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Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.

Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-β pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific multisubunit tethering complex, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We have demonstrated previously that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1-Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. To map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly but, rather, binds both the binary Sec9-Sso1 and ternary Sec9-Sso1-Snc2 SNARE complexes. Together, these results suggest a new model in which Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.

Primary cilia are microtubule-based solitary membrane projections on the cell surface that play important roles in signaling and development. Recent studies have demonstrated that polarized vesicular trafficking involving the small GTPase Rab8 and its guanine nucleotide exchange factor Rabin8 is essential for primary ciliogenesis. In this study, we show that a highly conserved region of Rabin8 is pivotal for its activation as a guanine nucleotide exchange factor for Rab8. In addition, in its activated conformation, Rabin8 interacts with Sec15, a subunit of the exocyst and downstream effector of Rab8. Expression of constitutively activated Rab8 promotes the association of Sec15 with Rabin8. Using immunofluorescence microscopy, we found that Sec15 co-localized with Rab8 along the primary cilium. Inhibition of Sec15 function in cells led to defects in primary ciliogenesis. The Rabin8-Rab8-Sec15 interaction may couple the activation of Rab8 to the recruitment of the Rab8 effector and is involved in the regulation of vesicular trafficking for primary cilium formation.

Primary cilia are found on many epithelial cell types, including renal tubular epithelial cells, where they participate in flow sensing. Disruption of cilia function has been linked to the pathogenesis of polycystic kidney disease. We demonstrated previously that the exocyst, a highly conserved eight-protein membrane trafficking complex, localizes to primary cilia of renal tubular epithelial cells, is required for ciliogenesis, biochemically and genetically interacts with polycystin-2 (the protein product of the polycystic kidney disease 2 gene), and, when disrupted, results in MAPK pathway activation both in vitro and in vivo. The small GTPase Cdc42 is a candidate for regulation of the exocyst at the primary cilium. Here, we demonstrate that Cdc42 biochemically interacts with Sec10, a crucial component of the exocyst complex, and that Cdc42 colocalizes with Sec10 at the primary cilium. Expression of dominant negative Cdc42 and shRNA-mediated knockdown of both Cdc42 and Tuba, a Cdc42 guanine nucleotide exchange factor, inhibit ciliogenesis in Madin-Darby canine kidney cells. Furthermore, exocyst Sec8 and polycystin-2 no longer localize to primary cilia or the ciliary region following Cdc42 and Tuba knockdown. We also show that Sec10 directly interacts with Par6, a member of the Par complex that itself directly interacts with Cdc42. Finally, we show that Cdc42 knockdown results in activation of the MAPK pathway, something observed in cells with dysfunctional primary cilia. These data support a model in which Cdc42 localizes the exocyst to the primary cilium, whereupon the exocyst then targets and docks vesicles carrying proteins necessary for ciliogenesis.

Insulin regulates cellular glucose uptake by changing the amount of glucose transporter-4 (GLUT4) in the plasma membrane through stimulation of GLUT4 exocytosis. However, how the particular trafficking, tethering, and fusion steps are regulated by insulin is still debated. In a 3T3-L1 adipocyte cell line, the Exocyst complex and its Exo70 subunit were shown to critically affect GLUT4 exocytosis. Here we investigated the effects of Exo70 on tethering and fusion of GLUT4 vesicles in primary isolated rat adipose cells. We found that Exo70 wild type was sequestered away from the plasma membrane in non-stimulated cells, and its overexpression had no effect on GLUT4 trafficking. The addition of insulin increased the amount of Exo70 in the vicinity of the plasma membrane and stimulated the tethering and fusion of GLUT4 vesicles, but the rates of fusion and GLUT4 exposure were not affected by overexpression of Exo70. Surprisingly, the Exo70-N mutant induced insulin-independent tethering of GLUT4 vesicles, which, however, did not lead to fusion and exposure of GLUT4 at the plasma membrane. Upon insulin stimulation, the stationary pretethered GLUT4 vesicles in Exo70-N mutant cells underwent fusion without relocation. Taken together, our data suggest that fusion of GLUT4 vesicles is the rate-limiting step regulated by insulin downstream of Exo70-mediated tethering.

RalA is a small GTPase that is thought to facilitate exocytosis through its direct interaction with the mammalian exocyst complex. In this study, we report an essential role for RalA in regulated insulin secretion from pancreatic beta cells. We employed lentiviral-mediated delivery of RalA short hairpin RNAs to deplete endogenous RalA protein in mouse pancreatic islets and INS-1 beta cells. Perifusion of mouse islets depleted of RalA protein exhibited inhibition of both first and second phases of glucose-stimulated insulin secretion. Consistently, INS-1 cells depleted of RalA caused a severe inhibition of depolarization-induced insulin exocytosis determined by membrane capacitance, including a reduction in the size of the ready-releasable pool of insulin granules and a reduction in the subsequent mobilization and exocytosis of the reserve pool of granules. Collectively, these data suggest that RalA is a critical component in biphasic insulin release from pancreatic beta cells.

The movement of signal transduction enzymes in and out of multi-protein complexes coordinates the spatial and temporal resolution of cellular events. Anchoring and scaffolding proteins are key to this process because they sequester protein kinases and phosphatases with a subset of their preferred substrates. The protein kinase A-anchoring family of proteins (AKAPs), which target the cAMP-dependent protein kinase (PKA) and other enzymes to defined subcellular microenvironments, represent a well studied group of these signal-organizing molecules. In this report we demonstrate that the Rab27a GTPase effector protein MyRIP is a member of the AKAP family. The zebrafish homolog of MyRIP (Ze-AKAP2) was initially detected in a two-hybrid screen for AKAPs. A combination of biochemical, cell-based, and immunofluorescence approaches demonstrate that the mouse MyRIP ortholog targets the type II PKA holoenzyme via an atypical mechanism to a specific perinuclear region of insulin-secreting cells. Similar approaches show that MyRIP interacts with the Sec6 and Sec8 components of the exocyst complex, an evolutionarily conserved protein unit that controls protein trafficking and exocytosis. These data indicate that MyRIP functions as a scaffolding protein that links PKA to components of the exocytosis machinery.

In eukaryotic cells, recycling endosome-mediated trafficking contributes to the completion of cytokinesis, in a manner under the control of the centrosome. We report that the exocyst complex and its interacting GTPase RalA play a critical role in this polarized trafficking process. RalA resides in the recycling endosome and relocates from the pericentrosomal region to key cytokinetic structures including the cleavage furrow, and later, the abscission site. This event is coupled to the dynamic redistribution of the exocyst proteins. These associate with the centrosome in interphase and concentrate on the central spindle/midbody during cytokinesis. Disruption of RalA-exocyst function leads to cytokinesis failure in late stages, particularly abscission, resembling the cytokinesis defects induced by loss of centrosome function. These data suggest that RalA and the exocyst may regulate vesicle delivery to the centrosome-related abscission site during the terminal stage of cytokinesis, implicating RalA as a critical regulator of cell cycle progression.

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.

Glucose stimulates insulin secretion from pancreatic beta cells by inducing the recruitment and fusion of insulin vesicles to the plasma membrane. However, little is currently known about the mechanism of the initial docking or tethering of insulin vesicles prior to fusion. Here, we examined the role of the SEC6-SEC8 (exocyst) complex, implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane in yeast and in regulating glucose-stimulated insulin secretion from pancreatic MIN6 beta cells. We show first that SEC6 is concentrated on insulin-positive vesicles, whereas SEC5 and SEC8 are largely confined to the cytoplasm and the plasma membrane, respectively. Overexpression of truncated, dominant-negative SEC8 or SEC10 mutants decreased the number of vesicles at the plasma membrane, whereas expression of truncated SEC6 or SEC8 inhibited overall insulin secretion. When single exocytotic events were imaged by total internal reflection fluorescence microscopy, the fluorescence of the insulin surrogate, neuropeptide Y-monomeric red fluorescent protein brightened, diffused, and then vanished with kinetics that were unaffected by overexpression of truncated SEC8 or SEC10. Together, these data suggest that the exocyst complex serves to selectively regulate the docking of insulin-containing vesicles at sites of release close to the plasma membrane.

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.

The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.

Like all members of the Na(+)/Cl(-)-dependent neurotransmitter transporter family, the rat gamma-aminobutyric acid transporter-1 (GAT1) is sorted and targeted to specialized domains of the cell surface. Here we identify two discontinuous signals in the carboxyl terminus of GAT1 that cooperate to drive surface expression. This conclusion is based on the following observations. Upon deletion of the last 37 amino acids, the resulting GAT1-Delta37 remained trapped in the endoplasmic reticulum. The presence of 10 additional residues (GAT1-Delta27) sufficed to support the interaction with the coat protein complex II component Sec24D; surface expression of GAT1-Delta27 reached 50% of the wild type level. Additional extensions up to the position -3 (GAT1-Delta3) did not further enhance surface expression. Thus the last three amino acids (AYI) comprise a second distal signal. The sequence AYI is reminiscent of a type II PDZ-binding motif; accordingly substituting Glu for Ile abrogated the effect of this motif. Neither the AYI motif nor the last 10 residues rescued the protein from intracellular retention when grafted onto GAT1-Delta37 and GAT1-Delta32; the AYI motif was dispensable for targeting of GAT1 to the growth cone of differentiating PC12 cells. We therefore conclude that the two segments act in a hierarchical manner such that the proximal motif ((569)VMI(571)) supports endoplasmic reticulum export of the protein and the distal AYI motif places GAT1 under the control of the exocyst.

Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA(E38R)) and the Sec5 (Sec5(T11A)) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.

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

Polarized delivery and incorporation of proteins and lipids to specific domains of the plasma membrane is fundamental to a wide range of biological processes such as neuronal synaptogenesis and epithelial cell polarization. The exocyst complex is specifically localized to sites of active exocytosis and plays essential roles in secretory vesicle targeting and docking at the plasma membrane. Sec3p, a component of the exocyst, is thought to be a spatial landmark for polarized exocytosis. In a search for proteins that regulate the localization of the exocyst in the budding yeast Saccharomyces cerevisiae, we found that certain cdc42 mutants affect the polarized localization of the exocyst proteins. In addition, we found that these mutant cells have a randomized protein secretion pattern on the cell surface. Biochemical experiments indicated that Sec3p directly interacts with Cdc42 in its GTP-bound form. Genetic studies demonstrated synthetically lethal interactions between cdc42 and several exocyst mutants. These results have revealed a role for Cdc42 in exocytosis. We propose that Cdc42 coordinates the vesicle docking machinery and the actin cytoskeleton for polarized secretion.

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