Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth

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.     

Rho GTPase regulation of exocytosis in yeast is independent of GTP hydrolysis and polarization of the exocyst complex

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.     

The Microtubule-Associated Rho Activating Factor GEF-H1 Interacts with Exocyst Complex to Regulate Vesicle Traffic

The exocyst complex plays a critical role in targeting and tethering vesicles to specific sites of the plasma membrane. These events are crucial for polarized delivery of membrane components to the cell surface, which is critical for cell motility and division. Though Rho GTPases are involved in regulating actin dynamics and membrane trafficking, their role in exocyst-mediated vesicle targeting is not very clear. Herein, we present evidence that depletion of GEF-H1, a guanine nucleotide exchange factor for Rho proteins, affects vesicle trafficking. Interestingly, we found that GEF-H1 directly binds to exocyst component Sec5 in a Ral GTPase-dependent manner. This interaction promotes RhoA activation, which then regulates exocyst assembly/localization and exocytosis. Taken together, our work defines a mechanism for RhoA activation in response to RalA-Sec5 signaling and involvement of GEF-H1/RhoA pathway in the regulation of vesicle trafficking.     

Cdc42 interacts with the exocyst and regulates polarized secretion

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 yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants 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 between cdc42 and 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.     

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

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

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.     

Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.     

Membrane association and functional regulation of Sec3 by phospholipids and Cdc42

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

The sterol-binding protein Kes1/Osh4p is a regulator of polarized exocytosis

Oxysterol-binding protein (OSBP)-related protein Kes1/ Osh4p is implicated in nonvesicular sterol transfer between membranes in Saccharomyces cerevisiae. However, we found that Osh4p associated with exocytic vesicles that move from the mother cell into the bud, where Osh4p facilitated vesicle docking by the exocyst tethering complex at sites of polarized growth on the plasma membrane. Osh4p formed complexes with the small GTPases Cdc42p, Rho1p and Sec4p, and the exocyst complex subunit Sec6p, which was also required for Osh4p association with vesicles. Although Osh4p directly affected polarized exocytosis, its role in sterol trafficking was less clear. Contrary to what is predicted for a sterol-transfer protein, inhibition of sterol binding by the Osh4p Y97F mutation did not cause its inactivation. Rather, OSH4(Y97F) is a gain-of-function mutation that causes dominant lethality. We propose that in response to sterol binding and release Osh4p promotes efficient exocytosis through the co-ordinate regulation of Sac1p, a phosphoinositide 4-phosphate (PI4P) phosphatase, and the exocyst complex. These results support a model in which Osh4p acts as a sterol-dependent regulator of polarized vesicle transport, as opposed to being a sterol-transfer protein.     

The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA

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.     

The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene.

Ral is a small GTPase involved in critical cellular signaling pathways. The two isoforms, RalA and RalB, are widely distributed in different tissues, with RalA being enriched in brain. The best characterized RalA signaling pathways involve RalBP1 and phospholipase D. To investigate RalA signaling in neuronal cells we searched for RalA-binding proteins in brain. We found at least eight proteins that bound RalA in a GTP-dependent manner. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified these as the components of the exocyst complex. The yeast exocyst is a regulator of polarized secretion, docking vesicles to regions of the plasma membrane involved in active exocytosis. We identified the human FLJ10893 protein as the mammalian homologue of the yeast exocyst protein Sec3p. The exocyst complex did not contain the previously identified exocyst component rSec15, but a new homologue of both yeast Sec15p and rSec15, called KIAA0919. Western blots confirmed that two rat exocyst proteins, rSec6 and rSec8, bound active RalA in nerve terminals, as did RalBP1. Phospholipase D bound RalA in a nucleotide-independent manner. This places the RalA signaling system in mammalian nerve terminals, where the exocyst may act as an effector for activated RalA in directing sites of exocytosis.     

Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.     

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.     

Pob1 ensures cylindrical cell shape by coupling two distinct rho signaling events during secretory vesicle targeting

Proper cell morphogenesis requires the co-ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1-664 has a curious lemon-like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin-polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon-like cell morphology similar to pob1-664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin-mediated vesicle transport and exocyst-mediated vesicle tethering during secretory vesicle targeting.     

Ultracentrifugation-based approaches to study regulation of Sec6/8 (exocyst) complex function during development of epithelial cell polarity

The Sec6/8 (exocyst) complex is an essential component of the exocytic apparatus and plays an evolutionarily conserved role in polarized membrane growth. During development of epithelial cell polarity, this cytosolic protein complex is recruited to plasma membrane sites of cell-cell contact, where it facilitates exocytosis to the lateral membrane domain. However, the identity of membrane binding sites for Sec6/8 complex, mechanisms regulating association of Sec6/8 complex with these sites, and the precise function of the complex in polarized trafficking are not known. Biochemical strategies involving differential, rate-zonal, and isopycnic density gradient ultracentrifugation are providing clues to these questions.     

Structure of the GTPase-binding domain of Sec5 and elucidation of its Ral binding site

The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock. The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation. Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst. This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles. We have determined the structure of the Ral-binding domain of Sec5 and shown that it adopts a fold that has not been observed in a G protein effector before. This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains. We have mapped the Ral binding site on this domain and found that it overlaps with protein-protein interaction sites on other IPT domains but that it is completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor. This mapping, along with available site-directed mutagenesis data, allows us to predict how Ral and Sec5 may interact.     

Rho GTPases and exocytosis: what are the molecular links?

Delivery of proteins or lipids to the plasma membrane or into the extracellular space occurs through exocytosis, a process that requires tethering, docking, priming and fusion of vesicles, as well as F-actin rearrangements in response to specific extracellular cues. GTPases of the Rho family have been implicated as important regulators of exocytosis, but how Rho proteins control this process is an open question. In this review, we focus on molecular connections that drive Rho-dependent exocytosis in polarized and regulated exocytosis. Specifically, we present data showing that Rho proteins interaction with the exocyst complex and IQGAP mediates polarized exocytosis, whereas interaction with actin-binding proteins like N-WASP mediates regulated exocytosis.     

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.     

Septin-Dependent Assembly of the Exocyst Is Essential for Plant Infection by Magnaporthe oryzae

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septin-mediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.     

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

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