increased vWF exocytosis compared to RalB-WT (69±4.6% increase). IF and EM confirmed co-localization of RalB and exocyst on WPBs. Unexpectedly, FLAG pulldown and protein identification by mass spectrometry demonstrated that RalB-WT (GDP-bound;“off”) but not RalB-G23V (GTP-bound;“on”) interacts with exocyst, confirmed by immunoblotting. IP of resting and thrombin-stimulated native HUVEC lysates with RalB antibodies, as well as reverse-IP with exocyst antibodies confirmed this differential binding. Exocyst binding site mutation (RalB-WTD49E) and transduction of these mutants in HUVECs resulted in significantly augmented vWF exocytosis (40±3% increase), like constitutively active RalB-G23V mutant. Moreover, IF showed uniform cytoplasmic distribution of WPB cigars in RalB-WT HUVECs, whereas in RalB-WTD49E mutants WPBs were found at the plasma membrane, like RalB-G23V. This confirms that RalB-free exocyst tethers WPB to the plasma membrane ( Figure 1). Serine phosphorylation site mutation (RalB-WTS198A), previously shown to regulate GTPase activity of RalB, hence GTP-GDP cycling, also significantly decreased RalB exocyst binding, in turn increasing vWF release. This shows that post-translational phosphorylation of RalB at serine 192, which is also accelerated by thrombin activation of endothelial cells, regulates RalB-exocyst interaction, hence vWF release. Conclusions: We have identified an important regulatory role of RalB in exocyst-dependent WPB secretion and vWF release. WPB localized exocyst is associated with RalB in resting endothelium. Endothelial cell activation switches RalB ‘on’, and exocyst, now uncoupled from GTP-bound RalB, is free to tether WPBs to the plasma membrane and facilitate vWF release.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Exo84c interacts with VAP27 to regulate exocytotic compartment degradation and stigma senescence

In plants, exocyst subunit isoforms exhibit significant functional diversity in that they are involved in either protein secretion or autophagy, both of which are essential for plant development and survival. Although the molecular basis of autophagy is widely reported, its contribution to plant reproduction is not very clear. Here, we have identified Exo84c, a higher plant-specific Exo84 isoform, as having a unique function in modulating exocytotic compartment degradation during stigmatic tissue senescence. This process is achieved through its interaction with the ER localised VAP27 proteins, which regulate the turnover of Exo84c through the autophagy pathway. VAP27 recruits Exo84c onto the ER membrane as well as numerous ER-derived autophagosomes that are labelled with ATG8. These Exo84c/exocyst and VAP27 positive structures are accumulated in the vacuole for degradation, and this process is partially perturbed in the exo84c knock-out mutants. Interestingly, the exo84c mutant showed a prolonged effective pollination period with higher seed sets, possibly because of the delayed stigmatic senescence when Exo84c regulated autophagy is blocked. In conclusion, our studies reveal a link between the exocyst complex and the ER network in regulating the degradation of exocytosis vesicles, a process that is essential for normal papilla cell senescence and flower receptivity.

Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects.

Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. We have previously demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. Here, we extend this analysis using LRRK2 drosophila models and show that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, our study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.

The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity.

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response (HR), in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers respectively, in order to characterize the in-vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases, and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309 and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that, XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1, but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with identifier PXD041405.

Genome-Wide Analysis of Exocyst Complex Subunit Exo70 Gene Family in Cucumber.

Cucumber (Cucumis sativus L.) is an important vegetable worldwide, but its yield is affected by a wide range of pathogens and pests. As the major subunit of the exocyst complex, the roles of Exo70 members have been shown in Arabidopsis and rice, but their function are unknown in cucumber. Here, we identified 18 CsExo70 members in cucumber, which were divided into three groups (Exo70.1-Exo70.3) and nine subgroups (Exo70A-Exo70I) based on the phylogenetic tree. Subsequently, systematical analyses were performed, including collinearity, gene structure, cis-acting elements, conserved motifs, expression patterns, and subcellular localization. Our results showed that CsExo70 genes were generally expressed in all tissues, and CsExo70C1 and CsExo70C2 were highly expressed in the stamen. Moreover, the expression levels of most CsExo70 genes were induced by Pseudomonas syringae pv. lachrymans (Psl) and Fusarium oxysporum f. sp. cucumerinum Owen (Foc), especially CsExo70E2 and CsExo70H3. In addition, these CsExo70s displayed similar location patterns with discrete and punctate signals in the cytoplasm. Together, our results indicate that CsExo70 members may be involved in plant development and resistance, and provide a reference for future in-depth studies of Exo70 genes in cucumber.

The EXO70 inhibitor Endosidin2 alters plasma membrane protein composition in Arabidopsis roots.

To sustain normal growth and allow rapid responses to environmental cues, plants alter the plasma membrane protein composition under different conditions presumably by regulation of delivery, stability, and internalization. Exocytosis is a conserved cellular process that delivers proteins and lipids to the plasma membrane or extracellular space in eukaryotes. The octameric exocyst complex contributes to exocytosis by tethering secretory vesicles to the correct site for membrane fusion; however, whether the exocyst complex acts universally for all secretory vesicle cargo or just for specialized subsets used during polarized growth and trafficking is currently unknown. In addition to its role in exocytosis, the exocyst complex is also known to participate in membrane recycling and autophagy. Using a previously identified small molecule inhibitor of the plant exocyst complex subunit EXO70A1, Endosidin2 (ES2), combined with a plasma membrane enrichment method and quantitative proteomic analysis, we examined the composition of plasma membrane proteins in the root of Arabidopsis seedlings, after inhibition of the ES2-targetted exocyst complex, and verified our findings by live imaging of GFP-tagged plasma membrane proteins in root epidermal cells. The abundance of 145 plasma membrane proteins was significantly reduced following short-term ES2 treatments and these likely represent candidate cargo proteins of exocyst-mediated trafficking. Gene Ontology analysis showed that these proteins play diverse functions in cell growth, cell wall biosynthesis, hormone signaling, stress response, membrane transport, and nutrient uptake. Additionally, we quantified the effect of ES2 on the spatial distribution of EXO70A1 with live-cell imaging. Our results indicate that the plant exocyst complex mediates constitutive dynamic transport of subsets of plasma membrane proteins during normal root growth.

TORC1 regulation of dendrite regrowth after pruning is linked to actin and exocytosis.

Neurite pruning and regrowth are important mechanisms to adapt neural circuits to distinct developmental stages. Neurite regrowth after pruning often depends on differential regulation of growth signaling pathways, but their precise mechanisms of action during regrowth are unclear. Here, we show that the PI3K/TORC1 pathway is required for dendrite regrowth after pruning in Drosophila peripheral neurons during metamorphosis. TORC1 impinges on translation initiation, and our analysis of 5' untranslated regions (UTRs) of remodeling factor mRNAs linked to actin suggests that TOR selectively stimulates the translation of regrowth over pruning factors. Furthermore, we find that dendrite regrowth also requires the GTPase RalA and the exocyst complex as regulators of polarized secretion, and we provide evidence that this pathway is also regulated by TOR. We propose that TORC1 coordinates dendrite regrowth after pruning by coordinately stimulating the translation of regrowth factors involved in cytoskeletal regulation and secretion.

The exocyst complex is required for the trafficking and delivery of KCa3.1 to the basolateral membrane of polarized epithelia.

Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein, we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. In addition, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, Soluble N-ethylmaleimide-sensitive factor (SNF) Attachment Receptors (SNARE) proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.NEW & NOTEWORTHY Our Ussing chamber and immunoblot experiments demonstrate that when subunits of the exocyst complex were transiently knocked down, this significantly reduced the basolateral population and functional expression of KCa3.1. These data suggest, combined with our protein association experiments, that the exocyst complex regulates the tethering of KCa3.1-containing vesicles to the basolateral membrane prior to the SNARE-dependent insertion of channels into the basolateral membrane of epithelial cells.

The exocyst complex in neurological disorders.

Exocytosis is the process by which secretory vesicles fuse with the plasma membrane to deliver materials to the cell surface or to release cargoes to the extracellular space. The exocyst-an evolutionarily conserved octameric protein complex-mediates spatiotemporal control of SNARE complex assembly for vesicle fusion and tethering the secretory vesicles to the plasma membrane. The exocyst participates in diverse cellular functions, including protein trafficking to the plasma membrane, membrane extension, cell polarity, neurite outgrowth, ciliogenesis, cytokinesis, cell migration, autophagy, host defense, and tumorigenesis. Exocyst subunits are essential for cell viability; and mutations or variants in several exocyst subunits have been implicated in human diseases, mostly neurodevelopmental disorders and ciliopathies. These conditions often share common features such as developmental delay, intellectual disability, and brain abnormalities. In this review, we summarize the mutations and variants in exocyst subunits that have been linked to disease and discuss the implications of exocyst dysfunction in other disorders.

The exocyst complex is an essential component of the mammalian constitutive secretory pathway.

Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

Septin-mediated RhoA activation engages the exocyst complex to recruit the cilium transition zone.

Septins are filamentous GTPases that play important but poorly characterized roles in ciliogenesis. Here, we show that SEPTIN9 regulates RhoA signaling at the base of cilia by binding and activating the RhoA guanine nucleotide exchange factor, ARHGEF18. GTP-RhoA is known to activate the membrane targeting exocyst complex, and suppression of SEPTIN9 causes disruption of ciliogenesis and mislocalization of an exocyst subunit, SEC8. Using basal body-targeted proteins, we show that upregulating RhoA signaling at the cilium can rescue ciliary defects and mislocalization of SEC8 caused by global SEPTIN9 depletion. Moreover, we demonstrate that the transition zone components, RPGRIP1L and TCTN2, fail to accumulate at the transition zone in cells lacking SEPTIN9 or depleted of the exocyst complex. Thus, SEPTIN9 regulates the recruitment of transition zone proteins on Golgi-derived vesicles by activating the exocyst via RhoA to allow the formation of primary cilia.

Regulation of cargo exocytosis by a Reps1-Ralbp1-RalA module.

Surface levels of membrane proteins are determined by a dynamic balance between exocytosis-mediated surface delivery and endocytosis-dependent retrieval from the cell surface. Imbalances in surface protein levels perturb surface protein homeostasis and cause major forms of human disease such as type 2 diabetes and neurological disorders. Here, we found a Reps1-Ralbp1-RalA module in the exocytic pathway broadly regulating surface protein levels. Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex. RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex. Ralbp1 selectively recognizes GTP-bound RalA but is not a RalA effector. Instead, Ralbp1 binding maintains RalA in an active GTP-bound state. These studies uncovered a segment in the exocytic pathway and, more broadly, revealed a previously unrecognized regulatory mechanism for small GTPases, GTP state stabilization.

Functional hierarchy among different Rab27 effectors involved in secretory granule exocytosis.

The Rab27 effectors are known to play versatile roles in regulated exocytosis. In pancreatic beta cells, exophilin-8 anchors granules in the peripheral actin cortex, whereas granuphilin and melanophilin mediate granule fusion with and without stable docking to the plasma membrane, respectively. However, it is unknown whether these coexisting effectors function in parallel or in sequence to support the whole insulin secretory process. Here, we investigate their functional relationships by comparing the exocytic phenotypes in mouse beta cells simultaneously lacking two effectors with those lacking just one of them. Analyses of prefusion profiles by total internal reflection fluorescence microscopy suggest that melanophilin exclusively functions downstream of exophilin-8 to mobilize granules for fusion from the actin network to the plasma membrane after stimulation. The two effectors are physically linked via the exocyst complex. Downregulation of the exocyst component affects granule exocytosis only in the presence of exophilin-8. The exocyst and exophilin-8 also promote fusion of granules residing beneath the plasma membrane prior to stimulation, although they differentially act on freely diffusible granules and those stably docked to the plasma membrane by granuphilin, respectively. This is the first study to diagram the multiple intracellular pathways of granule exocytosis and the functional hierarchy among different Rab27 effectors within the same cell.

Exploitation of the host exocyst complex by bacterial pathogens.

Intracellular bacterial pathogens remodel the plasma membrane of eukaryotic cells in order to establish infection. A common and well-studied mechanism of plasma membrane remodelling involves bacterial stimulation of polymerization of the host actin cytoskeleton. Here, we discuss recent results showing that several bacterial pathogens also exploit the host vesicular trafficking pathway of 'polarized exocytosis' to expand and reshape specific regions in the plasma membrane during infection. Polarized exocytosis is mediated by an evolutionarily conserved octameric protein complex termed the exocyst. We describe examples in which the bacteria Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri co-opt the exocyst to promote internalization into human cells or intercellular spread within host tissues. We also discuss results showing that Legionella pneumophila or S. flexneri manipulate exocyst components to modify membrane vacuoles to favour intracellular replication or motility of bacteria. Finally, we propose potential ways that pathogens manipulate exocyst function, discuss how polarized exocytosis might promote infection and highlight the importance of future studies to determine how actin polymerization and polarized exocytosis are coordinated to achieve optimal bacterial infection.

Allosteric regulation of exocyst: Discrete activation of tethering by two spatial signals.

The exocyst imparts spatial control during exocytic vesicle tethering through its interactions with proteins and lipids on the vesicle and the plasma membrane. One such interaction is with the vesicle tether Sro7, although the outcome of this interaction is poorly understood. Here, we describe how Sro7 binding to the Exo84 subunit results in activation of the exocyst complex which leads to an increase in avidity for the Rab GTPase Sec4 and an increase in exocyst-mediated vesicle tethering. Gain-of-function (GOF) mutations in Exo84 that mimic Sro7 activation replicate these biochemical changes and result in allosteric changes within the complex. Direct comparison of GOF mutants which mimic Sro7- and Rho/Cdc42-activation of the exocyst reveals distinct mechanisms and outcomes. We propose a model by which these two activation pathways reside within the same tethering complex but remain insulated from one another. Structural modeling suggests a related mechanism for Sro7 activation of the exocyst in yeast and Ral GTPase activation of the exocyst in animal cells.