The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

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.     

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

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

The Neurospora crassa exocyst complex tethers Spitzenk?rper vesicles to the apical plasma membrane during polarized growth

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.     

The Plant Exocyst

The exocyst is an octameric vesicle tethering complex that functions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. All proteins in the complex have been conserved during evolution, and genes that encode the exocyst subunits are present in the genomes of all plants investigated to date. Although the plant exocyst has not been studied in great detail, it is likely that the basic function of the exocyst in vesicle tethering is conserved. Nevertheless, genomic and genetic studie...     

Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex

Vesicle transport requires four steps: vesicle formation, movement, tethering, and fusion. In yeast, two Rab GTPases, Ypt31/32, are required for post-Golgi vesicle formation. A third Rab GTPase, Sec4, and the exocyst act in tethering and fusion of these vesicles. Vesicle production is coupled to transport via direct interaction between Ypt31/32 and the yeast myosin V, Myo2. Here we show that Myo2 interacts directly with Sec4 and the exocyst subunit Sec15. Disruption of these interactions results in compromised growth and the accumulation of secretory vesicles. We identified the Sec15-binding region on Myo2 and also identified residues on Sec15 required for interaction with Myo2. That Myo2 interacts with Sec15 uncovers additional roles for the exocyst as an adaptor for molecular motors and implies similar roles for structurally related tethering complexes. Moreover, these studies predict that for many pathways, molecular motors attach to vesicles prior to their formation and remain attached until fusion.     

The Plant Exocyst

The exocyst is an octameric vesicle tethering complex that func-tions upstream of SNARE mediated exocytotic vesicle fusion with the plasma membrane. All proteins in the complex have been conserved during evolution, and genes that encode the exocyst subunits are present in the genomes of all plants investigated to date. Although the plant exocyst has not been studied in great detail, it is likely that the basic function of the exocyst in vesicle tethering is conserved. Nevertheless, genomic and genetic studies suggest that the exocyst complex in plants may have more diversified roles than that in budding yeast. In this review, we compare the knowledge about the exocyst in plant cells to the well-studied exocyst in budding yeast, in order to explore similarities and differences in expression and function between these organisms, both of which have walled cells.     

Arabidopsis thaliana myosin XIK is involved in root hair as well as trichome morphogenesis on stems and leaves

Myosins form a large superfamily of molecular motors that move along actin filaments. The functions of myosins in plant cells are thought to be related to various processes: cell division, movement of mitochondria and chloroplasts, cytoplasmic streaming, rearrangement of transvacuolar strands, and statolith positioning. Class VIII and XI myosins are represented in the Arabidopsis thaliana genome by 4 and 13 potential genes, respectively. The roles of individual class XI myosins and their cellular targets in A. thaliana are still unclear. In this work we implemented a reverse genetic approach to analyse the loss-of-function mutants of XIK, a representative of class XI myosins in A. thaliana. Three different T-DNA insertion mutants in the myosin XIK gene showed similar phenotypes: impaired growth of root hair cells, twisted shape of stem trichomes, and irregular size, branch positioning, and branch expansion of leaf trichomes. Morphometric analysis of mutant seedlings showed that the average length of root hairs was reduced up to 50% in comparison with wild-type root hairs, suggesting an involvement of the class XI myosin XIK in tip growth. On leaves, the proportion of trichomes with short branches was doubleed in mutant plants, and the mutant trichomes possessed a mildly twisted shape. Therefore, we concluded that myosin XIK is involved also in the elongation of stalks and branches of trichomes.     

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.     

The exocyst meets the translocon: a regulatory circuit for secretion and protein synthesis?

The translocon is responsible for the translocation of proteins across the membrane of the endoplasmic reticulum into its lumen, whereas the exocyst acts at the other end of the secretory pathway, tethering secretory vesicles to the sites of exocytosis. Here, we discuss three independent lines of evidence that indicate surprising genetic, physical and functional interactions between the two complexes. Although much of the existing evidence is rather preliminary in nature, these interactions might serve to coordinate the biosynthetic capacity of the cell with the function of the secretory machinery.     

The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis

Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that the exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion.     

Conservation and specialization. The role of the exocyst in neuronal exocytosis

The exocyst is an octameric complex mediating vesicle targeting and tethering at the plasma membrane for exocytosis. The role of exocyst in nervous system is unclear. In this issue of Neuron, Murthy et al. provide important insights: defects in the exocyst inhibit neurite outgrowth and neuromuscular junction formation; however, synaptic transmission persists.     

The rab exchange factor Sec2p reversibly associates with the exocyst

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.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

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

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

Exorcising the exocyst complex

The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate. Recent progress demonstrates that the exocyst is also important for additional trafficking steps and cellular processes beyond exocytosis, with links to development and disease. In this review, we discuss current knowledge of exocyst architecture, assembly, regulation and its roles in a variety of cellular trafficking pathways.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

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

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

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.     

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

Vesicle clustering by the molecular motor myosin XI enhances actin polymerization-dependent motility and polarized vesicle accumulation in tip-growing cells.