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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Ral GTPases regulate exocyst assembly through dual subunit interactions

Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. Here we report that an additional component of the exocyst, Exo84, is a direct target of activated Ral. We provide evidence that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane and that Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84.     

The critical role of Exo84p in the organization and polarized localization of the exocyst complex

The exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis. However, how the exocyst complex is assembled and targeted to sites of secretion is unclear. Here, we have investigated the role of the exocyst component Exo84p in these processes. We have generated an array of temperature-sensitive yeast exo84 mutants. Electron microscopy and cargo protein traffic analyses of these mutants indicated that Exo84p is specifically involved in the post-Golgi stage of secretion. Using various yeast mutants, we systematically studied the localization of Exo84p and other exocyst proteins by fluorescence microscopy. We found that pre-Golgi traffic and polarized actin organization are required for Exo84p localization. However, none of the exocyst proteins controls Exo84p polarization. In addition, Sec3p is not responsible for the polarization of Exo84p or any other exocyst component to the daughter cell. On the other hand, several exocyst members, including Sec10p, Sec15p, and Exo70p, clearly require Exo84p for their polarization. Biochemical analyses of the exocyst composition indicated that the assembly of Sec10p, Sec15p, and Exo70p with the rest of the complex requires Exo84p. We propose that there are at least two distinct regulatory mechanisms for exocyst polarization, one for Sec3p and one for the other members, including Exo84p. Exo84p plays a critical role in both the assembly of the exocyst and its targeting to sites of secretion.     

The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles

The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins.     

An internal domain of Exo70p is required for actin-independent localization and mediates assembly of specific exocyst components

The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.     

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.     

Membrane association and functional regulation of Sec3 by phospholipids and Cdc42

The exocyst is an octameric protein complex implicated in tethering post-Golgi secretory vesicles at the plasma membrane in preparation for fusion. However, it is not clear how the exocyst is targeted to and physically associates with specific domains of the plasma membrane and how its functions are regulated at those regions. We demonstrate that the N terminus of the exocyst component Sec3 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues in Sec3 that are critical for its binding to the guanosine triphosphate–bound form of Cdc42. Genetic analyses indicate that the dual interactions of Sec3 with phospholipids and Cdc42 control its function in yeast cells. Disrupting these interactions not only blocks exocytosis and affects exocyst polarization but also leads to defects in cell morphogenesis. We propose that the interactions of Sec3 with phospholipids and Cdc42 play important roles in exocytosis and polarized cell growth.     

Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.     

Snapin interacts with the Exo70 subunit of the exocyst and modulates GLUT4 trafficking

The exocyst is a multisubunit complex that has been implicated in the transport of vesicles from the Golgi complex to the plasma membrane, possibly acting as a vesicle tether and contributing to the specificity of membrane fusion. Here we characterize a novel interaction between the Exo70 subunit of the exocyst and Snapin, a ubiquitous protein known to associate with at least two t-SNAREs, SNAP23 and SNAP25. The interaction between Exo70 and Snapin is mediated via an N-terminal coil-coil domain in Exo70 and a C-terminal helical region in Snapin. Exo70 competes with SNAP23 for Snapin binding, suggesting that Snapin does not provide a direct link between the exocyst and the SNARE complex but, rather, mediates cross-talk between the two complexes by sequential interactions. The insulin-regulated trafficking of GLUT4 to the plasma membrane serves to facilitate glucose uptake in adipocytes, and both SNAP23 and the exocyst have been implicated in this process. In this study, depletion of Snapin in adipocytes using RNA interference inhibits insulin-stimulated glucose uptake. Thus, Snapin interacts with the exocyst and plays a modulatory role in GLUT4 vesicle trafficking.     

Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis

The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.     

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.     

植物EXO70基因家族的研究进展

胞吐是存在于所有真核生物的一种极其重要的细胞活动,直接参与了激素和神经信号的分泌、细胞生长、细胞极性的建立,细胞分裂和细胞壁的形成等多项生理过程。在胞吐过程中,高尔基后转运膜泡与靶膜的识别是由进化上高度保守的胞泌复合体（exocyst）介导的。该复合体由8个蛋白亚基构成,其中EXO70是组成胞泌复合体功能的关键亚基,可与小G蛋白和膜脂互作,参与复合体在靶膜组装。目前,对植物胞泌复合体功能的了解非常有限,已有证据显示其广泛参与了细胞生长,细胞壁形成、细胞分裂等多种生物学过程。与酵母和动物相比,植物胞泌复合体的一个显著特征是：EXO70在高等植物基因组中存在多个同源基因,其具体生物学功能尚不清楚。本文综述胞泌复合体的研究进展,重点讨论植物EXO70的多基因家族,推测不同的EXO70可能参与了组织细胞或运载底物特异的膜泡转运过程。