The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation

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.     

植物EXO70基因家族的研究进展

胞吐是存在于所有真核生物的一种极其重要的细胞活动,直接参与了激素和神经信号的分泌、细胞生长、细胞极性的建立,细胞分裂和细胞壁的形成等多项生理过程。在胞吐过程中,高尔基后转运膜泡与靶膜的识别是由进化上高度保守的胞泌复合体（exocyst）介导的。该复合体由8个蛋白亚基构成,其中EXO70是组成胞泌复合体功能的关键亚基,可与小G蛋白和膜脂互作,参与复合体在靶膜组装。目前,对植物胞泌复合体功能的了解非常有限,已有证据显示其广泛参与了细胞生长,细胞壁形成、细胞分裂等多种生物学过程。与酵母和动物相比,植物胞泌复合体的一个显著特征是：EXO70在高等植物基因组中存在多个同源基因,其具体生物学功能尚不清楚。本文综述胞泌复合体的研究进展,重点讨论植物EXO70的多基因家族,推测不同的EXO70可能参与了组织细胞或运载底物特异的膜泡转运过程。     

RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis

Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance.In flowering plants, sexual reproduction occurs as a result of constant communication between the male gametophyte and the female reproductive organ, from the initial acceptance of compatible pollen to final step of successful fertilization (for review, see Beale and Johnson, 2013; Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015). In the Brassicaceae, the stigmas that present a receptive surface for pollen are categorized as dry and covered with unicellular papillae (Heslop-Harrison and Shivanna, 1977). Communication is initiated rapidly following contact of a pollen grain with a stigmatic papilla, as the role of the papillae is to regulate the early cellular responses leading to compatible pollen germination. The basal compatible pollen recognition response also presents a barrier to foreign pollen or is inhibited with self-incompatible pollen (for review, see Dickinson, 1995; Hiscock and Allen, 2008; Chapman and Goring, 2010; Indriolo et al., 2014b).The initial adhesive interaction between the pollen grain and the papilla cell in the Brassicaceae is mediated by the exine of the pollen grain and the surface of the stigmatic papilla (Preuss et al., 1993; Zinkl et al., 1999). A stronger connection results between the adhered pollen grain and the stigmatic papilla with the formation of a lipid-protein interface (foot) derived from the pollen coat and the stigmatic papillar surface (Mattson et al., 1974; Stead et al., 1980; Gaude and Dumas, 1986; Elleman and Dickinson, 1990; Elleman et al., 1992; Preuss et al., 1993; Mayfield et al., 2001). It is at this point that a Brassicaceae-specific recognition of compatible pollen is proposed to occur (Hülskamp et al., 1995; Pruitt, 1999), though the nature of this recognition system is not clearly defined. Two stigma-specific Brassica oleracea glycoproteins, the S-Locus Glycoprotein and S-Locus Related1 (SLR1) protein, play a role in compatible pollen adhesion (Luu et al., 1997, 1999), potentially through interactions with the pollen coat proteins, PCP-A1 and SLR1-BP, respectively (Doughty et al., 1998; Takayama et al., 2000). The simultaneous recognition of self-incompatible pollen would also take place at this stage (for review, see Dresselhaus and Franklin-Tong, 2013; Indriolo et al., 2014b; Sawada et al., 2014). Thus, this interface not only provides a strengthened bond between the pollen grain and stigmatic papilla, but likely facilitates the interaction of signaling proteins from both partners to promote specific cellular responses in the stigmatic papilla toward the pollen grain.One response regulated by these interactions is the release of water from the stigmatic papilla to the adhered compatible pollen grain to enable the pollen grain to rehydrate, germinate, and produce a pollen tube (Zuberi and Dickinson, 1985; Preuss et al., 1993). Upon hydration, the pollen tube emerges at the site of pollen-papilla contact and penetrates the stigma surface between the plasma membrane and the overlaying cell wall (Elleman et al., 1992; Kandasamy et al., 1994). Pollen tube entry into the stigmatic surface represents a second barrier, selecting compatible pollen tubes. Subsequently, the compatible pollen tubes traverse down to the base of the stigma, enter the transmitting tract, and grow intracellularly toward ovules for fertilization. Pollen-pistil interactions at these later stages are also highly regulated (for review, see Beale and Johnson, 2013; Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015).EXO70A1, a subunit of the exocyst, was identified as a factor involved in early pollen-stigma interactions, where it is required in the stigma for the acceptance of compatible pollen and inhibited by the self-incompatibility response (Samuel et al., 2009). Stigmas from the Arabidopsis (Arabidopsis thaliana) exo70A1 mutant display constitutive rejection of wild-type-compatible pollen (Samuel et al., 2009; Safavian et al., 2014). This stigmatic defect was rescued by the stigma-specific expression of an Red Fluorescent Protein (RFP):EXO70A1 transgene (Samuel et al., 2009) or partially rescued by providing a high relative humidity environment (Safavian et al., 2014). In addition, the stigma-specific expression of an EXO70A1 RNA interference construct in Brassica napus ‘Westar’ resulted in impaired compatible pollen acceptance and a corresponding reduction in seed production compared with compatible pollinations with wild-type B. napus ‘Westar’ pistils (Samuel et al., 2009). From these studies, EXO70A1 was found to be a critical component in stigmatic papillae to promote compatible pollen hydration and pollen tube entry through the stigma surface. One of the functions of the exocyst is to mediate polar secretion (for review, see Heider and Munson, 2012; Zárský et al., 2013; Synek et al., 2014). Consistent with this, previous studies have observed vesicle-like structures in proximity to the stigmatic papillar plasma membrane in response to compatible pollen in both Brassica spp. and Arabidopsis species (Elleman and Dickinson, 1990, 1996; Dickinson, 1995; Safavian and Goring, 2013; Indriolo et al., 2014a). The secretory activity is predicted to promote pollen hydration and pollen tube entry. As well, consistent with the proposed inhibition of EXO70A1 by the self-incompatibility pathway (Samuel et al., 2009), a complete absence or a significant reduction of vesicle-like structures at the stigmatic papillar plasma membrane was observed in the exo70A1 mutant and with self-incompatible pollen (Safavian and Goring, 2013; Indriolo et al., 2014a).The exocyst is a well-defined complex in yeast (Saccharomyces cerevisiae) and animal systems, consisting of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Exocyst subunit mutants were first identified in yeast as secretory mutants displaying a cytosolic accumulation of secretory vesicles (Novick et al., 1980). Subsequent work defined roles for the exocyst in vesicle docking at target membranes in processes such as regulated secretion, polarized exocytosis, and cytokinesis to facilitate membrane fusion by Soluble NSF Attachment protein Receptor (SNARE) complexes (for review, see Heider and Munson, 2012; Liu and Guo, 2012). In plants, genes encoding all eight exocyst subunits have been identified, and many of these genes exist as multiple copies. For example, the Arabidopsis genome contains single copy genes for SEC6 and SEC8, two copies each for SECRETORY3 (SEC3), SEC5, SEC10, and SEC15, three EXO84 genes, and 23 EXO70 genes (Chong et al., 2010; Cvrčková et al., 2012; Vukašinović et al., 2014). Ultrastructural studies using electron tomography uncovered the existence of a structure resembling the exocyst in Arabidopsis (Otegui and Staehelin, 2004; Seguí-Simarro et al., 2004). Localization studies of specific Arabidopsis exocyst subunits also supported conserved roles in polarized exocytosis and cytokinesis in plants. Localization studies have shown EXO70, SEC6, and SEC8 at the growing tip of pollen tubes (Hála et al., 2008), EXO70A1 at the stigmatic papillar plasma membrane (Samuel et al., 2009), SEC3a, SEC6, SEC8, SEC15b, EXO70A1, and EXO84b at the root epidermal cell plasma membrane and developing cell plate (Fendrych et al., 2010, 2013; Wu et al., 2013; Zhang et al., 2013; Rybak et al., 2014), and SEC3a at the plasma membrane in the embryo and root hair (Zhang et al., 2013). Similar to the yeast exocyst mutants, vesicle accumulation has also been observed in the exo70A1 and exo84b mutants (Fendrych et al., 2010; Safavian and Goring, 2013). Taken together, these findings strongly support that plant exocyst subunits function in vivo in vesicle docking at sites of polarized secretion and cytokinesis (for review, see Zárský et al., 2013). In support of this, a recent study investigating Transport Protein Particle (TRAPP)II and exocyst complexes during cytokinesis in Arabidopsis has identified all eight exocyst components in immunoprecipitated complexes (SEC3a/SEC3b, SEC5a, SEC6, SEC8, SEC10, SEC15b, EXO70A1, EXO70H2, and EXO84b; Rybak et al., 2014).Several plant exocyst subunit genes have been implicated in biological processes that rely on regulated vesicle trafficking, where corresponding mutants have displayed a range of growth defects. At the cellular level, these phenotypes have been associated with decreased cell elongation and polar growth (Cole et al., 2005, 2014; Wen et al., 2005; Synek et al., 2006), defects in cytokinesis and cell plate formation (Fendrych et al., 2010; Wu et al., 2013; Rybak et al., 2014), and disrupted Pin-Formed (PIN) auxin efflux carrier recycling and polar auxin transport (Drdová et al., 2013). Several Arabidopsis subunit mutants display strong growth defects such as the sec3a mutant with an embryo-lethal phenotype (Zhang et al., 2013), sec6, sec8, and exo84b mutants with severely dwarfed phenotypes and defects in root growth (Fendrych et al., 2010; Wu et al., 2013; Cole et al., 2014), and exo70A1 with a milder dwarf phenotype (Synek et al., 2006). The Arabidopsis exo70A1 mutant has also been reported to have defects in root hair elongation, hypocotyl elongation, compatible pollen acceptance, seed coat deposition, and tracheary element differentiation (Synek et al., 2006; Samuel et al., 2009; Kulich et al., 2010; Li et al., 2013). Essential roles for other exocyst subunits include Arabidopsis SEC5a/SEC5b, SEC6, SEC8, and SEC15a/SEC15b in male gametophyte development and pollen tube growth (Cole et al., 2005; Hála et al., 2008; Wu et al., 2013), SEC8 in seed coat deposition (Kulich et al., 2010), SEC5a, SEC8, EXO70A1, and EXO84b in root meristem size and root cell elongation (Cole et al., 2014), and a maize (Zea mays) SEC3 homolog in root hair elongation (Wen et al., 2005). Finally, the Arabidopsis EXO70B1, EXO70B2, and EXO70H1 subunits have been implicated in plant defense responses (Pecenková et al., 2011; Stegmann et al., 2012; Kulich et al., 2013; Stegmann et al., 2013).Even with these detailed studies on the functions of exocyst subunits in plants, a systematic demonstration of the requirement of all eight exocyst subunits in a specific plant biological process is currently lacking. EXO70A1 was previously identified as an essential factor in the stigma for compatible pollen-pistil interactions in Arabidopsis and B. napus (Samuel et al., 2009), and we hypothesized that this protein functions as part of the exocyst complex to tether post-Golgi secretory vesicles to stigmatic papillar plasma membrane (Safavian and Goring, 2013). To provide support for the proposed biological role of the exocyst in the stigma for compatible pollen acceptance, we investigated the roles of the remaining seven subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmatic papillae. Given that some Arabidopsis exocyst subunits were previously determined to be essential at earlier growth stages, stigma-specific RNA-silencing constructs were used for each exocyst subunit, and the early postpollination stages were analyzed for these transgenic lines. Our collective data demonstrates that all eight exocyst subunits are required in the stigma for the early stages of compatible pollen-pistil interactions.     

An exocyst complex functions in plant cell growth in Arabidopsis and tobacco

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.     

The Candida albicans Exocyst Subunit Sec6 Contributes to Cell Wall Integrity and Is a Determinant of Hyphal Branching

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans.     

Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion

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.     

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.     

Analysis of exocyst function in endodermis reveals its widespread contribution and specificity of action

The exocyst is the main plasma membrane vesicle-tethering complex in eukaryotes and is composed of eight different subunits. Yet, in plant genomes, many subunits display multiple copies, thought to reflect evolution of complex subtypes with divergent functions. In Arabidopsis thaliana root endodermal cells, the isoform EXO70A1 is required for positioning of CASP1 at the Casparian Strip Domain, but not for its non-targeted secretion to the plasma membrane. Here, we show that exo84b resembles exo70a1 mutants regarding CASP1 mistargeting and secretion of apoplastic proteins, but exo84b additionally affects secretion of other integral plasma membrane proteins. Moreover, conditional, cell-type-specific gene editing of the single-copy core component SEC6 allows visualization of secretion defects in plant cells with a complete lack of exocyst complex function. Our approach opens avenues for deciphering the complexity/diversity of exocyst functions in plant cells and enables analysis of central trafficking components with lethal phenotypes.

Genetic analysis of exocyst isoforms reveals their distinct roles in cargo secretion.     

Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit

The exocyst is a well‐known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast‐two‐hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1‐PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3‐GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle‐tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P₂ in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit‐specific function, and provided data and tools for further characterization of PATL family proteins.     

Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.     

In Candida albicans,phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension

The exocyst, a conserved multiprotein complex, tethers secretory vesicles before fusion with the plasma membrane; thus it is essential for cell surface expansion. In both Saccharomyces cerevisiae and mammalian cells, cell surface expansion is halted during mitosis. In S. cerevisiae, phosphorylation of the exocyst component Exo84 by Cdk1-Clb2 during mitosis causes the exocyst to disassemble. Here we show that the hyphae of the human fungal pathogen Candida albicans continue to extend throughout the whole of mitosis. We show that CaExo84 is phosphorylated by Cdk1, which is necessary for efficient hyphal extension. This action of Cdk1 depends on the hyphal-specific cyclin Hgc1, the homologue of G1 cyclins in budding yeast. Phosphorylation of CaExo84 does not alter its localization but does alter its affinity for phosphatidylserine, allowing it to recycle at the plasma membrane. The different action of Cdk1 on CaExo84 and ScExo84 is consistent with the different locations of the Cdk1 target sites in the two proteins. Thus this conserved component of polarized growth has evolved so that its phosphoregulation mediates the dramatically different patterns of growth shown by these two organisms.     

Arabidopsis Exocyst Subcomplex Containing Subunit EXO70B1 Is Involved in Autophagy‐Related Transport to the Vacuole

Autophagic transport to the vacuole represents an endomembrane trafficking route, which is widely used in plants, not only during stress situations, but also for vacuole biogenesis and during developmental processes. Here we report a role in autophagic membrane transport for EXO70B1—one of 23 paralogs of Arabidopsis EXO70 exocyst subunits. EXO70B1 positive compartments are internalized into the central vacuole and co‐localize with autophagosomal marker ATG8f. This internalization is boosted by induction of autophagy. Loss of function (LOF) mutations in exo70B1 cause reduction of internalized autopagic bodies in the vacuole. Mutant plants also show ectopic hypersensitive response (HR) mediated by salicylic acid (SA) accumulation, increased nitrogen starvation susceptibility and anthocyanin accumulation defects. Anthocyanin accumulation defect persists in npr1x exo70B1 double mutants with SA signaling compromised, while ectopic HR is suppressed. EXO70B1 interacts with SEC5 and EXO84 and forms an exocyst subcomplex involved in autophagy‐related, Golgi‐independent membrane traffic to the vacuole. We show that EXO70B1 is functionally completely different from EXO70A1 exocyst subunit and adopted a specific role in autophagic transport .     

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.     

SEC1A and SEC6 synergistically regulate pollen tube polar growth

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles. The exocyst and Sec1/Munc18 (SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through co-immunoprecipitation-mass spectrometry screening, protein interaction analysis and in vivo microscopy, we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.     

Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission

The terminal step in cytokinesis, called abscission, requires resolution of the membrane connection between two prospective daughter cells. Our previous studies demonstrated that the coiled-coil protein centriolin localized to the midbody during cytokinesis and was required for abscission. Here we show that centriolin interacts with proteins of vesicle-targeting exocyst complexes and vesicle-fusion SNARE complexes. These complexes require centriolin for localization to a unique midbody-ring structure, and disruption of either complex inhibits abscission. Exocyst disruption induces accumulation of v-SNARE-containing vesicles at the midbody ring. In control cells, these v-SNARE vesicles colocalize with a GFP-tagged secreted polypeptide. The vesicles move to the midbody ring asymmetrically from one prospective daughter cell; the GFP signal is rapidly lost, suggesting membrane fusion; and subsequently the cell cleaves at the site of vesicle delivery/fusion. We propose that centriolin anchors protein complexes required for vesicle targeting and fusion and integrates membrane-vesicle fusion with abscission.     

Roles of the TRAPP-II Complex and the Exocyst in Membrane Deposition during Fission Yeast Cytokinesis

The cleavage-furrow tip adjacent to the actomyosin contractile ring is believed to be the predominant site for plasma-membrane insertion through exocyst-tethered vesicles during cytokinesis. Here we found that most secretory vesicles are delivered by myosin-V on linear actin cables in fission yeast cytokinesis. Surprisingly, by tracking individual exocytic and endocytic events, we found that vesicles with new membrane are deposited to the cleavage furrow relatively evenly during contractile-ring constriction, but the rim of the cleavage furrow is the main site for endocytosis. Fusion of vesicles with the plasma membrane requires vesicle tethers. Our data suggest that the transport particle protein II (TRAPP-II) complex and Rab11 GTPase Ypt3 help to tether secretory vesicles or tubulovesicular structures along the cleavage furrow while the exocyst tethers vesicles at the rim of the division plane. We conclude that the exocyst and TRAPP-II complex have distinct localizations at the division site, but both are important for membrane expansion and exocytosis during cytokinesis.     

Exo84p is an exocyst protein essential for secretion.

The exocyst is a multiprotein complex that plays an important role in secretory vesicle targeting and docking at the plasma membrane. Here we report the identification and characterization of a new component of the exocyst, Exo84p, in the yeast Saccharomyces cerevisiae. Yeast cells depleted of Exo84p cannot survive. These cells are defective in invertase secretion and accumulate vesicles similar to those in the late sec mutants. Exo84p co-immunoprecipitates with the exocyst components, and a portion of the Exo84p co-sediments with the exocyst complex in velocity gradients. The assembly of Exo84p into the exocyst complex requires two other subunits, Sec5p and Sec10p. Exo84p interacts with both Sec5p and Sec10p in a two-hybrid assay. Overexpression of Exo84p selectively suppresses the temperature sensitivity of a sec5 mutant. Exo84p specifically localizes to the bud tip or mother/daughter connection, sites of polarized secretion in the yeast S. cerevisiae. Exo84p is mislocalized in a sec5 mutant. These studies suggest that Exo84p is an essential protein that plays an important role in polarized secretion.     

Prospore Membrane Formation Defines a Developmentally Regulated Branch of the Secretory Pathway in Yeast

Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.