The exocyst complex in polarized exocytosis

doi:10.1016/j.ceb.2009.04.007

Current Opinion in Cell Biology

Volume 21, Issue 4, August 2009, Pages 537-542

https://doi.org/10.1016/j.ceb.2009.04.007 Get rights and content

The exocyst is an octameric protein complex, which mediates the tethering of post-Golgi secretory vesicles to the plasma membrane before exocytic fusion. The exocyst assembles by side-by-side packing of rod-shaped subunits composed of helical bundles. The targeting of secretory vesicles to the plasma membrane involves direct interactions of the exocyst with PI(4,5)P₂. In addition, a number of small GTP-binding proteins interact with components of the exocyst and regulate the assembly, localization, and function of this complex. Here we review the recent advances in the field, focusing on the function of the exocyst in polarized exocytosis.

Section snippets

The structure of the exocyst

Understanding the structure of the exocyst may provide important insight into the molecular mechanism of the exocyst function. So far, partial crystal structures of four exocyst components have been solved [3]. These include the near full-length yeast and mouse Exo70 [4••, 5, 6], and the C-terminal domains of Drosophila Sec15 [7], yeast Exo84 [4^••], and yeast Sec6 [8^••]. While these exocyst components share little sequence homology, they all display rod-like structures composed of two or more

Polarized localization and activation of the exocyst at the plasma membrane

In order to understand how the exocyst tethers vesicles at the plasma membrane, it is important to elucidate how the exocyst itself is targeted to the plasma membrane. In yeast, although all the exocyst components are localized to the growing end of the daughter cells (‘bud tip’), their targeting involves different mechanisms. Sec3 is localized to the bud tip independent of actin cables, along which the vesicles are transported [12•, 13••]. Exo70 polarization seems to be partially

Communication with the SNAREs

Vesicle tethering precedes fusion. In exocyst mutants, assembly of the SNARE complex is blocked [25]. The exocyst is likely to interact with the SNAREs and regulate SNARE assembly. It was reported that the brain exocyst complex co-immunoprecipitated with syntaxin [2^••]. Yeast Sec6 was shown to interact with the t-SNARE Sec9 in vitro and may inhibit Sec9 interaction with the other t-SNARE protein Sso1 [26]. The exocyst was also found to co-immunoprecipitate with Sec1 [27], a Sec1/Munc18 (SM)

Function of the exocyst in exocytosis and beyond

In yeast, the exocyst mutants exhibit blockages of secretion and show intracellular accumulation of secretory vesicles [34••, 35, 15, 36•]. In animal cells, the role of the exocyst in exocytosis and cell surface expansion has been demonstrated in a variety of cell types, such as protein targeting in epithelial cells [37••, 38•], dendritic delivery of NMDA receptors at postsynaptic membranes [39], and insulin-induced exocytosis of glucose transporter in adipocytes [40^•]. It is worth noting that

Regulation of the exocyst by small GTP-binding proteins

Functioning at a step before SNARE-mediated fusion, the exocyst is a target of a number of regulators that spatially and kinetically regulate exocytosis in cells. In particular, several small GTP-binding proteins directly interact with the exocyst. In yeast, Sec15 is a downstream effector of the Rab GTPase Sec4, which regulates the assembly of the exocyst complex [46^••]. In higher eukaryotes, Sec15 is a downstream effector of the Rab GTPase Rab11, which regulates vesicle transport to the plasma

Future perspectives

Recent decade saw exciting progresses toward our understanding of the exocyst. However, a number of important questions remain unanswered: What are the kinetics of exocyst assembly and disassembly? How are the exocyst components associated with secretory vesicles? While it is thought that the exocyst serves as a vesicle tether, can the tethering step be observed and characterized by microscopy? Is the exocyst just a physical tether or does it also activate the assembly of the SNARE complex? Is

References and recommended reading

Papers of particular interest published within the period of review have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

Owing to space limitations and the focus on exocytosis in this review, we were unable to provide a complete survey of the field, especially exciting new progresses related to cytokinesis, cell migration, and development. We apologize for any references we may have left out. Research in Wei Guo's lab is supported by grants from the National Institutes of Health, American Heart Association, and the Pew Scholars Program.

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CpEXO70A1/3 regulate root development via affecting auxin accumulation in Chinese herbal plant Codonopsis pilosula
2024, Scientia Horticulturae
Codonopsis pilosula is an herbal plant that belongs to the Campanulaceae family, the dried roots (Dangshen) of which have long been used in traditional Chinese medicine to treat dyspepsia, hypertension, and hyperglycemia. The development of roots is very crucial for producing high-quality Dangshen due to their main medicinal components. However, i the regulation mechanism of root development remains limited. Here, we identified two subunits CpEXO70A1 and CpEXO70A3, of the evolutionarily conserved exocyst complex in C. pilosula. Either CpEXO70A1 or CpEXO70A3 responded to IAA, ABA, and drought treatments. The subcellular localization and GUS-staining assay indicated that CpEXO70A1 and CpEXO70A3 were located in the plasma membrane and exhibited temporal and spatial expression patterns. Overexpression or CRISPR knock-out of the two genes led to reduced biomass in C. pilosula hairy roots, correlating with changes in auxin concentration. Besides, a model predicting the impact of auxin-PIN-EXO70A3 onon root growth and development was proposed. Our study aims to enhance Dangshen yield through molecular breeding in the future.
Exocyst complex component 2 is a potential host factor for SARS-CoV-2 infection
2022, iScience
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused an epidemic and spread rapidly all over the world. Because the analysis of host factors other than receptors and proteases has not been sufficiently performed, we attempted to identify and characterize host factors essential for SARS-CoV-2 infection using iPS cells and airway organoids (AO). Based on previous CRISPR screening and RNA-seq data, we found that exocyst complex component 2 (EXOC2) is one important host factor for SARS-CoV-2 infection. The intracellular SARS-CoV-2 nucleocapsid (N) expression level was decreased to 3.7% and the virus copy number in cell culture medium was decreased to 1.6% by EXOC2 knockdown. Consistently, immunostaining results showed that N protein-positive cells were significantly decreased by EXOC2 knockdown. We also found that EXOC2 knockdown downregulates SARS-CoV-2 infection by regulating IFNW1 expression. In conclusion, controlling the EXOC2 expression level may prevent SARS-CoV-2 infection and deserves further study.
A mechanism for exocyst-mediated tethering via Arf6 and PIP5K1C-driven phosphoinositide conversion
2022, Current Biology
Citation Excerpt :
Exocyst-regulatory factors are linked to phosphoinositide signaling, most notably evidenced by Rab1114 and Arf6.15–19 Indeed, exocyst conservation20 and connection of lipid kinases to polarized secretion21–25 suggest a similar interdependence of phosphoinositides and exocyst function. Phosphoinositides are a family of phospholipids that are signposts for membrane identity.26
Polarized trafficking is necessary for the development of eukaryotes and is regulated by a conserved molecular machinery. Late steps of cargo delivery are mediated by the exocyst complex, which integrates lipid and protein components to tether vesicles for plasma membrane fusion. However, the molecular mechanisms of this process are poorly defined. Here, we reconstitute functional octameric human exocyst, demonstrating the basis for holocomplex coalescence and biochemically stable subcomplexes. We determine that each subcomplex independently binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), which is minimally sufficient for membrane tethering. Through reconstitution and epithelial cell biology experiments, we show that Arf6-mediated recruitment of the lipid kinase PIP5K1C rapidly converts phosphatidylinositol 4-phosphate (PI(4)P) to PI(4,5)P₂, driving exocyst recruitment and membrane tethering. These results provide a molecular mechanism of exocyst-mediated tethering and a unique functional requirement for phosphoinositide signaling on late-stage vesicles in the vicinity of the plasma membrane.
Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration
2022, iScience
Citation Excerpt :
Exoc6 is one of the subunits of the exocyst complex. This complex is required for polarized exocytosis and mediates the tethering of post-Golgi secretory vesicles to the plasma membrane before exocytic fusion (He and Guo, 2009; Mei and Guo, 2018). The exocyst complex is also important for cell migration, as it is implicated in the delivery of proteins, including adhesion molecules and proteases for extracellular matrix (ECM) degradation, to the leading edge of migrating cells (Liu and Guo, 2012).
Rab proteins are well known regulators of intracellular trafficking; however, more and more studies point to their function also in other cellular processes, including cell migration. In this work, we have performed an siRNA screen to identify Rab proteins that influence cell migration. The screen revealed Rab33b as the strongest candidate that affected cell motility. Rab33b has been previously reported to localize at the Golgi apparatus to regulate Golgi-to-ER retrograde trafficking and Golgi homeostasis. We revealed that Rab33b also mediates post-Golgi transport to the plasma membrane. We further identified Exoc6, a subunit of the exocyst complex, as an interactor of Rab33b. Moreover, our data indicate that Rab33b regulates focal adhesion dynamics by modulating the delivery of cargo such as integrins to focal adhesions. Altogether, our results demonstrate a role for Rab33b in cell migration by regulating the delivery of integrins to focal adhesions through the interaction with Exoc6.
Arabidopsis exocyst subunit SEC6 is involved in cell plate formation during Microgametogenesis
2022, Biochemical and Biophysical Research Communications
Cytokinesis during pollen mitosis I is critical for cell division and differentiation in the male gametophyte development, but the vesicle trafficking mechanisms in this process are largely unknown. Exocyst is an octameric tethering complex which plays multiple important roles in plant cell vesicle trafficking. Here we report the characterization of exocyst subunit SEC6 in the cytokinesis during pollen mitosis I. We found that significantly amount of pollen from two sec6/+ mutant alleles arrested at the transition from unicelluar stage microspore to bicellular stage. Further analysis showed that sec6 mutation impaired cell plate formation and led to vesicles accumulation in cytoplasm. The localization of KNOLLE on the cell plate was compromised. Consistently, SEC6 gene was expressed start from early pollen development stage and SEC6-GFP localized to the cell plate. These results indicated that SEC6 participated in the cell plate formation during pollen mitosis I, where it might help to tether the vesicles before fusion.
The small GTPase RABA2a recruits SNARE proteins to regulate the secretory pathway in parallel with the exocyst complex in Arabidopsis
2022, Molecular Plant
Citation Excerpt :
Together, these results support the notion that neither the GTPase RABA2a nor the downstream effector SNARE proteins interact with the exocyst complex, which is consistent with our MS analysis. The evolutionarily conserved Ypt31/32-Sec2p-Sec4p-exocyst pathway in yeast and the Rab11-Rabin8-Rab8-exocyst pathway in mammals form a GTPase activation cascade to coordinate vesicle transport with membrane tethering at the PM (Feng et al., 2012; He and Guo, 2009; Knodler et al., 2010; Ortiz et al., 2002; Sultana et al., 2011). In a comparable pathway in plants, the DENN domain-containing proteins SCD1 and SCD2 directly interact with both RABE1 GTPases and SEC15b (Mayers et al., 2017).
Delivery of proteins to the plasma membrane occurs via secretion, which requires tethering, docking, priming, and fusion of vesicles. In yeast and mammalian cells, an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane. However, it is unclear whether this is the case in plants. In this study, we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion. Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays, we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a. Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex. Intriguingly, we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments. Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly, suggesting that the RABA2a-SNARE- and exocyst-mediated secretory pathways are largely independent. Consistently, our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes, and genetic and cell biologyanalyses indicate that the two pathways select different cargos. Finally, we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings. Collectively, our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.

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The exocyst complex in polarized exocytosis

Section snippets

The structure of the exocyst

Polarized localization and activation of the exocyst at the plasma membrane

Communication with the SNAREs

Function of the exocyst in exocytosis and beyond

Regulation of the exocyst by small GTP-binding proteins

Future perspectives

References and recommended reading

Acknowledgements

Nat Struct Mol Biol

Nat Struct Mol Biol

Dev Cell

J Cell Biol

Mol Biol Cell

J Biol Chem

Mol Biol Cell

J Cell Biol

J Cell Biol

Neuron

J Cell Biol

J Biol Chem

Mol Biol Cell

The exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae

EMBO J

The mammalian brain rsec6/8 complex

Neuron

The structures of exocyst subunit Exo70p and the Exo84p C-terminal domains reveal a common motif

Nat Struct Mol Biol

Crystal structure of the S. cerevisiae exocyst component Exo70p

J Mol Biol

The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst

J Mol Biol

Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo

Nat Struct Mol Biol

Structural analysis of conserved oligomeric Golgi complex subunit 2

J Biol Chem

Subunit composition, protein interactions, and structures of the mammalian brain sec6/8 complex and septin filaments

Neuron

Sec3p is a spatial landmark for polarized secretion in budding yeast

Cell

Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane

EMBO J

Membrane association and functional regulation of Sec3 by phospholipids and Cdc42

J Cell Biol

Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p

EMBO J

Cdc42 interacts with the exocyst and regulates polarized secretion

J Biol Chem