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1.  The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p 
The Journal of Cell Biology  2013;202(3):509-526.
The exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins to confer both specificity and directionality to vesicular traffic.
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.
doi:10.1083/jcb.201211148
PMCID: PMC3734085  PMID: 23897890
2.  Regulation of the Vitellogenin Receptor during Drosophila melanogaster Oogenesis 
Molecular Biology of the Cell  2000;11(2):511-521.
In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by the Drosophila melanogaster vitellogenin receptor gene yolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that yl RNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.
PMCID: PMC14789  PMID: 10679010
3.  The role of Sec3p in secretory vesicle targeting and exocyst complex assembly 
Molecular Biology of the Cell  2014;25(23):3813-3822.
The exocyst has been speculated to mediate the tethering of secretory vesicles to the plasma membrane. However, there has been no direct experimental evidence for this notion. An ectopic targeting strategy is used to provide experimental support for this model and investigate the regulators of exocyst assembly and vesicle targeting.
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.
doi:10.1091/mbc.E14-04-0907
PMCID: PMC4230786  PMID: 25232005
4.  Fission Yeast Sec3 Bridges the Exocyst Complex to the Actin Cytoskeleton 
Traffic (Copenhagen, Denmark)  2012;13(11):1481-1495.
The exocyst complex tethers post-Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co-ordinator of cell morphology in fission yeast.
doi:10.1111/j.1600-0854.2012.01408.x
PMCID: PMC3531892  PMID: 22891673
actin; endocytosis; exocyst; morphology; Schizosaccharomyces pombe
5.  The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers 
Molecular Biology of the Cell  2012;23(9):1742-1764.
During oogenesis in Drosophila, the phagocytic engulfment protein Ced-6 recognizes the atypical endocytic sorting signal within the vitellogenin receptor Yolkless. Because Ced-6 displays all of the features of an authentic clathrin adaptor, an unrecognized clathrin dependence for Ced-6/Gulp operation during phagocytosis is possible.
Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called “eat-me” signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6–null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.
doi:10.1091/mbc.E11-11-0939
PMCID: PMC3338440  PMID: 22398720
6.  Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells 
Molecular Biology of the Cell  2010;21(1):152-164.
In epithelial cells, Sec3 associates with Exocyst complexes enriched at desmosomes and centrosomes, distinct from Sec6/8 complexes at the apical junctional complex. RNAi-mediated suppression of Sec3 alters trafficking of desmosomal cadherins and impairs desmosome morphology and function, without noticeable effect on adherens junctions.
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.
doi:10.1091/mbc.E09-06-0459
PMCID: PMC2801709  PMID: 19889837
7.  Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1 
Molecular Biology of the Cell  2012;23(2):337-346.
The Sec6 subunit of the multisubunit exocyst tethering complex interacts with the Sec1/Munc18 protein Sec1 and with the t-SNARE Sec9. Assembly of the exocyst upon vesicle arrival at sites of secretion is proposed to release Sec9 for SNARE complex assembly and to recruit Sec1 for interaction with SNARE complexes to facilitate fusion.
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.
doi:10.1091/mbc.E11-08-0670
PMCID: PMC3258177  PMID: 22114349
8.  Cyclical Regulation of the Exocyst and Cell Polarity Determinants for Polarized Cell Growth 
Molecular Biology of the Cell  2005;16(3):1500-1512.
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.
doi:10.1091/mbc.E04-10-0896
PMCID: PMC551511  PMID: 15647373
9.  Developmentally distinct activities of the exocyst enable rapid cell elongation and determine meristem size during primary root growth in Arabidopsis 
BMC Plant Biology  2014;14(1):386.
Background
Exocytosis is integral to root growth: trafficking components of systems that control growth (e.g., PIN auxin transport proteins) to the plasma membrane, and secreting materials that expand the cell wall to the apoplast. Spatiotemporal regulation of exocytosis in eukaryotes often involves the exocyst, an octameric complex that tethers selected secretory vesicles to specific sites on the plasma membrane and facilitates their exocytosis. We evaluated Arabidopsis lines with mutations in four exocyst components (SEC5, SEC8, EXO70A1 and EXO84B) to explore exocyst function in primary root growth.
Results
The mutants have root growth rates that are 82% to 11% of wild-type. Even in lines with the most severe defects, the organization of the quiescent center and tissue layers at the root tips appears similar to wild-type, although meristematic, transition, and elongation zones are shorter. Reduced cell production rates in the mutants are due to the shorter meristems, but not to lengthened cell cycles. Additionally, mutants demonstrate reduced anisotropic cell expansion in the elongation zone, but not the meristematic zone, resulting in shorter mature cells that are similar in shape to wild-type. As expected, hypersensitivity to brefeldin A links the mutant root growth defect to altered vesicular trafficking. Several experimental approaches (e.g., dose–response measurements, localization of signaling components) failed to identify aberrant auxin or brassinosteroid signaling as a primary driver for reduced root growth in exocyst mutants.
Conclusions
The exocyst participates in two spatially distinct developmental processes, apparently by mechanisms not directly linked to auxin or brassinosteroid signaling pathways, to help establish root meristem size, and to facilitate rapid cell expansion in the elongation zone.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0386-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0386-0
PMCID: PMC4302519  PMID: 25551204
Exocyst; Root growth; Meristem; Cell expansion; Auxin; Brassinosteroid
10.  Dominant Negative Alleles of SEC10 Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast 
Molecular Biology of the Cell  1998;9(7):1725-1739.
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.
PMCID: PMC25411  PMID: 9658167
11.  The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst 
Molecular Biology of the Cell  2006;17(6):2757-2769.
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.
doi:10.1091/mbc.E05-10-0917
PMCID: PMC1474791  PMID: 16611746
12.  Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation 
Retrovirology  2012;9:33.
Background
HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown.
Results
To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery.
Conclusions
Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.
doi:10.1186/1742-4690-9-33
PMCID: PMC3382630  PMID: 22534017
HIV; Nef; Exocyst complex; Intercellular nanotubes; Pak2 kinase; Fluorescence confocal microscopy
13.  Sec3p Is Needed for the Spatial Regulation of Secretion and for the Inheritance of the Cortical Endoplasmic ReticulumV⃞ 
Molecular Biology of the Cell  2003;14(12):4770-4782.
Sec3p is a component of the exocyst complex that tethers secretory vesicles to the plasma membrane at exocytic sites in preparation for fusion. Unlike all other exocyst structural genes, SEC3 is not essential for growth. Cells lacking Sec3p grow and secrete surprisingly well at 25°C; however, late markers of secretion, such as the vesicle marker Sec4p and the exocyst subunit Sec8p, localize more diffusely within the bud. Furthermore, sec3Δ cells are strikingly round relative to wild-type cells and are unable to form pointed mating projections in response to α factor. These phenotypes support the proposed role of Sec3p as a spatial landmark for secretion. We also find that cells lacking Sec3p exhibit a dramatic defect in the inheritance of cortical ER into the bud, whereas the inheritance of mitochondria and Golgi is unaffected. Overexpression of Sec3p results in a prominent patch of the endoplasmic reticulum (ER) marker Sec61p-GFP at the bud tip. Cortical ER inheritance in yeast has been suggested to involve the capture of ER tubules at the bud tip. Sec3p may act in this process as a spatial landmark for cortical ER inheritance.
doi:10.1091/mbc.E03-04-0229
PMCID: PMC284782  PMID: 12960429
14.  Fission Yeast Sec3 and Exo70 Are Transported on Actin Cables and Localize the Exocyst Complex to Cell Poles 
PLoS ONE  2012;7(6):e40248.
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 PIP2 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.
doi:10.1371/journal.pone.0040248
PMCID: PMC3386988  PMID: 22768263
15.  Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells 
Molecular Biology of the Cell  2007;18(10):3978-3992.
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.
doi:10.1091/mbc.E07-02-0097
PMCID: PMC1995710  PMID: 17686995
16.  Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 
The Journal of Cell Biology  2008;180(1):145-158.
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.
doi:10.1083/jcb.200704128
PMCID: PMC2213614  PMID: 18195105
17.  ARF6 controls post-endocytic recycling through its downstream exocyst complex effector 
The Journal of Cell Biology  2003;163(5):1111-1121.
The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.
doi:10.1083/jcb.200305029
PMCID: PMC2173613  PMID: 14662749
ARF6; exocyst complex; recycling; endocytosis; small GTP-binding protein
18.  Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes 
The Journal of Cell Biology  1987;105(1):199-206.
Ultrastructural analysis of developing oocytes produced by the recessive female sterile mutant, yolkless (yl), in Drosophila melanogaster shows that yl+ gene activity is necessary for coated pit and coated vesicle formation within these oocytes. 29 alleles of the mutation are known to exist, and they fall either within a strongly affected class or a weakly affected class. Analysis of oocytes produced by females homozygous for the strongly affected class of alleles shows a greater than 90% reduction in the numbers of coated pits and coated vesicles. These oocytes have very little proteinaceous yolk, and the females accumulate vitellogenin (the yolk protein precursor) within their hemolymph. Moreover, females homozygous or hemizygous for a given strong allele produce mature oocytes that are flaccid. Alternatively, females homozygous or hemizygous for weak alleles produce yolk-filled oocytes, but the number of coated pits and coated vesicles within these oocytes is 50% of that found in the oocytes of wild-type females. Despite the presence of yolk within these oocytes, females homozygous for weak yl- alleles remain sterile, and their mature oviposited eggs collapse with time.
PMCID: PMC2114887  PMID: 2886508
19.  The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation 
Molecular Biology of the Cell  2009;20(16):3763-3771.
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.
doi:10.1091/mbc.E08-09-0967
PMCID: PMC2777935  PMID: 19535457
20.  Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana 
Molecular Biology of the Cell  2013;24(4):510-520.
The exocyst complex localizes to distinct foci at the plasma membrane of Arabidopsis thaliana cells. Their localization at the plasma membrane is insensitive to BFA treatment but is decreased in an exocyst-subunit mutant. In turn, exocyst-subunit mutants show decreased exocytosis.
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.
doi:10.1091/mbc.E12-06-0492
PMCID: PMC3571873  PMID: 23283982
21.  Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila 
Molecular Biology of the Cell  2010;21(9):1546-1555.
This study identifies phosphoinositides as key regulators of spermatid cell polarity. Polarization and elongation of spermatids in Drosophila are regulated through local synthesis of PIP2 by Sktl, which drives polarized localization of the exocyst complex to promote targeted membrane delivery and polarization of the elongating spermatid cysts.
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.
doi:10.1091/mbc.E09-07-0582
PMCID: PMC2861613  PMID: 20237161
22.  The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth 
Molecular Biology of the Cell  2014;25(8):1312-1326.
The Neurospora crassa exocyst presents two distinct localization patterns. EXO-70 and -84 colocalize with a region of the Spitzenkörper occupied by secretory macrovesicles. In contrast, SEC-3, -5, -6, -8, and -15 localize distinctively at the apical plasma membrane.
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.
doi:10.1091/mbc.E13-06-0299
PMCID: PMC3982996  PMID: 24523289
23.  Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion 
Molecular Biology of the Cell  2009;20(3):973-982.
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.
doi:10.1091/mbc.E08-09-0968
PMCID: PMC2633393  PMID: 19073882
24.  Vesicular Stomatitis Virus Enters Cells through Vesicles Incompletely Coated with Clathrin That Depend upon Actin for Internalization 
PLoS Pathogens  2009;5(4):e1000394.
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.
Author Summary
Clathrin-dependent endocytosis accounts for the majority of uptake from the plasma membrane. However, many viruses that infect cells through an endocytic route are larger than the dimensions of a typical clathrin-coated vesicle. Working with vesicular stomatitis virus, we determined how this cargo enters cells. We present evidence that VSV induces its own uptake by the clathrin-dependent endocytic machinery following binding to the plasma membrane. The clathrin-coated vesicles that contain virus differ from vesicles that internalize conventional clathrin dependent cargo such as LDL and transferrin. Specifically, we show that VSV particles are internalized by vesicles that are only partially coated with clathrin, rather than the complete coat found on conventional vesicles. We show that the clathrin-dependent endocytic adaptor AP-2 is required for entry. Finally, we show for the first time that actin is recruited to virus-containing pits and that particle internalization depends upon actin function. Our work provides new mechanistic insights into VSV entry that may be directly relevant in understanding the clathrin dependent uptake of other viruses.
doi:10.1371/journal.ppat.1000394
PMCID: PMC2667253  PMID: 19390604
25.  Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits 
eLife  2014;3:e03311.
Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.
DOI: http://dx.doi.org/10.7554/eLife.03311.001
eLife digest
Clathrin-dependent endocytosis is one of the mechanisms used by cells to internalize specific proteins (cargo) from their surface. First, the cargo interacts with adaptor proteins that help cluster them in the cell's outer membrane, called the plasma membrane. This causes the protein clathrin to assemble into a lattice at the cytosolic side of the plasma membrane and deform the membrane into a pit. The pit grows deeper over time as more clathrin molecules assemble, eventually resulting in a deeply invaginated clathrin-coated pit that encloses the cargo to be taken up by the cell. The clathrin-coated pit then pinches off inside the cell in a process called fission to form a bubble-like structure called a vesicle, which transports the molecule to its destination.
The deep invagination of clathrin-coated pits that leads to fission is assisted by actin, a protein that assembles into filaments that are suggested to generate the forces needed for this process. Many other factors are also involved. One of them is epsin, the collective name for a family of three very similar proteins in mammalian cells. Epsin binds to several other proteins implicated in clathrin-dependent endocytosis, including clathrin itself, and to plasma membrane proteins specifically ‘tagged’ for internalization. In addition, a portion of the epsin molecule can insert into the plasma membrane and help it to curve, which is important for forming the invaginated pit. However, due to the number of possible functions epsin could perform, its main role has remained elusive.
Messa et al. created mouse cells that lack all three epsin proteins. Although these cells can form clathrin-coated pits, they struggle to develop into vesicles. The normal linking of the actin filaments to the clathrin coat does not occur, and another protein called Hip1R that also participates in clathrin-mediated endocytosis and links clathrin to actin, no longer accumulates at the clathrin-coated pits. Messa et al. also find that epsins can bind directly to actin. Overall, these results suggest that a main role of epsin is to help actin interact with the clathrin-coated pits and generate the force required for a pit to develop into a vesicle. However, epsin also performs many other roles, including recruiting a membrane protein (a so-called SNARE) that directs the fate of the vesicle to the clathrin-coated pit.
Additionally, Messa et al. find that cells lacking all three epsins have problems dividing correctly. More research is required to establish whether this effect is also due to epsin's interaction with the cell's actin cytoskeleton.
DOI: http://dx.doi.org/10.7554/eLife.03311.002
doi:10.7554/eLife.03311
PMCID: PMC4161027  PMID: 25122462
epsin; actin; clathrin-mediated endocytosis; SNARE; Hip1R; cytokinesis; mouse

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