Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane

The exocyst is an octameric protein complex implicated in the tethering of post-Golgi secretory vesicles to the plasma membrane before fusion. The function of individual exocyst components and the mechanism by which this tethering complex is targeted to sites of secretion are not clear. In this study, we report that the exocyst subunit Exo70 functions in concert with Sec3 to anchor the exocyst to the plasma membrane. We found that the C-terminal Domain D of Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues on Exo70 that are critical for its interaction with phospholipids and the small GTPase Rho3. Further genetic and cell biological analyses suggest that the interaction of Exo70 with phospholipids, but not Rho3, is essential for the membrane association of the exocyst complex. We propose that Exo70 mediates the assembly of the exocyst complex at the plasma membrane, which is a crucial step in the tethering of post-Golgi secretory vesicles for exocytosis.     

Exo70-Mediated Recruitment of Nucleoporin Nup62 at the Leading Edge of Migrating Cells is Required for Cell Migration

Nucleoporin Nup62 localizes at the central channel of the nuclear pore complex and is essential for nucleocytoplasmic transport. Through its FG-repeat domain, Nup62 regulates nuclear pore permeability and binds nuclear transport receptors. Here, we report that Nup62 interacts directly with Exo70 and colocalizes with Exo70 at the leading edge of migrating cells. Nup62 binds the N-terminal domain of Exo70 through its coiled-coil domain but not through its FG-repeat domain. Selective inhibition of leading edge Nup62 using RNA interference significantly reduces cell migration. Furthermore, Exo70 recruits Nup62 at the plasma membrane and at filopodia. Removal of the Exo70-binding domain of Nup62 prevents leading edge localization of Nup62. Analogous to Exo70, Nup62 cycles between the plasma membrane and the perinuclear recycling compartment. Altogether, we propose that Nup62 not solely regulates access to the cell nucleus, but additionally functions in conjunction with Exo70, a key regulator of exocytosis and actin dynamics, at the leading edge of migrating cells.     

The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst

The exocyst is a eukaryotic tethering complex necessary for the fusion of exocytic vesicles with the plasma membrane. Its function in vivo is tightly regulated by interactions with multiple small GTPases. Exo70, one of the eight subunits of the exocyst, is important for the localization of the exocyst to the plasma membrane. It interacts with TC10 and Rho3 GTPases in mammals and yeast, respectively, and has been shown recently to bind to the actin-polymerization complex Arp2/3. Here, we present the crystal structure of Mus musculus Exo70 at 2.25 A resolution. Exo70 is composed of alpha-helices in a series of right-handed helix-turn-helix motifs organized into a long rod of length 170 A and width 35 A. Although the alpha-helical organization of this molecule is similar to that in Saccharomyces cerevisiae Exo70, major structural differences are observed on the surface of the molecule, at the domain boundaries, and in various loop structures. In particular, the C-terminal domain of M. musculus Exo70 adopts a new orientation relative to the N-terminal half not seen in S. cerevisiae Exo70 structures. Given the low level of sequence conservation within Exo70, this structure provides new insights into our understanding of many species-specific functions of the exocyst.     

EXPO, an exocyst-positive organelle distinct from multivesicular endosomes and autophagosomes, mediates cytosol to cell wall exocytosis in Arabidopsis and tobacco cells

The exocyst protein complex mediates vesicle fusion with the plasma membrane. By expressing an (X)FP-tagged Arabidopsis thaliana homolog of the exocyst protein Exo70 in suspension-cultured Arabidopsis and tobacco (Nicotiana tabacum) BY-2 cells, and using antibodies specific for Exo70, we detected a compartment, which we term EXPO (for exocyst positive organelles). Standard markers for the Golgi apparatus, the trans-Golgi network/early endosome, and the multivesicular body/late endosome in plants do not colocalize with EXPO. Inhibitors of the secretory and endocytic pathways also do not affect EXPO. Exo70E2-(X)FP also locates to the plasma membrane (PM) as discrete punctae and is secreted outside of the cells. Immunogold labeling of sections cut from high-pressure frozen samples reveal EXPO to be spherical double membrane structures resembling autophagosomes. However, unlike autophagosomes, EXPOs are not induced by starvation and do not fuse with the lytic compartment or with endosomes. Instead, they fuse with the PM, releasing a single membrane vesicle into the cell wall. EXPOs are also found in other cell types, including root tips, root hair cells, and pollen grains. EXPOs therefore represent a form of unconventional secretion unique to plants.     

Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P(2). Disrupting Exo70-PI(4,5)P(2) interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P(2)-binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P(2) interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.     

Crystal structure of the S.cerevisiae exocyst component Exo70p

The exocyst is an evolutionarily conserved multiprotein complex required for the targeting and docking of post-Golgi vesicles to the plasma membrane. Through its interactions with a variety of proteins, including small GTPases, the exocyst is thought to integrate signals from the cell and signal that vesicles arriving at the plasma membrane are ready for fusion. Here we describe the three-dimensional crystal structure of one of the components of the exocyst, Exo70p, from Saccharomyces cerevisiae at 3.5A resolution. Exo70p binds the small GTPase Rho3p in a GTP-dependent manner with an equilibrium dissociation constant of approximately 70 microM. Exo70p is an extended rod approximately 155 angstroms in length composed principally of alpha helices, and is a novel fold. The structure provides a first view of the Exo70 protein family and provides a framework to study the molecular function of this exocyst component.     

ERK1/2 regulate exocytosis through direct phosphorylation of the exocyst component Exo70

The exocyst is a multiprotein complex essential for exocytosis and plasma membrane remodeling. The assembly of the exocyst complex mediates the tethering of post-Golgi secretory vesicles to the plasma membrane prior to fusion. Elucidating the mechanisms regulating exocyst assembly is important for the understanding of exocytosis. Here we show that the exocyst component Exo70 is a direct substrate of the extracellular signal-regulated kinases 1/2 (ERK1/2). ERK1/2 phosphorylation enhances the binding of Exo70 to other exocyst components and promotes the assembly of the exocyst complex in response to epidermal growth factor (EGF) signaling. We further demonstrate that ERK1/2 regulates exocytosis, because blocking ERK1/2 signaling by a chemical inhibitor or the expression of an Exo70 mutant defective in ERK1/2 phosphorylation inhibited exocytosis. In tumor cells, blocking Exo70 phosphorylation inhibits matrix metalloproteinase secretion and invadopodia formation. ERK1/2 phosphorylation of Exo70 may thus coordinate exocytosis with other cellular events in response to growth factor signaling.     

Exo70 stimulates the arp2/3 complex for lamellipodia formation and directional cell migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1, 2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in?vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.     

Exo70 interacts with the Arp2/3 complex and regulates cell migration

The exocyst is a multiprotein complex essential for tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. Here, we report that the exocyst component Exo70 interacts with the Arp2/3 complex, a key regulator of actin polymerization. We further show that the exocyst-Arp2/3 interaction is regulated by epidermal growth factor (EGF) signalling. Inhibition of Exo70 by RNA interference (RNAi) or antibody microinjection blocks the formation of actin-based membrane protrusions and affects various aspects of cell motility. We propose that Exo70, in addition to functioning in exocytosis, also regulates actin at the leading edges of migrating cells, therefore coordinating cytoskeleton and membrane traffic during cell migration.     

An association between type Iγ PI4P 5-kinase and Exo70 directs E-cadherin clustering and epithelial polarization

E-Cadherin-mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P(2) is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P(2) pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.     

The role of Exo70 in vascular smooth muscle cell migration

Background

As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70’s function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5.

Methods

Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay.

Results

The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration.

Conclusions

This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty.

     

Bem1p contributes to secretory pathway polarization through a direct interaction with Exo70p

The exocyst serves to tether secretory vesicles to cortical sites specified by polarity determinants, in preparation for fusion with the plasma membrane. Although most exocyst components are brought to these sites by riding on secretory vesicles as they are actively transported along actin cables, Exo70p displays actin-independent localization to these sites, implying an interaction with a polarity determinant. Here we show that Exo70p directly and specifically binds to the polarity determinant scaffold protein Bem1p. The interaction involves multiple domains of both Exo70p and Bem1p. Mutations in Exo70p that disrupt its interaction with Bem1, without impairing its interactions with other known binding partners, lead to the loss of actin-independent localization. Synthetic genetic interactions confirm the importance of the Exo70p–Bem1p interaction, although there is some possible redundancy with Sec3p and Sec15p, other exocyst components that also interact with polarity determinants. Similar to Sec3p, the actin-independent localization of Exo70p requires a synergistic interaction with the phosphoinositide PI(4,5)P₂.     

Membrane targeting of the yeast exocyst complex

The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits — Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP₂) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP₂. We found that PIP₂ is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP₂ in the adjacent leaflet. We further revealed that PIP₂ is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP₂ for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.     

Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.     

GTP Hydrolysis of TC10 Promotes Neurite Outgrowth through Exocytic Fusion of Rab11- and L1-Containing Vesicles by Releasing Exocyst Component Exo70

The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.     

Caveolae Internalization Regulates Integrin-Dependent Signaling Pathways

Integrin-mediated adhesion regulates trafficking of cholesterol-enriched membrane microdomains (CEMM). Upon cell detachment from the extracellular matrix (ECM), CEMMs undergo rapid internalization and are cleared from the plasma membrane. This pathway regulates integrin-mediated Rac membrane targeting, allowing coupling of Rac to downstream effectors. Internalization of CEMMs is mediated by Dynamin-2, a regulator of caveolae dynamics, and caveolin-1, an essential caveolae coat protein. Translocation of tyrosine phosphorylated caveolin-1 from focal adhesions to caveolae upon cell detachment induces CEMM internalization. Notably, integrin-mediated regulation of Erk, phosphatidylinositol-3-OH kinase (PI3K) and Rac pathways is dependent on caveolin-1. These results describe a novel pathway in which integrins prevent downregulation of Erk, PI3K and Rac-dependent pathways by inhibiting caveolin-1-dependent endocytosis. This pathway define a novel molecular mechanism for regulated cell growth and tumor suppression by caveolin-1.     

The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation

Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.     

Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth

In budding yeast, two classes of post-Golgi secretory vesicles carrying different sets of cargoes typified by Bgl2p and invertase are delivered to the plasma membrane for secretion. The exocyst is implicated in tethering these vesicles to the daughter cell membrane for exocytosis. In this study, we report that mutations in the exocyst component Exo70p predominantly block secretion of the Bgl2p vesicles. Furthermore, a defect in invertase vesicle trafficking caused by vps1Delta or pep12Delta in the exo70 mutant background is detrimental to the cell. The secretion defect in exo70 mutants was most pronounced during the early budding stage, which affected daughter cell growth. The selective secretion block does not occur at the vesicle formation or sorting stage because the exocytic vesicles are properly generated and protein processing is normal in the exo70 mutants. Our study suggests that Exo70p functions primarily at early stages of the cell cycle in Bgl2p vesicle secretion, which is critical for polarized cell growth.     

Membrane association of estrogen receptor alpha and beta influences 17beta-estradiol-mediated cancer cell proliferation

S-Palmitoylation is a widespread post-translational modification of integral and/or peripheral proteins occurring in all eukaryotic cells. The family of S-palmitoylated proteins is large and diverse and recently, estrogen receptor isoforms (ERalpha and ERbeta) belonging to the nuclear receptor superfamily have been added to the palmitoylproteoma. S-Palmitoylation allows ERalpha and ERbeta localization at the plasma membrane, where they associate with caveolin-1. Upon 17beta-estradiol (E2) stimulation, ERalpha dissociates from caveolin-1 allowing the activation of rapid signals relevant for cell proliferation. In contrast to ERalpha, E2 increases ERbeta association with caveolin-1 and activates p38 kinase and the downstream pro-apoptotic cascade (i.e., caspase-3 activation and PARP cleavage). These data highlight the physiological role of palmitoylation in modulating the ERalpha and ERbeta localization at the plasma membrane and the regulation of different E2-induced non-genomic functions relevant for controlling cell proliferation.     

Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.