Sec6: Biological Overview | References
Gene name - Sec6
Synonyms - FlyBase name: Sec6 ortholog (S. cerevisiae)
Cytological map position - 55E2-55E2
Function - signaling
Symbol - Sec6
FlyBase ID: FBgn0034367
Genetic map position - 2R:14,512,772..14,515,249 [+]
Classification - Exocyst complex component Sec6
Cellular location - cytoplasmic
|Recent literature||Swope, R. D., Hertzler, J. I., Stone, M. C., Kothe, G. O. and Rolls, M. M. (2022). The exocyst complex is required for developmental and regenerative neurite growth in vivo. Dev Biol 492: 1-13. PubMed ID: 36162553
The exocyst complex is an important regulator of intracellular trafficking and tethers secretory vesicles to the plasma membrane. Understanding of its role in neuron outgrowth remains incomplete, and previous studies have come to different conclusions about its importance for axon and dendrite growth, particularly in vivo. To investigate exocyst function in vivo Drosophila sensory neurons were used as a model system. To bypass early developmental requirements in other cell types, neuron-specific RNAi was used to target seven exocyst subunits. Initial neuronal development proceeded normally in these backgrounds, however, this was considered to be due to residual exocyst function. To probe neuronal growth capacity at later times after RNAi initiation, laser microsurgery was used to remove axons or dendrites and prompt regrowth. Exocyst subunit RNAi reduced axon regeneration, although new axons could be specified. In control neurons, a vesicle trafficking marker often concentrated in the new axon, but this pattern was disrupted in Sec6 RNAi neurons. Dendrite regeneration was also severely reduced by exocyst RNAi, even though the trafficking marker did not accumulate in a strongly polarized manner during normal dendrite regeneration. The requirement for the exocyst was not limited to injury contexts as exocyst subunit RNAi eliminated dendrite regrowth after developmental pruning. It is concluded that the exocyst is required for injury-induced and developmental neurite outgrowth, but that residual protein function can easily mask this requirement.
Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. This characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, Rab11 forms a complex with Sec5 and Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs (Beronja, 2005).
Plasma membrane domains of polarized cells display distinct protein and lipid compositions. One critical mechanism that contributes to the formation and maintenance of membrane domains is targeted exocytosis of transport vesicles from the biosynthetic pathway or the recycling endosome (RE). Genetic analysis in yeast has identified mutants in which bud growth is stalled and secretory vesicles accumulate below the bud site. Eight of these genes encode components of the exocyst (or Sec6/8) complex (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p) that localizes to the bud site and apparently promotes the tethering of exocytotic vesicles to the plasma membrane before SNARE-mediated fusion (Beronja, 2005).
Recent work has initiated the characterization of exocyst components in mammals and Drosophila. In contrast to yeast cells, where the exocyst contributes to all major secretory events, the metazoan exocyst appears to have more specialized functions. In neurons, for example, the exocyst has been implicated in neurite outgrowth and in the targeting of glutamate receptors to the synapse but a general role in neurotransmission has not been detected. Similarly, the exocyst is essential for transport of proteins to the basolateral membrane in mammalian epithelial cell culture systems, but not the apical membrane. Cooperative action of E-cadherin and nectin, the two adhesion receptors found at the epithelial zonula adherens (ZA), recruits the exocyst to the apical junctional complex rapidly after cell contact formation (Yeaman, 2004). During branching morphogenesis of MDCK epithelial cysts in 3D culture, the exocyst can relocalize away from the apical junctional complex toward regions on the basolateral membrane that undergo rapid growth (Lipschutz, 2000). Regulation of exocyst function in both yeast and mammals involves a number of small GTPases including members of the Rab and Rho families (Lipschut, 2002; Inoue, 2003; Prigent, 2003). Nevertheless, the function and regulation of the metazoan exocyst in plasma membrane remodeling remains largely unresolved (Beronja, 2005).
The yeast exocyst is not only involved in all major exocytotic events, but each of the eight exocyst components is essential for targeted exocytosis (Finger, 1998; Hsu, 2004). Whether such functional uniformity is also found in multicellular organisms remains unclear. Initial genetic analysis in Drosophila raises the possibility that significant functional diversification of exocyst components may have taken place. Although Sec5 is broadly required for exocytosis and cell survival in flies (Murthy, 2003; Murthy, 2004), Sec10 appears to have an essential function only in a very limited number of secretory events (Andrews, 2002). A genetic analysis has been conducted of Drosophila sec6 in order to study Sec6 function and the role of polarized exocytosis. Interactions were also shown between the small GTPase Rab11 and the exocyst suggesting that the exocyst acts as a Rab11 effector (Beronja, 2005).
Sec6 is critical for multiple secretory events during Drosophila development. PRCs provide a striking example. Secretory vesicles accumulate in PRCs with reduced Sec6 function and these cells fail to transport Rh1 and Chaoptin (Chp) to the rhabdomere. These defects in secretory activity lead to a corresponding failure in the growth of the membrane-rich rhabdomere. Sec6, similarly as previously reported for Sec5 (Murthy, 2003, Murthy, 2004), is also required for plasma membrane growth in female germline cells and cell survival. A recent independent analysis of sec6 mutants confirms the function of Sec6 in cell viability and plasma membrane growth in the female germline, and indicates similar requirements for Sec6 and Sec5 in neuronal exocytosis (Murthy, 2005). These findings, together with the physical interactions between Drosophila Sec6 and Sec5 and the largely overlapping profiles of these proteins in a membrane cofractionation experiment suggest that both proteins are core components of the Drosophila exocyst (Beronja, 2005).
The function of Sec6 in differentiating PRCs is specific to the targeting of secretory vesicles to the apical rhabdomere. The colocalization of Sec5, Sec6, and Sec8 at the rhabdomere suggests that all three exocyst components cooperate in this process. Although Sec6 is required for targeting Chp and Rh1 to the rhabdomere, it is not needed for DEcad and Crb localization to the ZA and stalk membrane, respectively, during the second half of pupal development (PD). An alternative explanation for the normal localization of DEcad and Crb in sec6(pr) PRCs could be that both proteins are transported to the membrane in the first half of PD but not subsequently. However, this seems highly improbable, as it would imply that both proteins do not turn over any more. Also, the apical membrane of PRCs, including the Crb containing stalk membrane and the ZA, increases dramatically in the second half of PD, an increase that is most likely supported by protein exocytosis. Additional protein delivery is relevant in particular for Crb as the concentration of Crb determines the size of the stalk membrane. Failure to transport Rh1 and Chp in the absence of Sec6 is accompanied by an extensive accumulation of secretory vesicles in the cytoplasm of PRCs, similar to yeast cells that lack exocyst functio. Rh1 transport is also reduced or abolished in PRCs that lack normal function of the small Rab GTPases, Rab1, Rab6, or Rab11. PRCs that lack Rab1 or Rab6 function do not accumulate secretory vesicles and these Rab proteins are believed to contribute to the ER to Golgi transport or inter-Golgi transport, respectively. Rab1 or Rab6 are therefore unlikely to directly interact with the exocyst in vesicle targeting to the rhabdomere (Beronja, 2005).
In contrast to Rab1 and Rab6, Rab11-depleted PRCs accumulate secretory vesicles (Satoh, 2005) similar to sec6(pr) mutant PRCs, and Sec5 coimmunoprecipitates with Rab11::GFP from PRC and embryo lysates. These findings suggest that Rab11 takes the place of yeast Sec4p as the transport vesicle-associated small GTPase that recruits the exocyst (Guo, 1999). Although Sec6 was detected in Sec5 immunoprecipitates, bi Sec6 was detected in Rab11::GFP precipitates. Two explanations are envisioned for this discrepancy. First, Rab11 may predominantly associate with a subcomplex of the exocyst that includes Sec5 but not Sec6. Second, in the yeast exocyst, Sec6p links to Sec4p through Sec15p, Sec10p, and Sec5p (Guo, 1999), suggesting that the Sec6 Rab11 interaction may involve several intermediates including Sec5 and therefore is more difficult to detect. Both explanations are consistent with the model that Sec5 connects Sec6 to Rab11, a relationship that is similar to the interactions of yeast exocyst components and Sec4p (Guo, 1999). These results together with those of Satoh (2005) suggest that the exocyst is a Rab11 effector complex in PRCs (Beronja, 2005).
A more general role of the interaction of Rab11 and the exocyst in regulating exocytosis of metazoan cells is supported by a number of recent findings. First, Rab11 is associated with the RE (in epithelial cells often referred to as the apical RE or subapical compartment) and is involved in recycling of proteins in mammalian and Drosophila cell. The RE has now been identified as a major intermediate for the biosynthetic, Rab11-dependent transport of basolateral proteins. For example, the majority of biosynthetic E-cadherin travels through the RE in a Rab11-dependent way in HeLa and MDCK cells. Drosophila Rab11 is also required for basolateral transport. Rab11 localizes to the RE in cellularizing embryos and facilitates the transport of proteins recycled from the apical membrane and biosynthetic proteins to the forming basolateral membrane (Beronja, 2005 and references therein).
Second, the exocyst is required for basolateral protein transport including the targeting of E-cadherin to the basolateral membrane of MDCK cells. Whether the accumulation of Drosophila Sec6 at the ZA signifies a role for the exocyst in basolateral transport similar to MDCK cells remains to be established. Although it was not possible to study the localization of basolateral markers in Sec6 mutant imaginal disc cells as they failed to grow, it was observed that DEcad and DN-cadherin accumulate in the cytoplasm of sec6 mutant epithelial follicle cells, which is consistent with a role of Sec6 in basolateral transport. Third, mammalian Sec15 was recently shown to directly bind to Rab11 in a GTP-dependent manner but did not interact with Rab4, Rab6 and Rab7, and Sec15 was found to colocalize with Rab11 in the RE of COS-7 cells. Together, these data are consistent with the hypothesis that the exocyst is a Rab11 effector in many different cell types in mammals and flies that facilitates RE to plasma membrane transport of recycled and biosynthetic cargo. In Drosophila PRCs, the Rab11/exocyst transport of Rh1 appears to be predominantly biosynthetic; a block in endocytosis does not affect Rh1 delivery to the rhabdomere (Beronja, 2005).
Considering the specific association of Sec6 with the ZA of early PRCs and a potentially broad role of Rab11/exocyst in basolateral transport it is tempting to speculate that during PRC development exocyst targeting specificity changes from basolateral to the apical rhabdomere. One possible explanation for this shift is that the exocyst associates with the actin cytoskeleton. As the vast majority of actin filaments in PRCs are found in the rhabdomere microvilli or the rhabdomere terminal web simple mass action via actin association could contribute to targeting specificity. The actin cytoskeleton is required in yeast to recruit the exocyst to secretory sites. Moreover, in the cells of pancreatic acini, exocyst proteins bind the actin cytoskeleton and this interaction is required for the association of the exocyst with Ca2+ signaling complexes that are targeted to the apical membrane. How shifts in exocyst targeting specificity are achieved is a major challenge for future research (Beronja, 2005).
A number of differences in the distribution of Sec6, Sec5, and Sec8 in PRCs and during oogenesis have been noted that raise the possibility that exocyst proteins do not always act together. Also cofractionation analysis shows a broader distribution of Sec6 than Sec5 and Sec8 indicating the association of Sec6 with additional membrane compartments. The possibility that the observed differences in protein distribution are the result of differences in epitope availability of distinct exocyst protein pools cannot be ruled out. This issue has been raised by Yeaman (2001), who reports that mAbs directed against mammalian Sec6 and Sec8 recognize protein pools with different subcellular localizations. It is believed that this is highly unlikely in this case as pAbs against Sec6 and Sec8 were used. Moreover, each antibody used recognizes cytoplasmic and plasma membrane-associated protein pools and either two or all three proteins are recognized when they colocalize as, for example, in the rhabdomere. Furthermore, inconsistencies in protein prevalence are also apparent by immunoblot and by cofractionation analysis. The ability of exocyst proteins to exist in subcomplexes was documented in yeast (Guo, 1999) and mammalian cells (Moskalenko, 2003). Also, biochemical studies of the interaction between Sec8, Sec6, and SAP102 in rat brain lysates suggested that Sec6 and Sec8 are not always present in the same complexes (Sans, 2003
To allow a detailed analysis of exocyst function in multicellular organisms, sec6 mutants were generated in Drosophila. These mutations were used to compare the phenotypes of sec6 and sec5 in the ovary and nervous system, and they were found to be similar. Sec5 is mislocalized in sec6 mutants. Additionally, an epitope-tagged Sec8 was generated that is localized with Sec5 on oocyte membranes and is mislocalized in sec5 and sec6 germ-line clones. This construct further revealed a genetic interaction of sec8 and sec5. These data, taken together, provide new information about the organization of the exocyst complex and suggest that Sec5, Sec6 and Sec8 act as a complex, each member dependent on the others for proper localization and function (Murthy, 2005).
Studies of the exocyst complex in yeast have benefited from an abundance of mutations in each member of the complex. The eight subunits of the exocyst (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) were first identified in a screen that isolated conditional mutations in genes required for exocytosis. Mutations of each have been shown to prevent exocytosis and to arrest growth of the daughter cell and cytokinesis. The similarities of the phenotypes and extensive biochemical characterization have led to a model in which the complex functions as an integral unit that can interact with both plasma membranes and transport vesicles, and that, as a unit, marks sites of membrane insertion (Murthy, 2005).
In higher organisms, the investigation of the exocyst has been hampered by a lack of mutations. A mutation in murine sec8 causes lethality shortly after gastrulation of the embryo, precluding a detailed analysis of the role of the complex. Recently, Drosophila sec5 mutations were characterized. As in yeast, Sec5 localization in Drosophila undergoes dynamic changes correlating with the sites at which it is required for the traffic of membrane proteins during oogenesis and cellularization. In homozygous sec5 larvae and germ-line clones of sec5 alleles, defects were observed in trafficking proteins to the plasma membrane (Murthy, 2003; Murthy, 2004; Murthy, 2005 and references therein).
In contrast to these genetic studies, investigation of other components of the exocyst has depended on the introduction of antibodies and the overexpression of wild-type or mutated forms of the proteins in wild-type genetic backgrounds. From these studies, some discrepancies in the localization of exocyst proteins and their phenotypes have emerged. Drosophila Sec5 concentrates specifically at sites of membrane addition in both ovaries and embryos but, in normal rat kidney (NRK) cells, different monoclonal antibodies to Sec6 and Sec8 recognized the exocyst complex at either the trans-Golgi network (TGN) or the plasma membrane (Yeaman, 2001). Furthermore, Exo70 associates with microtubules at the microtubule-organizing center in undifferentiated PC12 cells, and Sec10 has been found both at the endoplasmic reticulum (ER) and on tubulo-vesicular extensions of the TGN and recycling endosomes. Finally, an interaction between Sec8, SAP102 and the N-methyl-D-aspartate receptor (NMDAR) in mammalian neurons was found in the ER (Murthy, 2005 and references therein).
Indeed, although biochemical studies in yeast and neurons suggest the presence of only one copy of each subunit per complex and the isolation of the exocyst complex from all yeast exocyst mutants shows that its structure is altered, there is growing evidence that the members of the exocyst might not always act as a complex. For example, whereas Drosophila sec5 mutations blocked the transport of many proteins to the plasma membrane of neurons and developing oocytes, the addition of antibodies specific for TGN-bound exocyst complexes to semi-intact NRK cells resulted in cargo accumulation in a perinuclear region (Yeaman, 2001). Also, the introduction of a dominant negative Sec10 or Sec5 small interfering RNA to NRK cells causes morphological changes and phenotypes at the recycling endosome. Finally, the overexpression of Sec10 affects protein synthesis in MDCK cells by an interaction with an ER translocon and yeast Sec10p and Sec15p might form a subcomplex. These findings raise the possibility that different complex members have different functions within the cell and might not always function as a unit (Murthy, 2005 and references therein).
The present uncertainty about the significance of exocyst subunits in multicellular organisms might, in part, arise from a lack of loss-of-function mutations that can be directly compared. The present study reports the isolation of a sec6 mutation in Drosophila whose phenotype is comparable to that of sec5. Moreover, with antibodies to Sec5 and an epitope-tagged sec8 transgene, the interdependency of these complex members for their subcellular localization was determined (Murthy, 2005).
The distribution of Sec5 has been examined most closely in the ovary. In this tissue, it was present on all membranes early in the development of the egg chamber. At late stages, however, Sec5 acquires a characteristic distribution not reported for any other cellular component -- a progressive enrichment at the anterior end of the lateral oocyte membranes. HA-Sec8 has now been found to be similarly concentrated in this area, suggesting that several (and perhaps all) exocyst components will be similarly localized (Murthy, 2005).
Mutations in one complex member appear to disrupt the localization of others. Thus, in sec5E13 homozygous oocytes, HA-Sec8 is no longer membrane bound or concentrated at the anterior sites. Instead, it appears to fill the cytoplasm diffusely. Similarly, Sec5 is mislocalized within the nervous system of sec6 mutant larvae and Sec5 and HA-Sec8 are both mislocalized within germ lines homozygous for sec6. The mislocalization of Sec5 and HA-Sec8 in sec6 germ lines, however, is not identical to the mislocalization of HA-sec8 in sec5 germ lines. Whereas the latter involves a diffuse filling of the cytoplasm with immunoreactivity, the mislocalized Sec5 and HA-Sec8 remain punctate within the sec6 egg chambers. Because these puncta resemble syntaxin and lectin-staining in sec5 germ lines, it seems likely that they represent fragments of membrane or transport vesicles that have not fused with the plasma membrane. The difference in these two phenotypes might arise from any of several causes, including the perdurance of some Sec6 in the sec6Ex15 mutant germ lines. It is tempting to speculate, however, that the difference reflects the organization of proteins within the complex. Sec3p has been shown in yeast to bind to the plasma membrane at the bud tip even when other complex members are absent. This has been interpreted as indicating that Sec3p binds directly to a membrane protein and that the localization of other complex members is dependent on Sec3p. Sec5p is thought to bind directly to Sec3p and so it is plausible that, in the present study, Sec5 remains membrane bound via its direct interaction with Sec3 even in the absence of Sec6. Sec8, however, is not thought to interact directly with Sec3. Because Sec8 appears to remain membrane-associated in sec6 but not sec5 mutants, it is hypothesized that a partial complex consisting of Sec3, Sec5 and Sec8 remains on the membrane even in the absence of Sec6. The disposition of the remaining complex members in the sec5 and sec6 mutants must remain speculative until suitable reagents have been obtained for their localization (Murthy, 2005).
The interdependence of the complex members is also evident in the genetic interaction of Sec8 and Sec5: although germ-line expression of HA-tagged Sec8 has no phenotype of its own, it enhances the germ-line phenotype of sec5E13, making this partial loss-of-function allele more similar to the null allele. This observation requires that the epitope-tagged transgene be used with caution, because its expression might interfere with exocyst function owing either to an influence of the epitope tag or to unphysiological expression levels. Indeed, phenotypes have been associated with the overexpression of Sec10, another complex member (Murthy, 2005).
The phenotypes of sec6 and sec5 mutants can be compared in several regards. Like sec5, sec6 caused lethality at approximately 96 hours AEL and these larvae were stunted in their growth and do not progress beyond the first instar. In an assay of membrane-protein transport to the cell surface of identified neurons, trafficking defects were found for sec6 that were akin to those of sec5. In the germ line, it was found that membranes between cells disintegrate in sec6 clones, a phenotype observed for the null allele of sec5. For sec5, it is hypothesized that, as the cells of the germ line grow and expand, membrane addition cannot keep pace, and that membranes between nurse cells and the oocyte consequently fall apart. A similar explanation is likely for sec6. The mispositioning of the oocyte within the sec6 germ line was also observed. This phenotype occurs when either the germ line or the posterior follicles are mutant for sec5. Because the positioning of the oocyte is dependent on E-cadherin and cell-cell signaling between the oocyte and follicle cells, it is likely that this phenotype arises from a defect in the expression of E-cadherin or other signaling molecules on the oocyte surface. In fact, E-cadherin and Nectin 2a have been recently shown to be binding partners for the exocyst complex in MDCK cells (Murthy, 2005).
Although the similarities of their phenotypes suggest that Sec5 and Sec6 share functions, some differences are observed in the mutant phenotypes. sec6Ex15 larvae are smaller than sec5E10 larvae but germ-line clones of sec5E10 have a more severe phenotype in the ovary, arresting earlier and with fewer remaining membranes. The most intriguing difference arose in the mCD8-GFP expression assay: whereas sec5E10 larvae are capable of synthesizing the protein but not of expressing it at the cell surface, sec6Ex15 larvae express only low levels of the protein, which also appear to be blocked in their transport to the surface. Finally, whereas HA-Sec8 protein is mislocalized in both sec5 and sec6 germ-line clones, the patterns of mislocalized protein are distinct. The differences in the mutant phenotypes might arise from minor factors such as the degree of perdurance of protein in the homozygous germ-line clones or the amount or stability of maternal protein deposited in the egg. However, they might also represent legitimate functional distinctions. The most pronounced difference, the different levels of expression of the mCD8-GFP reporter protein, might reflect the fact that Sec6 is required at an earlier step in the synthesis of membrane proteins, in addition to its requirement (along with Sec5) for insertion at the plasma membrane. Such a role would be consistent with findings that Sec6 and Sec8 have been observed in the TGN, that Sec8 and Sec10 associate with proteins at the TGN and ER, and that overexpression of Sec10 alters membrane-protein synthesis. The general similarities between and severity of the sec6 and sec5 phenotypes also do not exclude the possibility that other components will have more restricted roles, particularly given that several GTPases have emerged as binding partners of particular members of the complex and might be either effectors or regulators of those components (Murthy, 2005).
In contrast to the cell lethality of the sec5 and sec6 phenotypes, a Sec10 RNA-interference construct in Drosophila has very little effect in most tissues, possibly affecting only the secretions of the ring gland cells. However, because no antibody is available for Drosophila Sec10 and because maternally contributed protein would be unaffected by this construct, the RNA interference might have been ineffective at reducing endogenous Sec10 levels. In light of the broad phenotypes of dominant negative and overexpressed Sec10 in other cell types, this is a likely explanation of the discrepancy (Murthy, 2005).
In summary, the similarity of localization of Sec5 and HA-Sec8, the interdependency of the complex members for proper localization in this study, the genetic interaction between HA-Sec8 and sec5, and the general similarity of the sec5 and sec6 phenotypes all suggest that Sec5, Sec6 and Sec8 associate as a complex in Drosophila, acting in concert, and that each is crucial for the function of the complex at the membrane. It will be important to examine the localization and phenotypes of the other complex members to determine whether all the complex members do indeed function primarily as part of the intact exocyst. Furthermore, the mutations in sec5 and sec6 should provide a useful genetic background for structure function studies with which to test the significance of their individual binding partners and regulators (Murthy, 2005).
Loss of function of the Drosophila exocyst components in epithelial cells results in E-Cadherin (Shotgun) accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits Shotgun delivery to the membrane. Rab11 and Armadillo interact with Sec15 and Sec10, respectively. These results support a model whereby the exocyst regulates E-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located (Langevin, 2005).
In budding yeast, the exocyst has been proposed to tether post-Golgi vesicles to the membrane of the growing bud prior to fusion. This model is supported by several observations. (1) Exocyst components localize both on post-Golgi vesicles and on the bud membrane (Boyd, 2004). Analogously in Drosophila, Sec5 and Sec15 localize along the lateral membrane and on the REs. (2) Mutations in genes encoding components of the exocyst complex lead to the accumulation of post-Golgi vesicles (Novick, 1980). Analogously, Sec5, Sec6, and Sec15 loss of function leads to an enlargement of the recycling endosome (RE) compartment; this enlargement interpreted as an accumulation of RE vesicles. (3) The localization of Sec8p and Exo70p at the growing bud, i.e., the site of polarized exocytosis, depends on the function of the other exocyst components. Analogously, Sec5 is localized along the lateral membrane, where E-Cadherin delivery is affected, and its localization along the cortex depends on Sec6. It is therefore proposed that in Drosophila epithelial cells, Sec5, Sec6, and Sec15 act by tethering vesicles originating from the recycling endosomal compartment to the lateral membrane of epithelial cells, as a prerequisite for their exocytosis (Langevin, 2005).
In epithelial cells, Arm and E-Cadherin colocalize to the AJs of the ZA as well as along the lateral membrane. In the absence of Sec5, Sec6, and Sec15 function, E-Cadherin trafficking is affected and E-Cadherin accumulates in the RE. Similarly, in the absence of arm, E-Cadherin fails to localize at the membrane and localizes in the RE. The identification of an interaction between Arm and Sec10 is therefore consistent with a model whereby this interaction provides a landmark at the site where Arm is enriched in order to deliver E-Cadherin from the recycling endosomes. Nevertheless, Arm may play an additional role in stabilizing E-Cadherin at the AJs. A direct demonstration of the function of Arm in regulating the delivery of E-Cadherin will therefore require the identification of arm mutant alleles that do not perturb its function as a regulator of E-Cadherin stabilization and only affects its interaction with Sec10 (Langevin, 2005).
In the absence of Sec5, Sec6, or Sec15 function, E-Cadherin delivery to the lateral membrane is inhibited and E-Cadherin accumulates in the REs. Furthermore, E-Cadherin was found to transcytose in a Sec5-dependent manner from the lateral membrane of epithelial cells to the apical AJs. Therefore, this study reveals at least a role of the exocyst in the recycling of E-Cadherin from the lateral membrane to the apical AJs. Furthermore, the strong reduction of E-Cadherin present on the lateral membrane is interpreted as a failure to recycle E-Cadherin from the lateral membrane back to the lateral membrane, which cannot be compensated for by the delivery of newly synthesized E-Cadherin to the lateral membrane. The loss of E-Cadherin on the lateral membrane may also lead to a reduction of E-Cadherin delivery at the AJs. This may have also contributed to the loss of epithelial cell polarity observed in some of the sec5 mutant epithelial cells (Langevin, 2005).
In polarized MDCK cells, the apical REs are well known as a site of sorting during endocytic and transcytotic transport. The REs have also been shown to serve as an intermediate during the transport of newly synthesized proteins from the Golgi to the plasma membrane in nonpolarized MDCK cells. Similarly, upon overexpression of GFP-E-Cad in HeLa cells, E-Cad transits from the Golgi to the Rab11 endosomes. Nevertheless, the existence of such a pathway remains to be established in polarized MDCK cells. In fact, the overexpression of a dominant-negative form of Rab11 leads to sequestration of E-Cadherin in the REs, but whether sequestered E-Cadherin represented newly synthesized or recycled E-Cadherin was not determined. The existence of such a Golgi-to-RE pathway also remains to be established in Drosophila epithelial cells. If so, a role of the exocyst in regulating the delivery of newly synthesized E-Cadherin from the Golgi to the lateral membrane via the REs remains plausible (Langevin, 2005).
Whether the exocyst regulates E-Cadherin localization in mammalian cells has not been directly analyzed. However, E-Cadherin is proposed to act as a regulator of the localization of the exocyst complex in polarizing mammalian cells since E-Cad- and Nectin-2α-dependent cell-cell contacts were proposed to recruit the exocyst complex in order to promote the growth of the lateral epithelial cell domain. The current study suggests that upon the recruitment of the exocyst complex by E-Cadherin, the exocyst promotes the delivery of more E-Cadherin to the lateral membrane during the establishment of apico-basal polarity. In fact, several reports can be reconciled with a function of the exocyst in regulating the transport of E-Cadherin in mammalian cells. Thus, polarized exocytosis of E-Cad to the lateral membrane is dependent upon its interaction with Arm. And, as stated above, REs have shown to serve as an intermediate during the transport of E-Cad from the Golgi to the lateral membrane where E-Cadherin, β-Catenin, and α-Catenin form the AJs. Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane. Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11. Finally, E-Cadherin and catenins are associated with exocyst components (Langevin, 2005 and references therein).
In conclusion, this work provides evidence for a conserved role of the exocyst in regulating the delivery of E-Cadherin from REs to sites on the plasma membrane and in thereby contributing to the maintenance of epithelial cell polarity (Langevin, 2005).
Search PubMed for articles about Drosophila Sec6
Andrews, H. K., Zhang, Y. Q., Trotta, N. and Broadie, K. (2002). Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission. Traffic 3: 906-921. PubMed ID: 12453153
Beronja, S., Laprise, P., Papoulas, O., Pellikka, M., Sisson, J. and Tepass, U. (2005). Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J. Cell Biol. 169(4): 635-46. PubMed ID: 15897260
Boyd, C., Hughes, T., Pypaert, M. and Novick, P. (2004). Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol. 167(5): 889-901. PubMed ID: 15583031
Finger, F. P. and Novick, P. (1998). Spatial regulation of exocytosis: lessons from yeast. J. Cell Biol. 142: 609-612. PubMed ID: 9700152
Guo, W., Roth, D., Walch-Solimena, C. and Novick, P. (1999). The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18: 1071-1080. PubMed ID: 10022848
Hsu, S.C., TerBush, D., Abraham, M. and Guo, W. (2004). The exocyst complex in polarized exocytosis. Int. Rev. Cytol. 233: 243-265. PubMed ID: 15037366
Inoue, M., et al. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422: 629-633. PubMed ID: 12687004
Langevin, J., et al. (2005). Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev Cell. 9(3): 365-76. PubMed ID: 16224820
Lipschutz, J. H., et al. (2000). Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins. Mol. Biol. Cell. 11: 4259-4275. PubMed ID: 11102522
Lipschutz, J. H. and Mostov, K. E. (2002). Exocytosis: the many masters of the exocyst. Curr. Biol. 12: R212-R214. PubMed ID: 11909549
Moskalenko, S., et al. (2003). Ral GTPases regulate exocyst assembly through dual subunit interactions. J. Biol. Chem. 278: 51743-51748. PubMed ID: 14525976
Prigent, M., et al. (2003). ARF6 controls post-endocytic recycling through its downstream exocyst complex effector. J. Cell Biol. 163: 1111-1121. PubMed ID: 14662749
Murthy, M., Garza, D., Scheller, R. H. and Schwarz, T. L. (2003). Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 37(3): 433-47. PubMed ID: 12575951
Murthy, M. and Schwarz, T. L. (2004). The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary. Development. 131(2): 377-88. PubMed ID: 14681190
Murthy, M., et al. (2005). Sec6 mutations and the Drosophila exocyst complex. J. Cell Sci. 118: 1139-1150. PubMed ID: 15728258
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date revised: 2 January 2023
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