Journal articles: 'Vesicle tethering complex exocyst'

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Wiederkehr, Andreas, Johan-Owen De Craene, Susan Ferro-Novick, and Peter Novick. "Functional specialization within a vesicle tethering complex." Journal of Cell Biology 167, no. 5 (December 6, 2004): 875–87. http://dx.doi.org/10.1083/jcb.200408001.

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The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Δ, sec5Δ, and exo70Δ strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.

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Luo, Guangzuo, Jian Zhang, and Wei Guo. "The role of Sec3p in secretory vesicle targeting and exocyst complex assembly." Molecular Biology of the Cell 25, no. 23 (November 15, 2014): 3813–22. http://dx.doi.org/10.1091/mbc.e14-04-0907.

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During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

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Eckardt, Nancy A. "An Exocyst Vesicle Tethering Complex in Plants." Plant Cell 20, no. 5 (May 2008): 1188. http://dx.doi.org/10.1105/tpc.108.200511.

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Zhang, Weiwei, Lei Huang, Chunhua Zhang, and Christopher J. Staiger. "Arabidopsis myosin XIK interacts with the exocyst complex to facilitate vesicle tethering during exocytosis." Plant Cell 33, no. 7 (April 19, 2021): 2454–78. http://dx.doi.org/10.1093/plcell/koab116.

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Abstract Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits, and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.

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Fendrych, Matyáš, Lukáš Synek, Tamara Pečenková, Edita Janková Drdová, Juraj Sekereš, Riet de Rycke, Moritz K. Nowack, and Viktor Žárský. "Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana." Molecular Biology of the Cell 24, no. 4 (February 15, 2013): 510–20. http://dx.doi.org/10.1091/mbc.e12-06-0492.

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The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6–green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

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Morgera, Francesca, Margaret R. Sallah, Michelle L. Dubuke, Pallavi Gandhi, Daniel N. Brewer, Chavela M. Carr, and Mary Munson. "Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1." Molecular Biology of the Cell 23, no. 2 (January 15, 2012): 337–46. http://dx.doi.org/10.1091/mbc.e11-08-0670.

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Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function—it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6–Sec1 interaction is exclusive of Sec6–Sec9 but compatible with Sec6–exocyst assembly. In contrast, the Sec6–exocyst interaction is incompatible with Sec6–Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6–exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.

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Riquelme, Meritxell, Erin L. Bredeweg, Olga Callejas-Negrete, Robert W. Roberson, Sarah Ludwig, Alejandro Beltrán-Aguilar, Stephan Seiler, Peter Novick, and Michael Freitag. "The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth." Molecular Biology of the Cell 25, no. 8 (April 15, 2014): 1312–26. http://dx.doi.org/10.1091/mbc.e13-06-0299.

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Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

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Medkova, Martina, Y. Ellen France, Jeff Coleman, and Peter Novick. "The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst." Molecular Biology of the Cell 17, no. 6 (June 2006): 2757–69. http://dx.doi.org/10.1091/mbc.e05-10-0917.

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Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.

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Lira, Matías, Rodrigo G. Mira, Francisco J. Carvajal, Pedro Zamorano, Nibaldo C. Inestrosa, and Waldo Cerpa. "Glutamatergic Receptor Trafficking and Delivery: Role of the Exocyst Complex." Cells 9, no. 11 (November 3, 2020): 2402. http://dx.doi.org/10.3390/cells9112402.

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Cells comprise several intracellular membrane compartments that allow them to function properly. One of these functions is cargo movement, typically proteins and membranes within cells. These cargoes ride microtubules through vesicles from Golgi and recycling endosomes to the plasma membrane in order to be delivered and exocytosed. In neurons, synaptic functions employ this cargo trafficking to maintain inter-neuronal communication optimally. One of the complexes that oversee vesicle trafficking and tethering is the exocyst. The exocyst is a protein complex containing eight subunits first identified in yeast and then characterized in multicellular organisms. This complex is related to several cellular processes, including cellular growth, division, migration, and morphogenesis, among others. It has been associated with glutamatergic receptor trafficking and tethering into the synapse, providing the molecular machinery to deliver receptor-containing vesicles into the plasma membrane in a constitutive manner. In this review, we discuss the evidence so far published regarding receptor trafficking and the exocyst complex in both basal and stimulated levels, comparing constitutive trafficking and long-term potentiation-related trafficking.

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Fais, Milena, Giovanna Sanna, Manuela Galioto, Thi Thanh Duyen Nguyen, Mai Uyên Thi Trần, Paola Sini, Franco Carta, et al. "LRRK2 Modulates the Exocyst Complex Assembly by Interacting with Sec8." Cells 10, no. 2 (January 20, 2021): 203. http://dx.doi.org/10.3390/cells10020203.

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Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson’s disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking, likely by Rab phosphorylation, that in turn may regulate different aspects of neuronal physiology. Here we show that LRRK2 interacts with Sec8, one of eight subunits of the exocyst complex. The exocyst complex is an evolutionarily conserved multisubunit protein complex mainly involved in tethering secretory vesicles to the plasma membrane and implicated in the regulation of multiple biological processes modulated by vesicle trafficking. Interestingly, Rabs and exocyst complex belong to the same protein network. Our experimental evidence indicates that LRRK2 kinase activity or the presence of the LRRK2 kinase domain regulate the assembly of exocyst subunits and that the over-expression of Sec8 significantly rescues the LRRK2 G2019S mutant pathological effect. Our findings strongly suggest an interesting molecular mechanism by which LRRK2 could modulate vesicle trafficking and may have important implications to decode the complex role that LRRK2 plays in neuronal physiology.

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Novick, P., M. Medkova, G. Dong, A. Hutagalung, K. Reinisch, and B. Grosshans. "Interactions between Rabs, tethers, SNAREs and their regulators in exocytosis." Biochemical Society Transactions 34, no. 5 (October 1, 2006): 683–86. http://dx.doi.org/10.1042/bst0340683.

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Sec2p is the exchange factor that activates Sec4p, the Rab GTPase controlling the final stage of the yeast exocytic pathway. Sec2p is recruited to secretory vesicles by Ypt32-GTP, a Rab controlling exit from the Golgi. Sec15p, a subunit of the octameric exocyst tethering complex and an effector of Sec4p, binds to Sec2p on secretory vesicles, displacing Ypt32p. Sec2p mutants defective in the region 450–508 amino acids bind to Sec15p more tightly. In these mutants, Sec2p accumulates in the cytosol in a complex with the exocyst and is not recruited to vesicles by Ypt32p. Thus the region 450–508 amino acids negatively regulates the association of Sec2p with the exocyst, allowing it to recycle on to new vesicles. The structures of one nearly full-length exocyst subunit and three partial subunits have been determined and, despite very low sequence identity, all form rod-like structures built of helical bundles stacked end to end. These rods may bind to each other along their sides to form the assembled complex. While Sec15p binds Sec4-GTP on the vesicle, other subunits bind Rho GTPases on the plasma membrane, thus tethering vesicles to exocytic sites. Sec4-GTP also binds Sro7p, a yeast homologue of the Drosophila lgl (lethal giant larvae) tumour suppressor. Sro7 also binds to Sec9p, a SNAP25 (25 kDa synaptosome-associated protein)-like t-SNARE [target-membrane-associated SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)], and can form a Sec4p–Sro7p–Sec9p ternary complex. Overexpression of Sec4p, Sro7p or Sec1p (another SNARE regulator) can bypass deletions of three different exocyst subunits. Thus promoting SNARE function can compensate for tethering defects.

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Rossi, Guendalina, Kelly Watson, Wade Kennedy, and Patrick Brennwald. "The tomosyn homologue, Sro7, is a direct effector of the Rab GTPase, Sec4, in post-Golgi vesicle tethering." Molecular Biology of the Cell 29, no. 12 (June 15, 2018): 1476–86. http://dx.doi.org/10.1091/mbc.e18-02-0138.

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The tomosyn/Sro7 family is thought to play an important role in cell surface trafficking both as an effector of Rab family GTPases and as a regulator of plasma-membrane SNARE function. Recent work has determined the binding site of GTP-bound Sec4 on Sro7. Here we examine the effect of mutations in Sro7 that block Sec4 binding in determining the role of this interaction in Sro7 function. Using an in vitro vesicle:vesicle tethering assay, we find that most of Sro7’s ability to tether vesicles is blocked by mutations that disrupt binding to Sec4-GTP. Similarly, genetic analysis demonstrates that the interaction with Sec4 is important for most of Sro7’s functions in vivo. The interaction of Sro7 with Sec4 appears to be particularly important when exocyst function is compromised. This provides strong evidence that Sro7 and the exocyst act as dual effector pathways downstream of Sec4. We also demonstrate that Sro7 tethering requires the presence of Sec4 on both opposing membranes and that homo-oligomerization of Sro7 occurs during vesicle tethering. This suggests a simple model for Sro7 function as a Rab effector in tethering post-Golgi vesicles to the plasma membrane in a pathway parallel to that of the exocyst complex.

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Inoue, Mayumi, Shian-Huey Chiang, Louise Chang, Xiao-Wei Chen, and Alan R. Saltiel. "Compartmentalization of the Exocyst Complex in Lipid Rafts Controls Glut4 Vesicle Tethering." Molecular Biology of the Cell 17, no. 5 (May 2006): 2303–11. http://dx.doi.org/10.1091/mbc.e06-01-0030.

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Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8. Knockdowns of these proteins block insulin-stimulated glucose uptake. Moreover, their targeting to lipid rafts is required for glucose uptake and Glut4 docking at the plasma membrane. The assembly of this complex also requires the PDZ domain protein SAP97, a member of the MAGUKs family, which binds to Sec8 upon its translocation to the lipid raft. Exocyst assembly at lipid rafts sets up targeting sites for Glut4 vesicles, which transiently associate with these microdomains upon stimulation of cells with insulin. These results suggest that the TC10/exocyst complex/SAP97 axis plays an important role in the tethering of Glut4 vesicles to the plasma membrane in adipocytes.

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Prigent, Magali, Thierry Dubois, Graça Raposo, Valérie Derrien, Danièle Tenza, Carine Rossé, Jacques Camonis, and Philippe Chavrier. "ARF6 controls post-endocytic recycling through its downstream exocyst complex effector." Journal of Cell Biology 163, no. 5 (December 8, 2003): 1111–21. http://dx.doi.org/10.1083/jcb.200305029.

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The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

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Rivera-Molina, Felix, and Derek Toomre. "Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion." Journal of Cell Biology 201, no. 5 (May 20, 2013): 673–80. http://dx.doi.org/10.1083/jcb.201212103.

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Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.

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Arasaki, Kohei, Hana Kimura, Mitsuo Tagaya, and Craig R. Roy. "Legionella remodels the plasma membrane–derived vacuole by utilizing exocyst components as tethers." Journal of Cell Biology 217, no. 11 (October 1, 2018): 3863–72. http://dx.doi.org/10.1083/jcb.201801208.

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During the initial stage of infection, Legionella pneumophila secretes effectors that promote the fusion of endoplasmic reticulum (ER)–derived vesicles with the Legionella-containing vacuole (LCV). This fusion leads to a remodeling of the plasma membrane (PM)–derived LCV into a specialized ER-like compartment that supports bacterial replication. Although the effector DrrA has been shown to activate the small GTPase Rab1, it remains unclear how DrrA promotes the tethering of host vesicles with the LCV. Here, we show that Sec5, Sec15, and perhaps Sec6, which are subunits of the exocyst that functions in the tethering of exocytic vesicles with the PM, are required for DrrA-mediated, ER-derived vesicle recruitment to the PM-derived LCV. These exocyst components were found to interact specifically with a complex containing DrrA, and the loss of Sec5 or Sec15 significantly suppressed the recruitment of ER-derived vesicles to the LCV and inhibited intracellular replication of Legionella. Importantly, Sec15 is recruited to the LCV, and Rab1 activation is necessary for this recruitment.

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Liu, Jianglan, Xiaofeng Zuo, Peng Yue, and Wei Guo. "Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells." Molecular Biology of the Cell 18, no. 11 (November 2007): 4483–92. http://dx.doi.org/10.1091/mbc.e07-05-0461.

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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)P2). 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)P2. Disrupting Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P2-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)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.

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Synek, Lukáš, Roman Pleskot, Juraj Sekereš, Natalia Serrano, Nemanja Vukašinović, Jitka Ortmannová, Martina Klejchová, et al. "Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit." Proceedings of the National Academy of Sciences 118, no. 36 (September 1, 2021): e2105287118. http://dx.doi.org/10.1073/pnas.2105287118.

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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.

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Mathieson, Erin M., Yasuyuki Suda, Mark Nickas, Brian Snydsman, Trisha N. Davis, Eric G. D. Muller, and Aaron M. Neiman. "Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation inSaccharomyces cerevisiae." Molecular Biology of the Cell 21, no. 21 (November 2010): 3693–707. http://dx.doi.org/10.1091/mbc.e10-07-0563.

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During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

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Marković, Vedrana, Ivan Kulich, and Viktor Žárský. "Functional Specialization within the EXO70 Gene Family in Arabidopsis." International Journal of Molecular Sciences 22, no. 14 (July 15, 2021): 7595. http://dx.doi.org/10.3390/ijms22147595.

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Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.

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Sakurai-Yageta, Mika, Chiara Recchi, Gaëlle Le Dez, Jean-Baptiste Sibarita, Laurent Daviet, Jacques Camonis, Crislyn D'Souza-Schorey, and Philippe Chavrier. "The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA." Journal of Cell Biology 181, no. 6 (June 9, 2008): 985–98. http://dx.doi.org/10.1083/jcb.200709076.

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Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.

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Conibear, Elizabeth, Jessica N. Cleck, and Tom H. Stevens. "Vps51p Mediates the Association of the GARP (Vps52/53/54) Complex with the Late Golgi t-SNARE Tlg1p." Molecular Biology of the Cell 14, no. 4 (April 2003): 1610–23. http://dx.doi.org/10.1091/mbc.e02-10-0654.

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Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN.vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.

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De Craene, Johan-Owen, Jeff Coleman, Paula Estrada de Martin, Marc Pypaert, Scott Anderson, John R. Yates, Susan Ferro-Novick, and Peter Novick. "Rtn1p Is Involved in Structuring the Cortical Endoplasmic Reticulum." Molecular Biology of the Cell 17, no. 7 (July 2006): 3009–20. http://dx.doi.org/10.1091/mbc.e06-01-0080.

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The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Δ in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Δ cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.

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VerPlank, Lynn, and Rong Li. "Cell Cycle-regulated Trafficking of Chs2 Controls Actomyosin Ring Stability during Cytokinesis." Molecular Biology of the Cell 16, no. 5 (May 2005): 2529–43. http://dx.doi.org/10.1091/mbc.e04-12-1090.

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Cytokinesis requires the coordination of many cellular complexes, particularly those involved in the constriction and reconstruction of the plasma membrane in the cleavage furrow. We have investigated the regulation and function of vesicle transport and fusion during cytokinesis in budding yeast. By using time-lapse confocal microscopy, we show that post-Golgi vesicles, as well as the exocyst, a complex required for the tethering and fusion of these vesicles, localize to the bud neck at a precise time just before spindle disassembly and actomyosin ring contraction. Using mutants affecting cyclin degradation and the mitotic exit network, we found that targeted secretion, in contrast to contractile ring activation, requires cyclin degradation but not the mitotic exit network. Analysis of cells in late anaphase bearing exocyst and myosin V mutations show that both vesicle transport and fusion machineries are required for the completion of cytokinesis, but this is not due to a delay in mitotic exit or assembly of the contractile ring. Further investigation of the dynamics of contractile rings in exocyst mutants shows these cells may be able to initiate contraction but often fail to complete the contraction due to premature disassembly during the contraction phase. This phenotype led us to identify Chs2, a transmembrane protein targeted to the bud neck through the exocytic pathway, as necessary for actomyosin ring stability during contraction. Chs2, as the chitin synthase that produces the primary septum, thus couples the assembly of the extracellular matrix with the dynamics of the contractile ring during cytokinesis.

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Luo, L., M. Hannemann, S. Koenig, J. Hegermann, M. Ailion, M. K. Cho, N. Sasidharan, M. Zweckstetter, S. A. Rensing, and S. Eimer. "The Caenorhabditis elegans GARP complex contains the conserved Vps51 subunit and is required to maintain lysosomal morphology." Molecular Biology of the Cell 22, no. 14 (July 15, 2011): 2564–78. http://dx.doi.org/10.1091/mbc.e10-06-0493.

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In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits—Vps51p, Vps52p, Vps53p, and Vps54p—and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.

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Grosshans, Bianka L., Anna Andreeva, Akanksha Gangar, Sherry Niessen, John R. Yates, Patrick Brennwald, and Peter Novick. "The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p." Journal of Cell Biology 172, no. 1 (January 2, 2006): 55–66. http://dx.doi.org/10.1083/jcb.200510016.

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Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation.

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Zhang, Xiaoyu, Kelly Orlando, Bing He, Fengong Xi, Jian Zhang, Allison Zajac, and Wei Guo. "Membrane association and functional regulation of Sec3 by phospholipids and Cdc42." Journal of Cell Biology 180, no. 1 (January 14, 2008): 145–58. http://dx.doi.org/10.1083/jcb.200704128.

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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.

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Liu, Jianglan, Peng Yue, Vira V. Artym, Susette C. Mueller, and Wei Guo. "The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation." Molecular Biology of the Cell 20, no. 16 (August 15, 2009): 3763–71. http://dx.doi.org/10.1091/mbc.e08-09-0967.

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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.

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Zajac, Allison, Xiaoli Sun, Jian Zhang, and Wei Guo. "Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth." Molecular Biology of the Cell 16, no. 3 (March 2005): 1500–1512. http://dx.doi.org/10.1091/mbc.e04-10-0896.

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Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, we systematically analyzed the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, in various mutants. We found that the polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. We also found that Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, we found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. We further report that Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. We propose that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast.

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Walsh, Tony G., Yong Li, Christopher M. Williams, Elizabeth W. Aitken, Robert K. Andrews, and Alastair W. Poole. "Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis." Blood Advances 5, no. 3 (February 1, 2021): 674–86. http://dx.doi.org/10.1182/bloodadvances.2020002515.

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Abstract The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

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Hou, Hongna, Jianbo Fang, Jiahui Liang, Zhijuan Diao, Wei Wang, Dewei Yang, Shengping Li, and Dingzhong Tang. "OsExo70B1 Positively Regulates Disease Resistance to Magnaporthe oryzae in Rice." International Journal of Molecular Sciences 21, no. 19 (September 25, 2020): 7049. http://dx.doi.org/10.3390/ijms21197049.

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The exocyst, an evolutionarily conserved octameric protein complex, mediates tethering of vesicles to the plasma membrane in the early stage of exocytosis. Arabidopsis Exo70, a subunit of the exocyst complex, has been found to be involved in plant immunity. Here, we characterize the function of OsExo70B1 in rice. OsExo70B1 mainly expresses in leaf and shoot and its expression is induced by pathogen-associated molecular patterns (PAMPs) and rice blast fungus Magnaporthe oryzae (M. oryzae). Knocking out OsExo70B1 results in significantly decreased resistance and defense responses to M. oryzae compared to the wild type, including more disease lesions and enhanced fungal growth, downregulated expression of pathogenesis-related (PR) genes, and decreased reactive oxygen species accumulation. In contrast, the exo70B1 mutant does not show any defects in growth and development. Furthermore, OsExo70B1 can interact with the receptor-like kinase OsCERK1, an essential component for chitin reception in rice. Taken together, our data demonstrate that OsExo70B1 functions as an important regulator in rice immunity.

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Žárský, Viktor, and Martin Potocký. "Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst." Biochemical Society Transactions 38, no. 2 (March 22, 2010): 723–28. http://dx.doi.org/10.1042/bst0380723.

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The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a ‘recycling domain’ (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase–effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

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Estey, Mathew P., Caterina Di Ciano-Oliveira, Carol D. Froese, Margaret T. Bejide, and William S. Trimble. "Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission." Journal of Cell Biology 191, no. 4 (November 8, 2010): 741–49. http://dx.doi.org/10.1083/jcb.201006031.

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Septins are a family of GTP-binding proteins implicated in mammalian cell division. Most studies examining the role of septins in this process have treated the family as a whole, thus neglecting the possibility that individual members may have diverse functions. To address this, we individually depleted each septin family member expressed in HeLa cells by siRNA and assayed for defects in cell division by immunofluorescence and time-lapse microscopy. Depletion of SEPT2, SEPT7, and SEPT11 causes defects in the early stages of cytokinesis, ultimately resulting in binucleation. In sharp contrast, SEPT9 is dispensable for the early stages of cell division, but is critical for the final separation of daughter cells. Rescue experiments indicate that SEPT9 isoforms containing the N-terminal region are sufficient to drive cytokinesis. We demonstrate that SEPT9 mediates the localization of the vesicle-tethering exocyst complex to the midbody, providing mechanistic insight into the role of SEPT9 during abscission.

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Arasaki, Kohei, Daichi Takagi, Akiko Furuno, Miwa Sohda, Yoshio Misumi, Yuichi Wakana, Hiroki Inoue, and Mitsuo Tagaya. "A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex." Molecular Biology of the Cell 24, no. 18 (September 15, 2013): 2907–17. http://dx.doi.org/10.1091/mbc.e13-01-0014.

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Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor–mediated initial contact followed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum–localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to–trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

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Elbert, Maya, Guendalina Rossi, and Patrick Brennwald. "The Yeast Par-1 Homologs Kin1 and Kin2 Show Genetic and Physical Interactions with Components of the Exocytic Machinery." Molecular Biology of the Cell 16, no. 2 (February 2005): 532–49. http://dx.doi.org/10.1091/mbc.e04-07-0549.

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Kin1 and Kin2 are Saccharomyces cerevisiae counterparts of Par-1, the Caenorhabditis elegans kinase essential for the establishment of polarity in the one cell embryo. Here, we present evidence for a novel link between Kin1, Kin2, and the secretory machinery of the budding yeast. We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking. Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst. We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis. Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway and implicates the conserved 42-amino acid tail at the carboxy terminal of the kinase in autoinhibition. Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane. In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9.

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Masgrau, Aina, Andrea Battola, Trinidad Sanmartin, Leszek P. Pryszcz, Toni Gabaldón, and Manuel Mendoza. "Distinct roles of the polarity factors Boi1 and Boi2 in the control of exocytosis and abscission in budding yeast." Molecular Biology of the Cell 28, no. 22 (November 2017): 3082–94. http://dx.doi.org/10.1091/mbc.e17-06-0404.

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Boi1 and Boi2 (Boi1/2) are budding yeast plasma membrane proteins that function in polarized growth, and in cytokinesis inhibition in response to chromosome bridges via the NoCut abscission checkpoint. How Boi1/2 act in these two distinct processes is not understood. We demonstrate that Boi1/2 are required for a late step in the fusion of secretory vesicles with the plasma membrane of the growing bud. Cells lacking Boi1/2 accumulate secretory vesicles and are defective in bud growth. In contrast, Boi2 is specifically required for abscission inhibition in cells with chromatin bridges. The SH3 domain of Boi2, which is dispensable for bud growth and targets Boi2 to the site of abscission, is necessary and sufficient for abscission inhibition. Gain of function of the exocyst, a conserved protein complex involved in tethering of exocytic vesicles to the plasma membrane, rescued secretion and bud growth defects in boi mutant cells, and abrogated NoCut checkpoint function. Thus Boi2 functions redundantly with Boi1 to promote the fusion of secretory vesicles with the plasma membrane at sites of polarized growth, and acts as an abscission inhibitor during cytokinesis in response to chromatin bridges.

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Nishida‐Fukuda, Hisayo. "The Exocyst: Dynamic Machine or Static Tethering Complex?" BioEssays 41, no. 8 (July 2019): 1900056. http://dx.doi.org/10.1002/bies.201900056.

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Donovan, Kirk W., and Anthony Bretscher. "Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process." Journal of Cell Biology 210, no. 2 (July 13, 2015): 181–89. http://dx.doi.org/10.1083/jcb.201501118.

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Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.

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Boehm, Cordula, and Mark C. Field. "Evolution of late steps in exocytosis: conservation and specialization of the exocyst complex." Wellcome Open Research 4 (November 29, 2019): 112. http://dx.doi.org/10.12688/wellcomeopenres.15142.2.

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Background: The eukaryotic endomembrane system most likely arose via paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist Trypanosoma brucei, and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. Methods: We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. Results: The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. Conclusions: These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.

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Boehm, Cordula, and Mark C. Field. "Evolution of late steps in exocytosis: conservation, specialization." Wellcome Open Research 4 (July 26, 2019): 112. http://dx.doi.org/10.12688/wellcomeopenres.15142.1.

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Background: The eukaryotic endomembrane system likely arose via paralogous expansion of genes encoding proteins specifying organelle identity, coat complexes and government of fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events molded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical subunits, emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis, and possibly additional pathways, and is a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family, which includes conserved oligomeric Golgi (COG), homotypic fusion and vacuole protein sorting (HOPS), class C core vacuole/endosome tethering (CORVET) and others. The exocyst is integrated into a complex GTPase signaling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist Trypanosoma brucei, and significantly increased genome sequence data, we examined evolution of the exocyst. Methods: We examined evolution of the exocyst by comparative genomics, phylogenetics and structure prediction. Results: The exocyst is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants and Metazoa. Significantly, few taxa retain a partial complex, suggesting that, in the main, all subunits are required for functionality. Further, the ninth exocyst subunit Exo99 is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. Conclusions: These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.

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He, Bing, Fengong Xi, Jian Zhang, Daniel TerBush, Xiaoyu Zhang, and Wei Guo. "Exo70p mediates the secretion of specific exocytic vesicles at early stages of the cell cycle for polarized cell growth." Journal of Cell Biology 176, no. 6 (March 5, 2007): 771–77. http://dx.doi.org/10.1083/jcb.200606134.

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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 vps1Δ or pep12Δ 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.

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Munson, Mary, Dante Lepore, Michael Feyder, Guendalina Rossi, Alexander B. Czuchra, Lillian Kenner, Leonora Martinez-Nunez, Jacqueline M. Forson, Adam Frost, and Patrick Brennwald. "Exocyst Tethering Complex Regulation of SNARE Proteins and Membrane Fusion." Biophysical Journal 118, no. 3 (February 2020): 340a—341a. http://dx.doi.org/10.1016/j.bpj.2019.11.1896.

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Munson, Mary, Dante Lepore, Michael Feyder, Guendalina Rossi, Alexander B. Czuchra, Lillian Kenner, Leonora Martínez-Núñez, Adam Frost, and Patrick Brennwald. "Activation of the Exocyst Tethering Complex for SNARE Complex Regulation and Membrane Fusion." FASEB Journal 34, S1 (April 2020): 1. http://dx.doi.org/10.1096/fasebj.2020.34.s1.00212.

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Lees, Joshua A., Calvin K. Yip, Thomas Walz, and Frederick M. Hughson. "Molecular organization of the COG vesicle tethering complex." Nature Structural & Molecular Biology 17, no. 11 (October 24, 2010): 1292–97. http://dx.doi.org/10.1038/nsmb.1917.

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Mukherjee, Debarati, Arpita Sen, and R. Claudio Aguilar. "RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking." Small GTPases 5, no. 4 (October 2, 2014): e983870. http://dx.doi.org/10.4161/sgtp.28453.

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Zhang, Xiaoyu, Erfei Bi, Peter Novick, Lilin Du, Keith G. Kozminski, Joshua H. Lipschutz, and Wei Guo. "Cdc42 Interacts with the Exocyst and Regulates Polarized Secretion." Journal of Biological Chemistry 276, no. 50 (October 10, 2001): 46745–50. http://dx.doi.org/10.1074/jbc.m107464200.

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Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeastSaccharomyces cerevisiae, we found that certaincdc42mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions betweencdc42and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.

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Jose, Mini, Sylvain Tollis, Deepak Nair, Romain Mitteau, Christophe Velours, Aurelie Massoni-Laporte, Anne Royou, Jean-Baptiste Sibarita, and Derek McCusker. "A quantitative imaging-based screen reveals the exocyst as a network hub connecting endocytosis and exocytosis." Molecular Biology of the Cell 26, no. 13 (July 2015): 2519–34. http://dx.doi.org/10.1091/mbc.e14-11-1527.

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The coupling of endocytosis and exocytosis underlies fundamental biological processes ranging from fertilization to neuronal activity and cellular polarity. However, the mechanisms governing the spatial organization of endocytosis and exocytosis require clarification. Using a quantitative imaging-based screen in budding yeast, we identified 89 mutants displaying defects in the localization of either one or both pathways. High-resolution single-vesicle tracking revealed that the endocytic and exocytic mutants she4∆ and bud6∆ alter post-Golgi vesicle dynamics in opposite ways. The endocytic and exocytic pathways display strong interdependence during polarity establishment while being more independent during polarity maintenance. Systems analysis identified the exocyst complex as a key network hub, rich in genetic interactions with endocytic and exocytic components. Exocyst mutants displayed altered endocytic and post-Golgi vesicle dynamics and interspersed endocytic and exocytic domains compared with control cells. These data are consistent with an important role for the exocyst in coordinating endocytosis and exocytosis.

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Essid, Miriam, Navin Gopaldass, Kunito Yoshida, Christien Merrifield, and Thierry Soldati. "Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole." Molecular Biology of the Cell 23, no. 7 (April 2012): 1267–82. http://dx.doi.org/10.1091/mbc.e11-06-0576.

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Water expulsion by the contractile vacuole (CV) in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion, and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a effector, Rab8a, the exocyst complex, and LvsA, a protein of the Chédiak–Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder, suggesting that a Rab8a guanine nucleotide exchange factor activity is associated with the complex. Absence of Drainin leads to overtethering and blocks fusion, whereas expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in postfusion detethering. Of interest, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype.

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Yu, Sidney, Ayano Satoh, Marc Pypaert, Karl Mullen, Jesse C. Hay, and Susan Ferro-Novick. "mBet3p is required for homotypic COPII vesicle tethering in mammalian cells." Journal of Cell Biology 174, no. 3 (July 31, 2006): 359–68. http://dx.doi.org/10.1083/jcb.200603044.

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TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.

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Mukherjee, Indrani, and Charles Barlowe. "Overexpression of Sly41 suppresses COPII vesicle–tethering deficiencies by elevating intracellular calcium levels." Molecular Biology of the Cell 27, no. 10 (May 15, 2016): 1635–49. http://dx.doi.org/10.1091/mbc.e15-10-0704.

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SLY41 was identified as a multicopy suppressor of loss of Ypt1, a Rab GTPase essential for COPII vesicle tethering at the Golgi complex. SLY41 encodes a polytopic membrane protein with homology to a class of solute transporter proteins, but how overexpression suppresses vesicle-tethering deficiencies is not known. Here we show that Sly41 is efficiently packaged into COPII vesicles and actively cycles between the ER and Golgi compartments. SLY41 displays synthetic negative genetic interactions with PMR1, which encodes the major Golgi-localized Ca2+/Mn2+transporter and suggests that Sly41 influences cellular Ca2+and Mn2+homeostasis. Experiments using the calcium probe aequorin to measure intracellular Ca2+concentrations in live cells reveal that Sly41 overexpression significantly increases cytosolic calcium levels. Although specific substrates of the Sly41 transporter were not identified, our findings indicate that localized overexpression of Sly41 to the early secretory pathway elevates cytosolic calcium levels to suppress vesicle-tethering mutants. In vitro SNARE cross-linking assays were used to directly monitor the influence of Ca2+on tethering and fusion of COPII vesicles with Golgi membranes. Strikingly, calcium at suppressive concentrations stimulated SNARE-dependent membrane fusion when vesicle-tethering activity was reduced. These results show that calcium positively regulates the SNARE-dependent fusion stage of ER–Golgi transport.

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Journal articles on the topic 'Vesicle tethering complex exocyst'

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