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• W2891887976 abstract "The exocyst is a multisubunit protein complex that was first identified and characterized in budding yeast. Later studies have demonstrated its conservation in eukaryotes, from plants to mammals. This complex mediates the tethering of secretory vesicles to the plasma membrane prior to fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The exocyst has been implicated in a variety of cellular processes, such as exocytosis, cell growth, cytokinesis, cell migration, primary ciliogenesis and tumorigenesis. Recent years have seen major progress in our understanding of this complex. In this Primer, we focus on some of the basic information about the exocyst complex, including its structure, assembly, molecular interactions, function in vesicle tethering and membrane fusion, and involvement in many physiological processes. The exocyst is a multisubunit protein complex that was first identified and characterized in budding yeast. Later studies have demonstrated its conservation in eukaryotes, from plants to mammals. This complex mediates the tethering of secretory vesicles to the plasma membrane prior to fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The exocyst has been implicated in a variety of cellular processes, such as exocytosis, cell growth, cytokinesis, cell migration, primary ciliogenesis and tumorigenesis. Recent years have seen major progress in our understanding of this complex. In this Primer, we focus on some of the basic information about the exocyst complex, including its structure, assembly, molecular interactions, function in vesicle tethering and membrane fusion, and involvement in many physiological processes. Despite the low sequence identities between the components of the exocyst complexes of different species, the structure and many functions of the exocyst are well conserved. The exocyst contains a single copy of each of the eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. Due to the large size and poor solubility of the individual subunits in isolation, structural studies of the exocyst are challenging. Combining cryo-EM and chemical cross-linking mass spectrometry, and benefiting from previous crystallography studies, we have recently built a model of the fully assembled yeast exocyst complex. The model consists of nearly full-length forms of Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, Exo84 and the carboxy-terminal half of Sec3. It shows that all of the eight subunits have a similar fold, with an extended coiled-coil near the amino terminus (termed a ‘CorEx’ motif; for Sec3, it is found in the middle of the protein) followed by an elongated rod-like helical bundle structure towards the carboxyl terminus (Figure 1). An exception to this is that, in Exo84, a Ral-binding domain with a β-barrel fold is inserted between the CorEx motif and the helical bundle structure. The structure of the exocyst complex also suggests that the intact complex is assembled in a hierarchical manner. Through antiparallel pairing of the CorEx motifs, Sec3–Sec5, Sec6–Sec8, Sec10–Sec15 and Exo70–Exo84 form four heterodimers. Then the two CorEx pairs from the first two heterodimers interlace with each other to form a four-helical bundle, resulting in subcomplex I, consisting of Sec3, Sec5, Sec6 and Sec8. In a similar manner, Sec10, Sec15, Exo70 and Exo84 assemble into subcomplex II. Subcomplex I and subcomplex II then interact with each other to form the holo-complex (Figure 1). The interactions between the two subcomplexes are mainly mediated by Sec8 and Sec5 from subcomplex I, both of which present extensive binding interfaces along the rod-like structures to bind to multiple subunits of subcomplex II. Sequence comparison and structural studies suggest that the mode of exocyst complex assembly through the CorEx motif is not only evolutionarily conserved for exocyst complexes from yeast to mammals, but also shared for other multisubunit tethering complexes (e.g. GARP and COG) that function at different stages of vesicular traffic. Yeast cells bearing exocyst mutations show an intracellular accumulation of secretory vesicles and defects in exocytosis. Secretory vesicles are transported by Myo2, a type V myosin, along the actin cables from the trans-Golgi network (TGN) to specific regions of the plasma membrane. Subsequent SNARE assembly leads to membrane fusion. In exocyst mutant cells, the accumulation of vesicles starts near sites of localized cell-surface expansion such as the yeast buds, where active exocytosis takes place, whereas mutations in Myo2 lead to the accumulation of the secretory vesicles in the mother cell, suggesting that the exocyst functions at a stage downstream of vesicle transport. When Sec3 is ectopically targeted to the mitochondrial outer membrane or peroxisomal membrane, the exocyst complex is assembled and secretory vesicles are tethered at these surrogate organelles, supporting the role of the exocyst in vesicle targeting and tethering. Live-cell imaging and fluorescence recovery after photobleaching (FRAP) analysis in yeast cells demonstrated that Sec3 and, in part, Exo70 localize to the plasma membrane independently of the actin cables, whereas the remaining six subunits, together with some Exo70, are carried to the plasma membrane by the vesicles. The localization of Sec3 to the plasma membrane is mediated by an interaction between its amino-terminal pleckstrin homology (PH) domain and phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). Sec15 is recruited to secretory vesicles via binding to the Rab protein Sec4 in its GTP-bound form. In addition, Sec6 interacts with the (vesicular) v-SNARE protein Snc2. When the partially assembled exocyst subunits including Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 arrive at the yeast daughter cell plasma membrane via the secretory vesicles, they interact with Sec3 to form the fully assembled exocyst complex at the plasma membrane. Along with the assembly of the exocyst complex and the binding of PI(4,5)P2 to Exo70, the secretory vesicle is tethered to the plasma membrane for subsequent membrane fusion (Figure 2). At the plasma membrane, the small GTPases Cdc42 and Rho1, in their GTP-bound form, interact with Sec3. In fact, these GTPases bind to the PH domain of Sec3 in a competitive manner, suggesting that they regulate the exocyst at different stages of cell growth. Exo70 also binds to Cdc42 and another Rho GTPase, Rho3. In mammals, Exo70 interacts with the Rho family protein TC10. The small GTPases and PI(4,5)P2 may synergistically regulate the localization and function of the exocyst. While in yeast Sec15 interacts with Sec4, in mammalian cells Sec15 interacts with Rab proteins, including Rab3, Rab8, Rab10, Rab11 and Rab27, in a GTP-dependent manner. Among them, Rab8 is the mammalian ortholog of Sec4. Interestingly, Sec15 also interacts with yeast Sec2, the homolog of mammalian Rabin8: Sec2 and Rabin8 are effectors of Ypt32/Rab11 and the guanine nucleotide exchange factors for Sec4/Rab8, respectively. The Ypt32/Rab11–Sec2/Rabin8–Sec4/Rab8 interactions form a GTPase cascade that coordinates vesicle exit from the donor membrane (TGN and recycling endosomes) with later tethering at the plasma membrane. In addition to the Sec4–Sec15 interaction, Sec6 interacts with the v-SNARE protein Snc2, and Exo84 and Sec5 bind to the Ral GTPases, which may also participate in recruiting the exocyst components to vesicles. In budding yeast, secretory vesicles are transported from the TGN to the plasma membrane along actin cables by the class V myosin Myo2, which interacts with the Rab GTPases and the exocyst protein Sec15. The Rab proteins Ypt31/32, which are required for the generation and exit of post-Golgi secretory vesicles, may initially recruit Myo2. Sec4, which recruits the exocyst to the vesicles, functions downstream of Ypt31/32. These Rab proteins bind to the same site on the cargo-binding domain of Myo2, whereas Sec15 binds to a different site on this domain. Myo2 may thus simultaneously interact with both the Rabs and Sec15. Immunoprecipitation experiments suggest that Myo2 binds to other members of the complex through Sec15. After the secretory vesicles are tethered to the plasma membrane, the SNARE proteins assemble to drive membrane fusion. In yeast, the t-SNARE proteins for fusion at the plasma membrane include Sso1/2 and Sec9, and the v-SNARE proteins include Snc1/2. Sso1/2 has an amino-terminal Habc domain that binds to its SNARE motif to keep it in an autoinhibited conformation. Sec3 binds to Sso2 through its amino terminus. Structural studies have shown that the Sec3–Sso2 interaction leads to the relaxation of a helix located in the loop connecting the Habc domain and the SNARE motif and may thus facilitate the relief of the autoinhibition of Sso2. Sec3 promotes the interaction of Sso2 with Sec9, which subsequently displaces Sec3 from Sso2. Liposomal assays showed that Sec3 promotes both the Sso2–Sec9 interaction to form the binary t-SNARE complex at the membrane and the subsequent fusion reaction. In addition to Sec3, Sec6 also interacts with SNAREs. Yeast Sec6 was reported to bind to Sec9, the Sec9–Sso1 binary complex as well as the fully assembled Sec9–Sso1–Snc2 ternary complex. Sec6 also interacts with Snc2. The interactions with both Sec9 and Snc2 are mediated by the amino-terminal region of Sec6. The implications of these interactions for membrane fusion are unclear, although studies support a role of Snc2 in recruiting the exocyst to the secretory vesicles. The assembly of the SNARE complex is regulated by a number of molecules. Pulldown assays showed that Sec6 binds to the Sec1/Munc18 family protein Sec1, leading to the speculation that Sec6 recruits Sec1 to sites of secretion to regulate SNARE complex formation and membrane fusion. In addition, Exo84 binds to Sro7 and Sro77, which regulate SNAREs through an interaction with Sec9. It is possible that these interactions are coordinated in cells to couple vesicle tethering and fusion. The Arp2/3 complex is the core machinery for the generation of a branched actin network that promotes the protrusion of the plasma membrane during morphogenesis and cell migration. Exo70 directly interacts with the ARPC1 subunit of the Arp2/3 complex and promotes actin branching in the presence of WAVE2, a nucleation-promoting factor for the Arp2/3 complex. The Exo70–Arp2/3 interaction is stimulated by epidermal growth factor (EGF) signaling, which is known to promote cell migration and tumor invasion. In addition to Exo70, other members of the exocyst also interact with components of the WAVE complex. The exocyst may be involved in the recruitment of WAVE proteins to the leading edge during cell migration. As well as modulating actin dynamics, Exo70 interacts with PI(4,5)P2 and induces negative curvature of the plasma membrane. Exo70 may couple actin polymerization and membrane deformation for membrane protrusion during cell migration and tumor invasion. As a basic secretion machinery, the exocyst complex functions in polarized exocytosis and is linked to many pathophysiological processes, ranging from neuronal development to tumor invasion. In neurons, the exocyst is enriched in axon growth cones and dendritic branches, and it functions in axon elongation and dendritic arborization. Drosophila sec5 null mutants are defective in neuron polarization and die as growth-arrested larvae that have a failure in the expansion of neuromuscular junctions. Epithelial cells establish apical–basolateral polarity through cell–cell junction formation, and the exocyst is implicated in cargo transport to different membrane domains. The polarity complex Par3–Par6 interacts with the exocyst to mediate vesicle trafficking to the apical surface of epithelial cells. Also, the adaptor protein AP-1B recruits the exocyst to specific vesicles and delivers adhesion proteins to the cell–cell junctions located at the basolateral surface. In addition to the above-mentioned direct interactions of the exocyst with actin regulators such as the Arp2/3 complex, the exocyst also mediates the trafficking of adhesion proteins such as integrins to the leading edge for cell migration. In addition, the exocyst mediates the release of matrix metalloproteinases for the degradation of extracellular matrix for tumor invasion. PI(4,5)P2 and the small GTPases (i.e. Ral, Rho and Rab) function upstream of the exocyst to control these processes. The primary cilium is a signaling organelle present on the surface of most cells. Defective ciliogenesis is implicated in multiple diseases, such as Bardet-Biedl syndrome, Joubert syndrome, and polycystic kidney disease. The exocyst localizes to both the basal body and cilia. As a result of regulation by the Rab GTPases (Rab8, Rab10 and Rab11), the exocyst is involved in the transport of transmembrane proteins required for ciliogenesis, likely through interactions with the Par3–Par6 complex and the intraflagellar transport (IFT) complex. Mutations in Exo84 have been linked to Joubert syndrome, a ciliopathy characterized by the pediatric onset of neurodevelopmental disease. The exocyst has also been implicated in tumorigenesis. Binding of RalB to Sec5 leads to activation of the kinase TBK, which helps the cancer cells to overcome apoptosis and initiates the innate immune response to viral infection, whereas RalB binding to Exo84 induces activation of the kinase ULK1 and autophagosome biogenesis under starvation conditions. The many functions of the exocyst in various cellular processes are under tight spatiotemporal regulation. Most studies of the regulation of the exocyst have focused on interactions with signaling molecules, such as small GTPases as mentioned above, and kinases. The cyclin-dependent kinase Cdk1 directly phosphorylates Exo84, disrupting the assembly of the exocyst complex and leading to an arrest in cell-surface expansion before the metaphase–anaphase transition in yeast. In contrast, ERK1/2 phosphorylates Exo70, promoting the assembly of the exocyst complex in response to EGF signaling. In addition to the interaction with signaling molecules, alternative splicing of the exocyst at the mRNA level could also serve as a means to diversify exocyst function. In humans, Exo70 has at least five splice variants. During epithelialization, epithelial splicing regulatory protein 1 and 2 (ESRP1/2) mediate the generation of an isoform of Exo70 (termed E-Exo70), which cannot interact with the Arp2/3 complex and fails to stimulate cell motility. Isoform switching of Exo70 takes place during epithelial-to-mesenchymal transition and has been implicated in breast cancer metastasis. In plants, dozens of Exo70 paralogs have been identified per genome, and some of the paralogs show unique functions. The existence of many isoforms may correlate with the diverse membrane structures observed in land plants and their adaptation to different environments, and could also allow for significant functional expansion of this evolutionarily conserved complex. The combined efforts in cell biology, genetics, biochemistry, and structural biology have shaped our understanding of the exocyst complex. The increased depth and expansion of our knowledge have also raised a number of new questions. First, to elucidate the fundamental mechanism of exocyst function in exocytosis, we must understand how the exocyst complex is assembled, and how this assembly is regulated. The recent cryo-EM analysis has provided important new information about the molecular organization of the complex that will not only direct future biochemical study of the hierarchal assembly of the exocyst, but also aid our understanding of the regulation of its assembly and disassembly by signaling molecules. Second, despite identification of the interactions of the exocyst with SNAREs, it is still not clear how the exocyst couples vesicle tethering to SNARE-mediated membrane fusion. Studying the functional implications of these interactions and the coordination of these interactions is crucial to our understanding of the molecular mechanism of exocytosis. Finally, the regulation of the exocyst in different physiological contexts needs to be further investigated at the cellular and tissue level. As the exocyst has been implicated in a number of diseases, such as diabetes and cancer, these studies will also provide molecular insight into these diseases." @default.
• W2891887976 created "2018-09-27" @default.
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• W2891887976 date "2018-09-01" @default.
• W2891887976 modified "2023-10-03" @default.
• W2891887976 title "The exocyst complex" @default.
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