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1
Pospekhova, N. A., and K. V. Regel. "Ultrastructure of the cercomer of the metacestodeMicrosomacanthus paraparvulaRegel, 1994 (Cestoda: Hymenolepididae)." Journal of Helminthology 87, no. 4 (October 17, 2012): 483–88. http://dx.doi.org/10.1017/s0022149x12000648.
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AbstractInvestigations were undertaken using light and transmission electron microscopy to clearly delineate the morphology of the cercomer, i.e. the protective envelopes and tail appendage, in cysticercoids ofMicrosomacanthus paraparvula, which develop in the haemocoel of the caddiswormGrensia praeterita(Insecta: Trichoptera). Two protective envelopes, the exocyst and endocyst, were identified. The non-cellular exocyst is found to consist of granular material and of thin, dense membrane-like layers, which are located parallel to each other. The exocyst of the mature metacestode tightly adjoins the outer surface of the endocyst, containing prospective parts (the scolex and the neck), except for the areas at its poles. A long tail appendage is located outside the exocyst. Evidence was found to indicate the existence of active synthetic processes occurring in the tail appendage. Non-cellular exocysts are widely distributed within metacestodes of the families Hymenolepididae and Dilepididae, and, presumably, are formed by means of glandular secretions from the oncosphere, given the early appearance of non-cellular exocysts in ontogeny.
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Chavez-Dozal, Alba A., Stella M. Bernardo, Hallie S. Rane, and Samuel A. Lee. "Functional Analysis of the Exocyst Subunit Sec15 in Candida albicans." Eukaryotic Cell 14, no. 12 (October 9, 2015): 1228–39. http://dx.doi.org/10.1128/ec.00147-15.
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ABSTRACTIn prior studies of exocyst-mediated late secretion inCandida albicans, we have determined that Sec6 contributes to cell wall integrity, secretion, and filamentation. A conditional mutant lackingSEC6expression exhibits markedly reduced lateral hyphal branching. In addition, lack of the related t-SNAREs Sso2 and Sec9 also leads to defects in secretion and filamentation. To further understand the role of the exocyst in the fundamental processes of polarized secretion and filamentation inC. albicans, we studied the exocyst subunit Sec15. SinceSaccharomyces cerevisiae SEC15is essential for viability, we generated aC. albicansconditional mutant strain in whichSEC15was placed under the control of a tetracycline-regulated promoter. In the repressed state, cell death occurred after 5 h in the tetR-SEC15 strain. Prior to this time point, the tetR-SEC15 mutant was markedly defective in Sap and lipase secretion and demonstrated increased sensitivity to Zymolyase and chitinase. Notably, tetR-SEC15 mutant hyphae were characterized by a hyperbranching phenotype, in direct contrast to strain tetR-SEC6, which had minimal lateral branching. We further studied the localization of the Spitzenkörper, polarisomes, and exocysts in the tetR-SEC15 and tetR-SEC6 mutants during filamentation. Mlc1-GFP (marking the Spitzenkörper), Spa2-GFP (the polarisome), and Exo70-GFP (exocyst) localizations were normal in the tetR-SEC6 mutant, whereas these structures were mislocalized in the tetR-SEC15 mutant. Following alleviation of gene repression by removing doxycycline, first Spitzenkörper, then polarisome, and finally exocyst localizations were recovered sequentially. These results indicate that the exocyst subunits Sec15 and Sec6 have distinct roles in mediating polarized secretion and filamentation inC. albicans.
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Beljakova, R. N., and S. V. Smirnova. "Stichosiphon subarcticus sp. nov. (Cyanoprokaryota, Chroococcales) from the White Sea." Novosti sistematiki nizshikh rastenii 52, no. 2 (2018): 235–44. http://dx.doi.org/10.31111/nsnr/2018.52.2.235.
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Representatives of the genus Stichosiphon live predominantly in fresh waterbodies epiphytic on algae and higher plants. Eight species of the genus have a tropical distribution, two species were found in central European countries, three species were described from the northwest of European Russia and one species lives in the highlands of Tibet. New species of the genus Stichosiphon, S. subarcticus is described. S. subarcticus was found in the desalinated pool at the upper littoral at Bolshoy Solovetskiy Island (Solovetskiy Archipelago, the White sea) growing on copepods. Morphological and ecological notes are given. Pseudofilaments solitary or in groups, straight or curved, uniseriate, not or sometimes slightly widened at the terminal part, mature pseudofilaments 25–55 μm long, 0.9–1.5(2.2) μm wide, exocytes and basal cells (0.6)0.85–1.45(2.2) μm long, 0.8–1.5(2.2) μm wide. This species is similar to S. sansibaricus var. marinus by short single-row and unbranched pseudofilaments, differs by width of pseudofilaments, exocyte dimensions and proportions (exocytes of S. sansibaricus var. marinus always longer than wide). Due to unstable conditions in the only known habitat salinity preferences of S. subarcticus are unclear. The location is the northernmost for this genus.
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Songer, Jennifer A., and Mary Munson. "Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion." Molecular Biology of the Cell 20, no. 3 (February 2009): 973–82. http://dx.doi.org/10.1091/mbc.e08-09-0968.
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The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.
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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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Shen, David, Hua Yuan, Alex Hutagalung, Avani Verma, Daniel Kümmel, Xudong Wu, Karin Reinisch, James A. McNew, and Peter Novick. "The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p." Journal of Cell Biology 202, no. 3 (July 29, 2013): 509–26. http://dx.doi.org/10.1083/jcb.201211148.
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A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.
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Sharda, Anish V., Alexandra M. Barr, Joshua A. Harrison, Adrian R. Wilkie, Chao Fang, Lourdes M. Mendez, Ionita C. Ghiran, Joseph E. Italiano, and Robert Flaumenhaft. "VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2." Blood 136, no. 24 (December 10, 2020): 2824–37. http://dx.doi.org/10.1182/blood.2020005300.
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Abstract von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2–dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2–interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release.
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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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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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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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Lipschutz, Joshua H., Wei Guo, Lucy E. O'Brien, Yen H. Nguyen, Peter Novick, and Keith E. Mostov. "Exocyst Is Involved in Cystogenesis and Tubulogenesis and Acts by Modulating Synthesis and Delivery of Basolateral Plasma Membrane and Secretory Proteins." Molecular Biology of the Cell 11, no. 12 (December 2000): 4259–75. http://dx.doi.org/10.1091/mbc.11.12.4259.
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Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.
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Fujimoto, Brent A., Madison Young, Lamar Carter, Alina P. S. Pang, Michael J. Corley, Ben Fogelgren, and Noemi Polgar. "The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells." American Journal of Physiology-Endocrinology and Metabolism 317, no. 6 (December 1, 2019): E957—E972. http://dx.doi.org/10.1152/ajpendo.00109.2019.
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Skeletal muscle handles ~80–90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.
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Fabian, Lacramioara, Ho-Chun Wei, Janet Rollins, Tatsuhiko Noguchi, J. Todd Blankenship, Kishan Bellamkonda, Gordon Polevoy, et al. "Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila." Molecular Biology of the Cell 21, no. 9 (May 2010): 1546–55. http://dx.doi.org/10.1091/mbc.e09-07-0582.
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During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by low-level expression of the PIP2 phosphatase SigD or mutation of the PIP2 biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP2 localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP2 levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP2 recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.
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Wang, Hongyan, Xie Tang, Jianhua Liu, Susanne Trautmann, David Balasundaram, Dannel McCollum, and Mohan K. Balasubramanian. "The Multiprotein Exocyst Complex Is Essential for Cell Separation in Schizosaccharomyces pombe." Molecular Biology of the Cell 13, no. 2 (February 2002): 515–29. http://dx.doi.org/10.1091/mbc.01-11-0542.
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Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. A mulitlayered division septum is assembled in concert with ring constriction. Finally, cleavage of the inner layer of the division septum results in the liberation of daughter cells. Although numerous studies have focused on actomyosin ring and division septum assembly, little information is available on the mechanism of cell separation. Here we describe a mutant, sec8-1, that is defective in cell separation but not in other aspects of cytokinesis.sec8-1 mutants accumulate ∼100-nm vesicles and have reduced secretion of acid phosphatase, suggesting that they are defective in exocytosis. Sec8p is a component of the exocyst complex. Using biochemical methods, we show that Sec8p physically interacts with other members of the exocyst complex, including Sec6p, Sec10p, and Exo70p. These exocyst proteins localize to regions of active exocytosis—at the growing ends of interphase cells and in the medial region of cells undergoing cytokinesis—in an F-actin–dependent and exocytosis-independent manner. Analysis of a number of mutations in various exocyst components has established that these components are essential for cell viability. Interestingly, all exocyst mutants analyzed appear to be able to elongate and to assemble division septa but are defective for cell separation. We therefore propose that the fission yeast exocyst is involved in targeting of enzymes responsible for septum cleavage. We further propose that cell elongation and division septum assembly can continue with minimal levels of exocyst function.
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Boyd, Charles, Thom Hughes, Marc Pypaert, and Peter Novick. "Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p." Journal of Cell Biology 167, no. 5 (December 6, 2004): 889–901. http://dx.doi.org/10.1083/jcb.200408124.
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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.
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Roumanie, Olivier, Hao Wu, Jeffrey N. Molk, Guendalina Rossi, Kerry Bloom, and Patrick Brennwald. "Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex." Journal of Cell Biology 170, no. 4 (August 15, 2005): 583–94. http://dx.doi.org/10.1083/jcb.200504108.
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Rho GTPases are important regulators of polarity in eukaryotic cells. In yeast they are involved in regulating the docking and fusion of secretory vesicles with the cell surface. Our analysis of a Rho3 mutant that is unable to interact with the Exo70 subunit of the exocyst reveals a normal polarization of the exocyst complex as well as other polarity markers. We also find that there is no redundancy between the Rho3–Exo70 and Rho1–Sec3 pathways in the localization of the exocyst. This suggests that Rho3 and Cdc42 act to polarize exocytosis by activating the exocytic machinery at the membrane without the need to first recruit it to sites of polarized growth. Consistent with this model, we find that the ability of Rho3 and Cdc42 to hydrolyze GTP is not required for their role in secretion. Moreover, our analysis of the Sec3 subunit of the exocyst suggests that polarization of the exocyst may be a consequence rather than a cause of polarized exocytosis.
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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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Torres, Michael J., Raj K. Pandita, Ozlem Kulak, Rakesh Kumar, Etienne Formstecher, Nobuo Horikoshi, Kalpana Mujoo, et al. "Role of the Exocyst Complex Component Sec6/8 in Genomic Stability." Molecular and Cellular Biology 35, no. 21 (August 17, 2015): 3633–45. http://dx.doi.org/10.1128/mcb.00768-15.
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The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.
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Dowd, Georgina C., Roman Mortuza, Manmeet Bhalla, Hoan Van Ngo, Yang Li, Luciano A. Rigano, and Keith Ireton. "Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread." Proceedings of the National Academy of Sciences 117, no. 7 (February 3, 2020): 3789–96. http://dx.doi.org/10.1073/pnas.1916676117.
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The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.
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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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Zhang, Ying, Chun-Ming Liu, Anne-Mie C. Emons, and Tijs Ketelaar. "The Plant Exocyst." Journal of Integrative Plant Biology 52, no. 2 (February 2010): 138–46. http://dx.doi.org/10.1111/j.1744-7909.2010.00929.x.
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Mei, Kunrong, and Wei Guo. "The exocyst complex." Current Biology 28, no. 17 (September 2018): R922—R925. http://dx.doi.org/10.1016/j.cub.2018.06.042.
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Sommer, Bernhard, Adrian Oprins, Catherine Rabouille, and Sean Munro. "The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster." Journal of Cell Biology 169, no. 6 (June 13, 2005): 953–63. http://dx.doi.org/10.1083/jcb.200411053.
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The exocyst is an octameric complex required for polarized secretion. Some components of the exocyst are found on the plasma membrane, whereas others are recruited to Golgi membranes, suggesting that exocyst assembly tethers vesicles to their site of fusion. We have found that in Drosophila melanogaster oocytes the majority of the exocyst component Sec5 is unexpectedly present in clathrin-coated pits and vesicles at the plasma membrane. In oocytes, the major substrate for clathrin-dependent endocytosis is the vitellogenin receptor Yolkless. A truncation mutant of Sec5 (sec5E13) allows the formation of normally sized oocytes but with greatly reduced yolk uptake. We find that in sec5E13 oocytes Yolkless accumulates aberrantly in late endocytic compartments, indicating a defect in the endocytic cycling of the receptor. An analogous truncation of the yeast SEC5 gene results in normal secretion but a temperature-sensitive defect in endocytic recycling. Thus, the exocyst may act in both Golgi to plasma membrane traffic and endocytic cycling, and hence in oocytes is recruited to clathrin-coated pits to facilitate the rapid recycling of Yolkless.
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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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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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Bendezú, Felipe O., and Sophie G. Martin. "Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast." Molecular Biology of the Cell 22, no. 1 (January 2011): 44–53. http://dx.doi.org/10.1091/mbc.e10-08-0720.
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Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2)–dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP2-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.
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Oztan, Asli, Mark Silvis, Ora A. Weisz, Neil A. Bradbury, Shu-Chan Hsu, James R. Goldenring, Charles Yeaman, and Gerard Apodaca. "Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells." Molecular Biology of the Cell 18, no. 10 (October 2007): 3978–92. http://dx.doi.org/10.1091/mbc.e07-02-0097.
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The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
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Zuo, Xiaofeng, Wei Guo, and Joshua H. Lipschutz. "The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro." Molecular Biology of the Cell 20, no. 10 (May 15, 2009): 2522–29. http://dx.doi.org/10.1091/mbc.e08-07-0772.
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Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, in which they are felt to participate in flow sensing and have been linked to the pathogenesis of cystic renal disorders such as autosomal dominant polycystic kidney disease. We previously localized the exocyst, an eight-protein complex involved in membrane trafficking, to the primary cilium of Madin-Darby canine kidney cells and showed that it was involved in cystogenesis. Here, using short hairpin RNA (shRNA) to knockdown exocyst expression and stable transfection to induce exocyst overexpression, we show that the exocyst protein Sec10 regulates primary ciliogenesis. Using immunofluorescence, scanning, and transmission electron microscopy, primary cilia containing only basal bodies are seen in the Sec10 knockdown cells, and increased ciliogenesis is seen in Sec10-overexpressing cells. These phenotypes do not seem to be because of gross changes in cell polarity, as apical, basolateral, and tight junction proteins remain properly localized. Sec10 knockdown prevents normal cyst morphogenesis when the cells are grown in a collagen matrix, whereas Sec10 overexpression results in increased cystogenesis. Transfection with human Sec10 resistant to the canine shRNA rescues the phenotype, demonstrating specificity. Finally, Par3 was recently shown to regulate primary cilia biogenesis. Par3 and the exocyst colocalized by immunofluorescence and coimmunoprecipitation, consistent with a role for the exocyst in targeting and docking vesicles carrying proteins necessary for primary ciliogenesis.
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Balakireva, Maria, Carine Rossé, Johanna Langevin, Yu-chen Chien, Michel Gho, Geneviève Gonzy-Treboul, Stéphanie Voegeling-Lemaire, et al. "The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster." Molecular and Cellular Biology 26, no. 23 (September 25, 2006): 8953–63. http://dx.doi.org/10.1128/mcb.00506-06.
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ABSTRACT Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK.
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Wang, Hongyan, Xie Tang, and Mohan K. Balasubramanian. "Rho3p Regulates Cell Separation by Modulating Exocyst Function in Schizosaccharomyces pombe." Genetics 164, no. 4 (August 1, 2003): 1323–31. http://dx.doi.org/10.1093/genetics/164.4.1323.
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Abstract Cytokinesis is the final stage of the cell division cycle in which the mother cell is physically divided into two daughters. In recent years the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis, since it divides using an actomyosin ring whose constriction is coordinated with the centripetal deposition of new membranes and a division septum. The final step of cytokinesis in S. pombe requires the digestion of the primary septum to liberate two daughters. We have previously shown that the multiprotein exocyst complex is essential for this process. Here we report the isolation of rho3+, encoding a Rho family GTPase, as a high-copy suppressor of an exocyst mutant, sec8-1. Overproduction of Rho3p also suppressed the temperature-sensitive growth phenotype observed in cells lacking Exo70p, another conserved component of the S. pombe exocyst complex. Cells deleted for rho3 arrest at higher growth temperatures with two or more nuclei and uncleaved division septa between pairs of nuclei. rho3Δ cells accumulate ∼100-nm vesicle-like structures. These phenotypes are all similar to those observed in exocyst component mutants, consistent with a role for Rho3p in modulation of exocyst function. Taken together, our results suggest the possibility that S. pombe Rho3p regulates cell separation by modulation of exocyst function.
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Wen, Pei, Fujian Zhang, Yulong Fu, Jun-yi Zhu, and Zhe Han. "Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes." Journal of the American Society of Nephrology 31, no. 5 (April 1, 2020): 1024–34. http://dx.doi.org/10.1681/asn.2019060591.
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BackgroundStudies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue.MethodsWe silenced genes encoding exocyst complex proteins specifically in Drosophila nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11.ResultsSilencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm’s highly organized surface structure—essential for filtration—was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named “exocyst rods,” which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11.ConclusionsOur findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.
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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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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.
Full textAbstract:
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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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.
Full textAbstract:
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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Andersen, Nicholas J., and Charles Yeaman. "Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells." Molecular Biology of the Cell 21, no. 1 (January 2010): 152–64. http://dx.doi.org/10.1091/mbc.e09-06-0459.
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The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.
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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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Jones, Laura A., and Peter E. Sudbery. "Spitzenkörper, Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties." Eukaryotic Cell 9, no. 10 (August 6, 2010): 1455–65. http://dx.doi.org/10.1128/ec.00109-10.
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ABSTRACT During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.
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Shipitsin, Michail, and Larry A. Feig. "RalA but Not RalB Enhances Polarized Delivery of Membrane Proteins to the Basolateral Surface of Epithelial Cells." Molecular and Cellular Biology 24, no. 13 (July 1, 2004): 5746–56. http://dx.doi.org/10.1128/mcb.24.13.5746-5756.2004.
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ABSTRACT RalA and RalB constitute a family of highly similar (85% identity) Ras-related GTPases. Recently, active forms of both RalA and RalB have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. However, we show here that only active RalA enhances the rate of delivery of E-cadherin and other proteins to their site in the basolateral membrane of MDCK cells, consistent with RalA being a regulator of exocyst function. One reason for this difference is that RalA binds more effectively to the exocyst complex than active RalB does both in vivo and in vitro. Another reason is that active RalA localizes to perinuclear recycling endosomes, where regulation of vesicle sorting is thought to take place, while active RalB does not. Strikingly, analysis of chimeras made between RalA and RalB reveals that high-affinity exocyst binding by RalA is due to unique amino acid sequences in RalA that are distal to the common effector-binding domains shared by RalA and RalB. Moreover, these chimeras show that the perinuclear localization of active RalA is due in part to its unique variable domain near the C terminus. This distinct localization appears to be important for RalA effects on secretion because all RalA mutants tested that failed to localize to the perinuclear region also failed to promote basolateral delivery of E-cadherin. Interestingly, one of these inactive mutants maintained binding to the exocyst complex, suggesting that RalA binding to the exocyst is necessary but not sufficient for RalA to promote basolateral delivery of membrane proteins.
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Hutagalung, Alex H., Jeff Coleman, Marc Pypaert, and Peter J. Novick. "An Internal Domain of Exo70p Is Required for Actin-independent Localization and Mediates Assembly of Specific Exocyst Components." Molecular Biology of the Cell 20, no. 1 (January 2009): 153–63. http://dx.doi.org/10.1091/mbc.e08-02-0157.
Full textAbstract:
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.
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Babbey, Clifford M., Robert L. Bacallao, and Kenneth W. Dunn. "Rab10 associates with primary cilia and the exocyst complex in renal epithelial cells." American Journal of Physiology-Renal Physiology 299, no. 3 (September 2010): F495—F506. http://dx.doi.org/10.1152/ajprenal.00198.2010.
Full textAbstract:
Rab10, a mammalian homolog of the yeast Sec4p protein, has previously been associated with endocytic recycling and biosynthetic membrane transport in cultured epithelia and with Glut4 translocation in adipocytes. Here, we report that Rab10 associates with primary cilia in renal epithelia in culture and in vivo. In addition, we find that Rab10 also colocalizes with exocyst proteins at the base of nascent cilia, and physically interacts with the exocyst complex, as detected with anti-Sec8 antibodies. These data suggest that membrane transport to the primary cilum may be mediated by interactions between Rab10 and an exocyst complex located at the cilium base.
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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.
Full textAbstract:
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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Zhang, Xiaoyu, Puyue Wang, Akanksha Gangar, Jian Zhang, Patrick Brennwald, Daniel TerBush, and Wei Guo. "Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis." Journal of Cell Biology 170, no. 2 (July 18, 2005): 273–83. http://dx.doi.org/10.1083/jcb.200502055.
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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.
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Zhang, Chunhua, Michelle Q. Brown, Wilhelmina van de Ven, Zhi-Min Zhang, Bin Wu, Michael C. Young, Lukáš Synek, et al. "Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis." Proceedings of the National Academy of Sciences 113, no. 1 (November 25, 2015): E41—E50. http://dx.doi.org/10.1073/pnas.1521248112.
Full textAbstract:
The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.
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van Gisbergen, Peter A. C., Shu-Zon Wu, Mingqin Chang, Kelli A. Pattavina, Madelaine E. Bartlett, and Magdalena Bezanilla. "An ancient Sec10–formin fusion provides insights into actin-mediated regulation of exocytosis." Journal of Cell Biology 217, no. 3 (January 26, 2018): 945–57. http://dx.doi.org/10.1083/jcb.201705084.
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Exocytosis, facilitated by the exocyst, is fundamentally important for remodeling cell walls and membranes. Here, we analyzed For1F, a novel gene that encodes a fusion of an exocyst subunit (Sec10) and an actin nucleation factor (formin). We showed that the fusion occurred early in moss evolution and has been retained for more than 170 million years. In Physcomitrella patens, For1F is essential, and the expressed protein is a fusion of Sec10 and formin. Reduction of For1F or actin filaments inhibits exocytosis, and For1F dynamically associates with Sec6, another exocyst subunit, in an actin-dependent manner. Complementation experiments demonstrate that constitutive expression of either half of the gene or the paralogous Sec10b rescues loss of For1F, suggesting that fusion of the two domains is not essential, consistent with findings in yeast, where formin and the exocyst are linked noncovalently. Although not essential, the fusion may have had selective advantages and provides a unique opportunity to probe actin regulation of exocytosis.
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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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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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Neto, Hélia, Gemma Balmer, and Gwyn Gould. "Exocyst proteins in cytokinesis." Communicative & Integrative Biology 6, no. 6 (November 9, 2013): e27635. http://dx.doi.org/10.4161/cib.27635.
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Heider, Margaret R., and Mary Munson. "Exorcising the Exocyst Complex." Traffic 13, no. 7 (April 8, 2012): 898–907. http://dx.doi.org/10.1111/j.1600-0854.2012.01353.x.
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Saeed, Bushra, Carla Brillada, and Marco Trujillo. "Dissecting the plant exocyst." Current Opinion in Plant Biology 52 (December 2019): 69–76. http://dx.doi.org/10.1016/j.pbi.2019.08.004.
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Zhu, Xiaoyu, Shundai Li, Songqin Pan, Xiaoran Xin, and Ying Gu. "CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane." Proceedings of the National Academy of Sciences 115, no. 15 (March 26, 2018): E3578—E3587. http://dx.doi.org/10.1073/pnas.1800182115.
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Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana. We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.