InteractiveFly: GeneBrief

Secretory 15 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized exocytosis/secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. An exocyst component, sec15, has been isolated in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. The data suggest a role for Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions (Mehta, 2005).

Exocyst components have been implicated in different developmental contexts where they appear to function in polarized exocytosis. Sec5 and Sec6 have been implicated in neurite growth, synaptic transmission, and polarization of the Drosophila oocyte (Beronja, 2005; Murthy, 2003; Murthy, 2004; Murthy, 2005). Furthermore, Sec6 was shown to regulate apical exocytosis in photoreceptor cells (Beronja, 2005). As with loss of sec15, loss of sec5, sec6, or sec15 function similarly disrupts the localization of Drosophila E-Cadherin, which accumulates in an enlarged recycling endosome (RE) compartment. Exocyst components play a role in regulating the localization of E-Cadherin in epithelial cells and the delivery of E-Cadherin from the REs. Sec5 regulates the recycling of basolateral E-Cadherin to the apical AJs. While a connection between exocyst and the Rab11 recycling endosomal compartment has been proposed both in Drosophila and mammalian cells, the characterization of sec15 phenotype demonstrates the functional significance of this interaction. Texocyst has been viewed as an important regulator of exocytosis, but the mechanisms by which the exocyst complex could regulate the exocytosis of specific cargoes to specific domains within the cell have remained poorly understood. A model is proposed whereby the interaction between Sec10 and Arm enables the exocyst to regulate E-Cadherin trafficking in Drosophila (Mehta, 2005 and references therein).

Sec15 plays multiple roles in recycling membrane components: (1) Studies with sec15 mutants point to a role of sec15 in recycling of Delta thus promoting Notch signaling during the asymmetric division of Drosophila sensory organ precursors (Jafar-Nejad, 2005); (2) Loss of function of sec15 in epithelial cells results in DE-Cad accumulation in an enlarged recycling endosomal compartment and inhibits DE-Cadherin delivery to the membrane (Langevin, 2005); (3) In fly photoreceptors Sec15 colocalizes with Rab11, acts as an effector of Rab11, and loss of Sec15 affects rhabdomere morphology (Wu, 2005); finally (4) sec15 is required for the delivery of specific cell adhesion and signaling molecules required for the establishment of synaptic specificity after the growth cones reach their target regions (Mehta, 2005)

More than twenty-five years ago, Novick (1980) identified 23 temperature-sensitive mutant complementation groups that caused a secretory defect in yeast. Many of the SEC mutants isolated in this work have now been studied in more detail (Schekman, 2004). Much is known about some of these genes, including SEC9, a SNAP-25 homolog, SEC17, an α-SNAP homolog, and SEC18, an NSF homolog, because they have been implicated in numerous secretory processes in yeast and many metazoans (Bennett, 1993). Another subset of SEC genes (SEC3, SEC5, SEC6, SEC8, SEC10, and SEC15) has been shown to encode members of a large protein complex, the exocyst or Sec6/8 complex, which also includes Exo70p and Exo84p. Although they have been studied quite extensively in yeast, their role in metazoans is ill-defined due to a dearth of functional studies (Mehta, 2005 and references therein).

In yeast, exocyst proteins are required for polarized exocytosis of secretory vesicles. Sec3p localizes to the membrane even when other exocyst members are lacking and is thought to represent a spatial landmark. Sec15p interacts with the secretory vesicle-associated rab GTPase, Sec4p, in its GTP bound form. A further interaction of Sec15p with Sec10p is thought to recruit other exocyst components and connect the complex to Sec3p (Guo, 1999). Hence, Sec15p is thought to target the vesicle to the correct exocytic site (Mehta, 2005).

In multicellular organisms, the exocyst proteins were found to be present in brain as well as most other tissues. In nonpolarized epithelial cells (Madin-Darby canine kidney [MDCK] cell culture), the complex is found in a soluble form in the cytoplasm. Upon cell-cell contact, it relocalizes to the interacting plasma membranes. After disruption of E-cadherin-mediated cell-cell contact, the complex dissociates again from the plasma membrane. In the same epithelial cell line, antibodies against Sec8 specifically inhibit vesicle delivery to the basolateral but not the apical membrane. These data suggest that the recruitment of the exocyst is a consequence of cell-cell adhesion and is essential for epithelial cell polarity (Mehta, 2005 and references therein).

In cultured hippocampal neurons, Sec6 immunoreactivity is present in the growth cone during neurite outgrowth as well as in periodic punctae on the axon prior to synaptogenesis. After formation of stable synapses, the Sec6 immunoreactivity disappears, suggesting that Sec6 protein and the corresponding complex are not required in mature synapses. In addition, overexpression of a dominant-negative Sec10 protein blocks neurite outgrowth in cultured PC12 cells. Taken together, the data from yeast and mammalian cells suggest that the complex may play a role in growth cone extension and possibly synaptogenesis (Mehta, 2005 are references therein).

To date, the only published mutants providing in vivo data in metazoans are knockouts of sec8 in mouse and sec5 in Drosophila. The sec8 knockout results in lethality at E7.5, precluding analyses of neuronal development. Loss of sec5 in Drosophila reveals no defect in neurotransmitter release, and larvae die soon after their maternal protein contribution is exhausted (Murthy, 2003). In cell culture, loss of sec5 blocks neurite outgrowth and incorporation of newly synthesized transmembrane proteins into the membrane, in agreement with a proposed role for the exocyst in neurite outgrowth (Hsu, 1999: Murthy, 2003). In the developing oocyte, sec5 is required for membrane trafficking and polarization (Murthy, 2004). Finally, clones of sec5 in the eye do not form eye tissue. Since Sec5 is a core component of the complex, Murthy (2003) proposes that the phenotype associated with the loss of sec5 represents the function of the entire exocyst complex. No functional data in yeast or metazoans so far indicate any role of individual exocyst components independent of the entire complex, suggesting that sec5 mutant phenotypes represent a generic consequence of mutations in exocyst members (Mehta, 2005).

This study describes the isolation of Drosophila sec15 mutants in a forward genetic screen designed to identify genes that affect synapse development. In contrast to the cell lethality associated with sec5 mutations, sec15 mutant photoreceptor neurons are viable and display surprisingly specific defects in a distinct neuronal targeting step. Loss of sec15 does not cause defects in neurite extension, but leads to the formation of synapses between inappropriate partners, causing a loss of synaptic specificity. The data indicate that sec15 is required for the delivery of specific cell adhesion and signaling molecules required for the establishment of synaptic specificity after the growth cones reach their target regions. They also suggest a model in which subcomplexes of the exocyst perform separable functions (Mehta, 2005).

It is surprising how many developmental processes are unaffected by the loss of sec15: neuronal differentiation, axonal outgrowth, and initial target recognition of the correct brain areas all appear normal. Subsequently, a specific neuronal sorting process that ensures synaptic specificity is disrupted. After the defective neuronal targeting step, the developmental program proceeds rather normally with synaptic partner selection and synapse formation. While it is known that these developmental processes are genetically separable, they have primarily been associated with cell adhesion molecules (CAMs). Based on the finding of targeting defects for specific CAMs and signaling molecules, it is proposed that a vesicular transport mechanism exists to spatiotemporally target certain CAMs as well as other proteins. Notably, soluble NSF-attachment receptors (SNAREs), which are required for most if not all vesicle docking and fusion processes, are unlikely to convey much spatiotemporal targeting information. This is primarily because target-SNAREs are distributed uniformly over membranes, even though vesicle fusion is restricted to limited subdomains, as is also the case for the target-SNARE syntaxin in photoreceptor terminals (Mehta, 2005).

Further evidence for targeting defects of a vesicular cargo transport mechanism comes from vertebrate cell culture experiments. Disrupting exocyst function caused defects in basolateral, but not apical, targeting of proteins in epithelial cells (Grindstaff, 1998). The exocyst has also been implicated in the trafficking of GLUT4 transporters in response to insulin stimulation (Inoue, 2003). Sec8 is involved in NMDA receptor insertion at dendrites through an interaction with SAP102 (Sans, 2003). In Drosophila photoreceptors, the basolateral compartment is the axon (Izaddoost, 2002), while in vertebrate neurons, the basolateral compartment is dendritic. It is therefore possible that the same subcomplex of exocyst (and other) components is required for correct targeting in the presynaptic compartment of Drosophila photoreceptors and vertebrate postsynaptic receptor targeting (Mehta, 2005).

The investigation of the establishment of specific synaptic contacts has largely been focused on CAMs and their regulating/modifying proteins, since these molecules are thought to convey information about spatiotemporal specificity. The finding of CAM misregulation in sec15 mutant neurons links the spatiotemporal regulation of CAMs to the cell biology of the neuron. Although vesicle trafficking of CAMs was not studied directly in this work, other studies in both yeast and vertebrate systems provides evidence that Sec15 and other exocyst members are associated with vesicles and play a role in vesicle trafficking (Ang, 2004 and Boyd, 2004). It is proposed that specificity may be established through unique vesicular trafficking mechanisms in addition to the transcriptional and posttranslational regulation of various isoforms of CAMs in certain cells at distinct times. Much is known about the specificity of the vesicular trafficking machinery at the ER and Golgi, where distinct cargoes need to be specifically targeted. In neurons, numerous synaptic proteins, including the CAM N-cadherin are specifically targeted to the active zones and other synaptic domains via a specialized vesicular transport mechanism. In Drosophila photoreceptors, the misregulation of CAMs prior to synapse formation has been observed in neurons that lack n-synaptobrevin, and specific vesicles for the targeted transport of synaptic components have been described in mammals. It is not known whether a distinct type of vesicle exists for the transport of CAMs and signaling molecules to the developing terminal, but it is proposed that specific subsets of CAMs are transported and integrated into the membrane by Sec15. This is not to say that synaptic specificity is the only role of Sec15 or the CAMs that were examined. The data shows mislocalization of CAMs in both the cell body as well as axons of sec15 mutant photoreceptors. The consequences of this cell body mislocalization were not investigated, except to make sure that such mislocalization does not impact cell viability, neuronal differentiation, axonal outgrowth, and initial target recognition of the correct brain areas by these photoreceptors. In this respect, it is interesting to note that some of the CAMs examined (N-cadherin, Flamingo) were correctly targeted in mutant photoreceptors while others (Dlar, Fas2, IrreC-rst) were not. Although the in vitro inhibition of microtubule polymerization by exocyst members (Wang, 2004) and defects in sec5 mutants (Murthy, 2003) with neurite extension may argue for a role in general protein or membrane trafficking for the exocyst, the curren data argue that this is not the case for Sec15. In addition to the correct localization of the CAMs mentioned above, sec15 mutant neurons are viable, extend axons, and assemble functional synapses. It seems unlikely that a loss of protein required for general protein trafficking would not affect these processes. In summary, the data describe a defect in synaptic specificity and concommitant mislocalization of proteins known to affect synaptic specificity in photoreceptors lacking Sec15. These defects can be explained by a Sec15-dependent intracellular vesicle trafficking mechanism for certain molecular components that are required for the establishment of synaptic specificity (Mehta, 2005).

Most studies consider individual exocyst components representative for the whole complex and assume that the entire eight member complex is responsible for the different roles proposed or demonstrated for the exocyst. Since mutations have been isolated in sec15, Drosophila is the first metazoan in which two independent gene disruptions of exocyst components allowed tests of this assumption. Furthermore, a gene disruption of Drosophila sec6 has recently been generated and, like sec5, observed to cause cell lethality in homozygous mutant eyes. The remarkable difference between sec5, sec6, and sec15 mutant phenotypes questions the idea of a mutation in an individual member having a 'generic exocyst' phenotype. One possibility that may explain this discrepancy would be an exocyst-independent function of sec15. The findings that Sec5 and Sec8 are mislocalized in sec15 mutant neuronal terminals argue against this possibility. Instead, it suggests that Sec15 participates with Sec5 and Sec8 in a specific developmental process. However, the sec15 phenotype is notably more specific than the sec5 or sec6 cell lethality. While Sec5 is suggested to have a role in general membrane trafficking (Murthy, 2003), the large number of normal processes observed in sec15 mutant neurons (cell viability, axon outgrowth, axon guidance, neurotransmitter release, etc.) argues against this being the case for Sec15. This implies that at least Sec5 and perhaps Sec8 sustain another essential function in the absence of Sec15. For the Sec15 interaction partner Sec10, a knockdown using RNAi as well as Sec10 overexpression were found to have no detectable phenotype in photoreceptors (Andrews, 2002). However, Sec10 overexpression in sec15 mutant photoreceptors causes cell lethality, indicating a genetic interaction. Moreover, the finding that Sec6 is normally localized in the absence of Sec15 is puzzling. Close investigation of the expression pattern in lamina cartridges revealed a clear postsynaptic localization of Sec6. This finding together with the differential expression patterns of Sec5, Sec6, Sec8, and Sec15 during development and adulthood argue against a single functional complex in Drosophila neurons. Similarly, immuno-EM in rat hippocampal neurons revealed that Sec6 localizes to secretory vesicles, while Sec8 has a diffuse, cytoplasmic distribution (Vik-Mo, 2003). Another precedent for subcomplexes of the exocyst having different functions can be found in the COG complex. The COG, exocyst, and GARP complexes share distant homology in the N termini of their subunits and are placed in a family of 'quatrefoil complexes' by Whyte because all three complexes have multiples of four subunits (Whyte, 2001). Distinct COG subcomplexes are proposed to act in intra-Golgi vesicle recycling and endosome to Golgi recycling (Whyte, 2002). A similar situation may exist for the exocyst, where subcomplexes are involved in trafficking different populations of vesicles. Although it is not known which assemblies of exocyst components exert which functions in vivo, the current results suggest that at least two different functional compositions of the known exocyst components exist. Given that Sec5 is considered the core member of the complex (Guo, 1999), loss of sec5 may represent either the loss of all exocyst functions as suggested by Murthy (2003), or of at least one essential function. Either way, the cell lethality associated with the loss of sec5 or sec6 masks possible later more specific functions of subcomplexes. Hence, the data indicate that a detailed analysis of the many proteins that are thought to be required for exocyst function will have to be initiated in order to understand their precise role in metazoans (Mehta, 2005).

REGULATION

Sec15 promotes Notch signaling during the asymmetric division of Drosophila sensory organ precursors

Asymmetric division of sensory organ precursors (SOPs) in Drosophila generates different cell types of the mature sensory organ. In a genetic screen designed to identify novel players in this process, a mutation was isolated in Drosophila sec15, which encodes a component of the exocyst, an evolutionarily conserved complex implicated in intracellular vesicle transport. sec15⁻ sensory organs contain extra neurons at the expense of support cells, a phenotype consistent with loss of Notch signaling. A vesicular compartment containing Notch, Sanpodo, and endocytosed Delta accumulates in basal areas of mutant SOPs. Based on the dynamic traffic of Sec15, its colocalization with the recycling endosomal marker Rab11, and the aberrant distribution of Rab11 in sec15 clones, it is proposed that a defect in Delta recycling causes cell fate transformation in sec15⁻ sensory lineages. The data indicate that Sec15 mediates a specific vesicle trafficking event to ensure proper neuronal fate specification in Drosophila (Jafar-Nejad, 2005).

In a genetic screen designed to identify novel players in Drosophila sensory organ development, a mutation in sec15 was isolated that caused a pIIa to pIIb transformation phenotype. Sec15 is a component of a multiprotein complex called the exocyst or Sec6/8 complex. Mutations in exocyst components were originally isolated in a yeast screen for secretion-defective mutants. Subsequent analysis of the exocyst complex in yeast and mammalian cell culture systems has indicated that it functions in intracellular vesicle transport. In yeast, the exocyst mediates the post-Golgi to membrane targeting of exocytic cargo via an interaction with the Rab GTPase Sec4p. In Madin-Darby canine kidney (MDCK) epithelial cells, the exocyst localizes to areas of cell-cell contact and is involved in basolateral delivery of vesicles. However, none of the studies on the exocyst components have implicated these proteins in cell fate determination. The data suggest that Sec15 mediates highly specific intracellular trafficking events that promote N signaling and thereby ensure proper cell fate specification in Drosophila mechanosensory organs (Jafar-Nejad, 2005).

The various cell types that form an adult sensory organ in Drosophila are generated via asymmetric divisions of a pI and its progeny. Differential activation of the N signaling pathway between the two daughter cells of each division ensures that each sensory organ acquires the proper complement of cell types necessary to function. Sec15, a component of the evolutionarily conserved exocyst complex as reported here, is required for proper cell fate specification of the pI progeny. Studies on sec15 mutations in the eye did not reveal any fate change in the photoreceptors. Loss-of-function mutations in three other exocyst components have been reported previously: sec5 and sec6 in flies and sec8 in mice. sec8 mutant mice die at day E7.5, before the development of specific neuronal populations can be studied. Also, sec5 and sec6 mutations are cell lethal in the Drosophila eye. Therefore, this report is the first to identify a role for an exocyst component in cell fate determination. At this point, it cannot be predicted if sec5 and sec6 also play a role in neuronal cell fate specification. However, given the data obtained from studies of the fly eye, the hypothesis is favored that components of the exocyst may form more than a single functional unit and/or have subunit-specific roles (Jafar-Nejad, 2005).

Live imaging of dividing pI cells indicates that Sec15 is associated with a vesicular compartment that traffics between apical and subapical areas. In sec15⁻ SOPs, an expanded compartment is observed that contains Spdo, N, and Dl. Unlike wt pI and pIIb cells, in which Spdo/N/Dl⁺ vesicles tend to reside at or above the level of septate junctions, in mutant SOPs these puncta accumulate at the basal side of the cell. Together, these observations suggest that Sec15 is involved in vesicle trafficking to the apical parts of the cell. The defect in the apical trafficking of proteins does not seem to be a general one, since localization of E-Cad and Arm at the adherens junction is not disrupted in mutant tissue. Therefore, the data link a specific vesicle trafficking event to a developmental decision made by sensory precursor cells (Jafar-Nejad, 2005).

Genetic experiments and immunohistochemical stainings strongly suggest that Sec15 and Spdo function in the same pathway in sensory cell fate determination process. It has been proposed, based on studies performed on the asymmetric divisions of Drosophila embryonic neuroblasts, that Spdo promotes N signaling at the membrane of the signal-receiving cell. In contrast, Numb and α-Adaptin in the signal-sending cell might promote endocytosis of Spdo and its removal from the membrane, thereby preventing the reception of signal by this cell. The subcellular distribution of Spdo in pIIa and pIIb cells is similar to its localization in embryonic neuroblast progeny, suggesting that this model might also apply to adult bristle formation. Notably, however, Spdo is observed at or close to the membrane of both pI progeny in sec15 clones. Therefore, while the proposed role for Spdo in promoting N signaling at the membrane of the signal-receiving cell cannot be ruled out, the data suggest a role for Spdo in Dl recycling in the signal-sending cell. It should be noted, though, that these two models are not mutually exclusive. Presence of a significantly higher number of vesicles containing both Dl and N in pIIb compared to the pIIa in wt sensory precursors has been implicated in the ability of the pIIb cell to send the Dl signal. Colocalization of Spdo with Dl in a significant fraction of these vesicles suggests that a defect in Spdo/Dl trafficking in pIIb contributes to the sec15 loss-of-function phenotype (Jafar-Nejad, 2005).

Presence of endocytosed Dl in vesicles that accumulate in sec15 clones implicates these vesicles in the endocytic traffic of Dl. This notion is further supported by the observation that in both wt and sec15⁻ SOPs, the Spdo/Dl/N puncta show a significant colocalization with the endosomal markers Rab5 and HRS. It has recently been proposed that in order to signal, Dl needs to traffic through a specific endocytic compartment, which will lead to recycling of the protein. A defect in Dl recycling is further suggested by the aberrant accumulation of the recycling endosomal marker Rab11 in sec15 clones. The Rab11⁺ endosomal compartment is thought to be a central trafficking intermediate in both exocytic and endocytic pathways and is shown to control the traffic of cargo from the perinuclear recycling endosomal compartment to the membrane. Interestingly, it has been shown that Sec15 meets the criteria of being an effector for Rab11 in mammalian cell lines: Sec15 physically binds Rab11 in a GTP-dependent manner; Sec15 colocalizes with Rab11 in the perinuclear region of the cells; Sec15 labels structures containing an endocytosed protein in immuno-EM experiments. Similarly, Drosophila Sec15 and Rab11 interact physically and show a high level of colocalization in SOPs. Altogether, these data are compatible with a model in which Sec15 regulates the traffic of a subset of endocytosed Dl to the membrane of the pIIb cell via a Rab11⁺ recycling endosomal compartment. Sec15 traffics symmetrically in pIIa and pIIb. Therefore, it is proposed that an intrinsic difference between the endocytic traffic of Dl in pIIa and pIIb allows the pIIb cell to employ the Sec15-Rab11 machinery differentially from the pIIa cell and thereby assume the role of signal-sending cell. The most likely mechanisms for the proposed intrinsic difference are unequal segregation of Neur into the pIIb, which promotes Dl endocytosis in this cell, and asymmetric distribution of the Rab11⁺ recycling endosomes in the pIIb versus pIIa, which is thought to specifically mediate Dl recycling in the pIIb (Jafar-Nejad, 2005).

These data suggest that at least some of the Spdo/N/Dl-containing vesicles that accumulate in the basal areas of sec15⁻ SOPs are of a mixed exo-endocytic nature. This is not unprecedented, since traffic from the TGN to an endosomal compartment has been documented. Accordingly, it has been proposed that some exocytic cargo might pass through the recycling endosome on its way from the TGN to the plasma membrane. Recently, it has been shown that upon exit from the Golgi apparatus, newly synthesized E-Cad fuses with a Rab11⁺ recycling endosomal compartment before it reaches the plasma membrane. It is interesting to note that members of the exocyst complex have been shown to localize to both the TGN and recycling endosomes in polarizing epithelial cells. Although the recycling endosome has been proposed as an intermediate to transfer the exocytic cargo to the plasma membrane, it is possible that passing through these vesicles somehow enhances the signaling ability of internalized Dl. In other words, presence of Spdo might be part of the specific environment that Dl needs to traffic through. Although Dl endocytosis and recycling are also implicated in N signaling during lateral inhibition, no lateral inhibition defects are observed in sec15 clones. It is proposed that the link to Spdo results in the specificity of the sec15 phenotype to the asymmetric divisions, since loss of spdo similarly does not affect lateral inhibition (Jafar-Nejad, 2005).

In summary, the data indicate that one component of the highly conserved exocyst complex affects the asymmetric division of the sensory precursors in the Drosophila PNS through specific vesicle trafficking events. Components of the exocyst complex are conserved from yeast to human, and several reports have shown parallels between the contribution of asymmetric divisions to Drosophila and vertebrate neurogenesis. Therefore, it is conceivable that Sec15, and perhaps other members of the exocyst complex, are involved in neural cell fate determination in other species (Jafar-Nejad, 2005).

Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth

Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches (Chabu, 2014).

Protein Interactions

Sec15, a component of the exocyst, recognizes vesicle-associated Rab GTPases, helps target transport vesicles to the budding sites in yeast and is thought to recruit other exocyst proteins. This study reports the characterization of a 35-kDa fragment that comprises most of the C-terminal half of Drosophila Sec15. This C-terminal domain binds a subset of Rab GTPases, especially Rab11, in a GTP-dependent manner. Evidence is provided that in fly photoreceptors Sec15 colocalizes with Rab11 and that loss of Sec15 affects rhabdomere morphology. Determination of the 2.5-Å crystal structure of the C-terminal domain revealed a novel fold consisting of ten alpha-helices equally distributed between two subdomains (N and C subdomains). The C subdomain, mainly via a single helix, is sufficient for Rab binding (Wu, 2005).

Sec15 plays multiple key roles in a variety of processes in exocytosis by interacting with vesicle-associated small Rab GTPases, assisting in targeting vesicles to budding sites and recruiting, by way of Sec10, other components of the exocyst. in vitro and in vivo studies have shed light on some of these roles. The relatively large size (85 kDa) of Sec15 ensures that there are enough docking sites, each probably residing in either a single domain or a combination of domains, for other protein components necessary for its diverse functions. These studies revealed a segment named the C-terminal domain, containing nearly the entire C-terminal half of the protein, possesses several interesting properties. This domain binds to a set of Rabs in a GTP-dependent manner. Its atomic structure is composed of two distinct subdomains, only one of which (the C subdomain) harbors the Rab-binding site. The finding of a bipartite C-terminal domain was unexpected; there was no hint of this feature even from the BLAST Conserved Domain Database search for domains. The all-helical structure of the domain, with its two different subdomains, has a novel fold. A search for overall structural similarities of the whole domain and of the N and C subdomains separately against the DALI database, a network tool for protein structure comparison, did not find any substantial matches (Wu, 2005).

Binding studies further show that Sec15 lacks stringent substrate specificity in vitro. The domain binds Rab11, Rab3, Rab8 and Rab27, but the binding data suggest that Rab11 is the major target. The ability of an effector protein to interact with different Rab proteins has been observed previously: rabphilin-3, originally identified as a Rab3-binding protein, has also been shown to interact with Rab8 and Rab27 in a cotransfection assay in COS-7 cells. In addition, Sec15 and Sec5 apparently do not have significant roles in regulating neurotransmitter release, a process in which Rab3 has been implicated. This suggests that the weaker in vitro binding of Sec15 with Rab3, Rab8 and Rab27 may have in vivo consequences. It remains to be investigated whether these interactions have any role in vivo (Wu, 2005).

The Rab-binding site is apparently confined mainly to the exposed middle three-fourths of one helix (alpha9) of the C subdomain, which contains mostly hydrophobic residues. The participation of hydrophobic residues in binding is a common feature observed in several crystal structures of complexes of effectors with their cognate small GTPases. The in vivo consequence of abolishing the Sec15-Rab11 interaction by using the Sec15 mutants is under investigation (Wu, 2005).

These studies further raise questions about the role of the ~15-kDa N subdomain, which is also composed entirely of helices, but helices with various lengths and with topology and geometry different from those in the C subdomain. The same questions could also apply to the function(s) of the ~40-kDa N-terminal half of Sec15. The combination of the N subdomain and the N-terminal half, which is approximately two-thirds of the entire Sec15 protein, seems too large for binding only the Sec10 component of the exocyst. This suggests that Sec15 may have additional partners that have yet to be identified (Wu, 2005).

There are informative differences between the phenotypes in rhodopsin1 trafficking and rhabdomere morphogenesis of rab11 and sec15 mutants. A defect in the initial delivery of rhodopsin in rab11 mutants has been associated with accumulations of rhodopsin outside the rhabdomere. No noticeable amounts of rhodopsin were observed outside the rhabdomeres in sec15 mutants. This observation, combined with the normal photoreceptor depolarization measured by electroretinography, suggests that initial delivery of rhodopsin occurs in sec15 mutants. However, there is also a significant defect in rhabdomere morphology in rab11 mutant photoreceptors. Hence, the photoreceptor staining data suggest that although targeted membrane delivery to the rhabdomere might occur in Sec15-mutant cells, defects in membrane recycling (evidenced by the strong accumulation of Rab11 in the rhabdomere) probably lead to disruptions in rhabdomere morphology over time. Together, these data suggest that the rab11 mutant phenotype is more severe than the sec15 mutant phenotype but that the two show some similarities. In summary, the interaction of Rab11 and Sec15 has functional consequences in vivo, in that Rab11 trafficking is disrupted in sec15 mutants and at least one aspect of the sec15 mutant phenotype, namely abnormal rhabdomere morphology, can be explained by loss of Rab11 function. An interaction between Sec15 and Rab11 has also been shown to have an important role in the asymmetric division of sensory organ precursors in Drosophila, in that Sec15 promotes Notch signaling during the asymmetric division of sensory organs, suggesting that Sec15/Rab11 interaction is required not only in rhodopsin targeting but also during cell-fate specification (Wu, 2005).

DEVELOPMENTAL BIOLOGY

Pupal

To determine the expression pattern and subcellular localization of Sec15, a polyclonal antibody was generated against a fragment of Sec15. The antibody is specific to Sec15; staining of sec15 homozygous mutant eye disc clones display a reduction of staining to background levels. During initial target recognition with glia (5% of pupal development) and during cartridge formation (30%), Sec15 immunoreactivity is highly enriched in the developing optic lobe neuropils, including the lamina and medulla. Costaining with mAb24B10 shows that photoreceptor terminals contain Sec15, as do synaptic terminals of other cells that contribute to the neuropil. In the second half of pupation, synapse formation commences in the lamina, and Sec15 immunoreactivity decreases in all neuropils. However, low levels of Sec15 immunoreactivity persist into adulthood in a very distinct punctate staining pattern. Sec15 punctae are localized within the glial border of each cartridge, indicating that they are present in photoreceptor terminals as well as pre- and post-synaptic endings of other cell types. These punctae mostly do not colocalize with n-synaptobrevin and synaptotagmin I and are located near active zones. In summary, these data indicate that Sec15 is present at the correct time and place during development to account for the neuronal targeting defects in the mutants and may serve other functions into adulthood by specifying distinct subdomains of synaptic terminals (Mehta, 2005).

EFFECTS OF MUTATION

To isolate new genes that play a role in synapse development or function, an F1 screen was carried out in the Drosophila visual system. Using the eyFLP system, 210,000 flies were created with eyes homozygous for a randomly induced chemical mutation while keeping the rest of the body heterozygous. Two assays were employed to identify mutations that cause a failure to evoke a postsynaptic response. To identify mutations that affect the accuracy of synaptic contacts (synaptic specificity), neuronal targeting defects were assessed with light microscopy for 450 mutants and synapse formation with electron microscopy for 40 complementation groups (Mehta, 2005).

Flies were selected with grossly normal eye morphology that phototax poorly or not at all. Control flies consistently walk toward light, while flies that do not synthesize the neurotransmitter histamine fail to phototax. Two independently isolated mutations that failed to complement each other, 3R41¹ and 3R41², display an aberrant response to light (red and blue). Electroretinograms (ERGs) were performed to identify mutations that cause a lack of 'on' and 'off' responses but display a normal depolarization profile. The lack of an on and off response is thought to indicate a lack of, or aberrant communication between, pre- and post-synaptic cells. This can be caused by (1) a defect in neurotransmission or (2) a developmental defect in synapse formation. To test for developmental defects at the level of light microscopy, adult brains were stained with the photoreceptor-specific antibody against chaoptin, mAb 24B10. The Drosophila compound eye consists of 800 unit eyes, called ommatidia, each with a complement of eight photoreceptor cells. mAb 24B10 staining reveals the morphology of photoreceptor terminals R1-R6 in the first optic neuropil, the lamina, as well as R7-R8 in the second optic neuropil, the medulla. 3D visualization of the R7/8 terminal field in the medulla of a control animal reveals a highly regular array of terminals. The terminals of R7 and R8 synapse in separate layers in the medulla. In contrast, 3R41¹ mutant photoreceptors display loss of the regular array of terminals in the medulla and highly aberrant R7 and R8 target layering. It was next analyzed whether these morphological disruptions are the result of long-range growth cone guidance defects or short-range wiring disruptions within the correct brain areas. Visualization of the adult optic neuropils with the synaptic marker N-cadherin revealed strong morphological disruptions of neuropil shape, but no alteration of their arrangement or size, indicating morphological disruptions only within the neuropils in mutant optic lobes. Visualization of only the R7 photoreceptor using R7-specific GFP expression in an eyFLP 3R41 mutant background revealed that all observable R7 terminals project into the distal medulla. While gross defects in R7 axon outgrowth were not detected, it cannot be rule out that tere are more subtle defects that are beyond the resolution of the described analyses. However, the data indicate that axons are not affected in long-range axonal pathfinding, axon extension, and the recognition of the correct brain areas or neuropils. Based on ERG results, mutant photoreceptors are able to sense light and depolarize normally after stimulation. Taken together, these data suggest that 3R41 mutant neurons do not exhibit disruptions in general cellular processes, but exhibit a severe and specific defect of neuronal terminals in establishing a normal, local wiring pattern within their correct neuropils (Mehta, 2005).

Sec15 is conserved from yeast to humans over the length of the protein. The second instar lethality associated with the loss of sec15 was rescueable using a 5 kb genomic fragment. Using expression of the sec15 cDNA in eyes in an eyFLP; sec15 background, both the R7–R8 terminal layering defect and the on and off transients of the ERG were rescued. These data show that phenotypes observed in the mutants are due to loss of Sec15. The identification of mutations in sec15 in a screen for synaptic specificity defects was unanticipated, given the proposed role of the exocyst in cellular polarization (Nelson, 2003) and neurite outgrowth, as well as the cell lethality associated with the loss of sec5 (Murthy, 2003) in photoreceptors (Mehta, 2005).

Mutations in sec15 cause defects in synaptic specificity and axon targeting

To determine whether R1-R6 photoreceptors display defects in synapse morphology, synapse formation, or vesicle morphology/number, wild-type and mutant laminae were analyzed by transmission electron microscopy (TEM). R1-R6 terminals from different ommatidia are organized into synaptic units called cartridges. During late larval stages, axons of R1-R6 invade the developing lamina plexus, where their growth cones are halted by interactions with glial cells. Initially, photoreceptors from the same ommatidium travel together in bundles. Later, during the first half of pupation, the R1-R6 terminals defasciculate and organize themselves into cartridges. In this sorting process, photoreceptors that receive light from the same point in space but reside in different ommatidia sort into a single cartridge according to the principle of neural superposition. Synaptogenesis does not start before cartridge formation is complete at the beginning of the second half of pupation. Quantitative ultrastructural studies of 1-day-old adults was performed to assess cartridge formation and synapse formation. Cartridges consist of six photoreceptor terminals that surround the processes of the L1 and L2 postsynaptic lamina monopolar cells. Photoreceptor terminals were identified based on the presence of capitate projections. The cartridges are delimited by epithelial glia. In eyFLP; 3R41¹ and eyFLP; 3R41² mutant laminae, cartridges are easily identifiable, but they contain highly variable numbers of photoreceptor terminals. Quantitative analysis of terminal number per cartridge revealed that eyFLP; 3R41 mutant laminae have a much broader distribution of photoreceptor terminals per cartridge than wild-type, indicating a defect in cartridge formation (Mehta, 2005).

Whether synapses are formed in these mis-sorted cartridges was analyzed. Drosophila photoreceptors form tetrad synapses in which the presynaptic active zone makes contacts with four postsynaptic dendrites from lamina monopolar and amacrine cells. The presynaptic active zone is identified by an electron-dense structure known as the 'T bar', and at least two of the four postsynaptic dendrites are identifiable in an ultrathin section at most angles. The typical configuration of tetrad synapses in which two T bars face each other and share postsynaptic processes is observed. In eyFLP; 3R41 mutant laminae, synapses appear morphologically normal and occur with a similar frequency as in controls, indicating that 3R41 mutants have no defect in synapse assembly. The synaptic vesicle content of mutant terminals appears to be normal, and immunohistochemical analyses of synaptic vesicle proteins including synaptotagmin and neuronal synaptobrevin revealed no altered distribution. It is concluded that 3R41 mutants exhibit a defect in synaptic specificity, since qualitatively and quantitatively normal synapses are formed in cartridges containing an incorrect complement of photoreceptor terminals (Mehta, 2005).

Homozygous mutant eyFLP; 3R41 eyes are generally smooth with only occasional irregularities in the ommatidial array. To ensure that the observed defects are not due to secondary defects in photoreceptor specification or differentiation, third instar larval imaginal discs containing marked 3R41 mutant clones at the time of axonal outgrowth were labeled with a variety of markers. No obvious defects were observed in patterning. Since early developing mutant photoreceptors are indistinguishable from wild-type and photoreceptors are able to respond to light stimuli (as evidenced by normal depolarization of the ERG), it is concluded that the targeting defects are not due to neuronal differentiation defects (Mehta, 2005).

Sec15 is required in photoreceptors for synaptic specificity but does not play an important role in neurotransmitter release

The eyFLP system generates homozygous mutant photoreceptors, as well as homozygous mutant lamina and medulla cells. This implies that some aspects of the sec15 mutant phenotype that were observe may not be caused by loss of Sec15 in photoreceptors. The finding that driving the sec15 cDNA only in eye tissue largely rescues the R7/R8 targeting defects as well as the ERG defect in eyFLP; sec15 mutant optic lobes indicates that Sec15 plays a critical role in photoreceptors. To further investigate the cell type-specific aspects of the observed phenotypes, two sets of experiments were devised (Mehta, 2005).

(1) The eyFLP system was used in combination with the MARCM technique to generate laminae in which 50% of the photoreceptors were homozygous mutant for sec15 in a random distribution. In these laminae, mutant photoreceptors express GFP, while other mutant optic lobe cells are not marked. Strong morphological disruptions are invariably and selectively seen in clones with marked mutant photoreceptors. Interestingly, areas with no mutant photoreceptors have very subtle or no morphological defects. These areas may contain mutant optic lobe cells despite not having any mutant photoreceptor terminals. Since an eye-specific driver was use to express GFP in mutant cells, the lamina cells that are mutant will not be marked. This finding indicates that the contribution of these cells to the overall morphological phenotype is minor. Also elevated levels of chaoptin were observed in isolated mutant terminals at the clone borders. To quantify this effect, Twelve cartridges at clone boundaries containing single mutant photoreceptor terminals were analyzed. 3D reconstructions were made of mutant terminals with one adjacent control terminal in each cartridge and pairwise mean fluorescence level ratios between mutant and control terminals were calculated. Isolated mutant terminals displayed a 62.3% (±11.7%) increase in chaoptin levels, indicating a cell-autonomous upregulation of chaoptin in sec15 mutant photoreceptor terminals (Mehta, 2005).

(2) In a second set of experiments, use was made of a new eyeless FLPase system developed by Iris Salecker and colleagues, called ey3.5FLP. This system uses a specific eyeless enhancer that only drives FLPase expression in eye imaginal discs (I. Salecker, personal communication to Mehta, 2005). This ensures that the only mutant terminals in the lamina are from photoreceptors. It was found that ey3.5FLP; sec15 mutant optic lobes still exhibit neuronal targeting defects. However, the ERGs exhibit on and off transients, albeit at reduced size. This clearly indicates that neurotransmitter release persists in photoreceptors lacking Sec15, even though TEM of the laminae of these flies revealed cartridges with abnormal numbers of terminals. However, the distribution of terminals per cartridge for sec15 mutants was less broad using the ey3.5FLP system compared to the eyFLP system. These data indicate that Sec15 is required for neuronal targeting in photoreceptors and also serves a function in other neurons. Since on and off transients in ERGs are field potential recordings of the synchronized firing of postsynaptic cells in the lamina, it is suspected that the loss of on and off transients in eyFLP; sec15¹ flies are secondary to the morphological defects. If only photoreceptors are made mutant using the ey3.5FLP system, the miswiring is less severe, causing small on and off responses to return. Likewise, when only nonphotoreceptor optic lobe cells are made mutant in the photoreceptor-specific sec15 cDNA rescue of the eyFLP; sec15¹ phenotype, on and off responses also persist and the R7/R8 targeting defect is greatly reduced. Sec15 must be removed from both populations of neurons (as in the eyFLP system) in order to eliminate the on and off responses, indicating that the loss is a cumulative effect secondary to morphological disruptions. These data argue that Sec15 is required in photoreceptors for correct neuronal targeting, but does not play an important role in regulating neurotransmission (Mehta, 2005).

Specific cell adhesion and signaling molecules are mislocalized in sec15 mutant photoreceptor terminals

Elevated levels of chaoptin in photoreceptor terminals have been described for another vesicle-trafficking mutant, the vesicle-SNARE neuronal-synaptobrevin (n-syb). This mutant also exhibits neuronal targeting defects. This observation raises the possibility that vesicle-dependent trafficking of transmembrane or other signaling molecules might be responsible for the neuronal targeting defects of sec15 mutant photoreceptors. Recently, Zhang (2004) identified Rab11 as an interacting partner of Sec15 in mammalian cell culture and proposed that Sec15 is an effector for some but not all Rabs. Indeed, an accumulation or upregulation of Rab11 immunoreactivity was seen in sec15 mutant photoreceptors, consistent with Rab11-positive vesicles failing to fuse with their target sites. To further test this hypothesis, the localization of cell adhesion and signaling molecules was examined in mutant photoreceptor cell bodies as well as terminals during photoreceptor development, precisely when target selection and cartridge formation occur (between P + 5% to P + 40% referring to time after pupation). Proteins were examined that have either been shown to be required for photoreceptor target selection, such as Dlar, N-cadherin, flamingo, and IrreC-rst, or that are likely to be required, based on work in other systems, such as Armadillo, Chaoptin, and Fasciclin II (Mehta, 2005).

Fasciclin II (Fas2) localization was examined in sec15 mutant photoreceptors, since chaoptin upregulation coincides with elevated levels of Fas2 in n-syb mutant photoreceptors. Fas2 appears to be present in aggregates in sec15 mutant photoreceptor cell bodies at P + 20%, in contrast to wild-type photoreceptors. In addition, the neuronal connections of the cell bodies exhibit Fas2 aggregated along the length of the mutant axons. Similarly, overexpression of Fas2 in photoreceptors causes neuronal targeting defects between P + 20% and P + 40%. In contrast to n-syb, however, no elevated levels of Fas2 are observed later in development. Hence, the data suggest that an aberrant localization of Fas2 in a specific developmental time window may at least partially underlie the observed phenotypes (Mehta, 2005).

Similar mislocalization phenotypes in photoreceptor cell bodies were also observed for other cell adhesion molecules such as Dlar and IrreC-rst during the developmental time window of photoreceptor target selection. Dlar is normally restricted apically in developing wild-type photoreceptors, at the center of the ommatidial array. In sec15 mutant photoreceptors it appears much more randomly distributed, such that a basal optical section through the eye shows Dlar at higher levels in mutant ommatidia. Although these results show mislocalization of cell adhesion molecules in the correct cell at the time when they are known to be required for proper target selection, no obvious defects were detected in the localization of Dlar or IrreC-rst in the developing lamina. This leaves open the question of whether mislocalization of Dlar and IrreC-rst beyond the resolution limit of confocal microscopy additionally contributes to the observed targeting defects (Mehta, 2005).

In vertebrates, Lar is known to localize to adherens junctions. Hence, a possible explanation for the mislocalization of Fas2, IrreC-rst, and Dlar in mutant photoreceptor cell bodies is a defect of adherens junctions. The subcellular localization of the adherens junction markers N-cadherin and armadillo was examined in the cell bodies as well as the terminals of mutant photoreceptors, but no mislocalization of N-cadherin was detected in either compartment. However, armadillo displayed localization defects selectively in the developing lamina, but not the photoreceptor cell bodies. Several other cell adhesion and signaling molecules, including flamingo, Crumbs, and Bazooka, were examined, all of which did not display aberrant localization at the level of light microscopy. It is concluded conclude that a specific subset of proteins is mislocalized in sec15 mutants (Mehta, 2005).

Sec15 is required for the localization of some but not all exocyst members to neuronal terminals

Does Sec15 exert an exocyst-dependent function at the neuronal terminal? To date, only one other mutant has been reported that affects a component of the exocyst in Drosophila, namely sec5 (Murthy, 2003). Murthy showed that Sec5 is required for cell polarization in the developing oocyte and neurite outgrowth in cell culture. In vivo, homozygous mutations in sec5 are lethal in photoreceptor neurons (Murthy, 2003), as are mutations in sec6 (S.B. and U.T., unpublished data). In contrast, it was observed that sec15 homozygous mutant photoreceptor neurons are viable, even in aged flies. Either Sec15 exerts a function independent of the exocyst at the neuronal terminal, or its developmental role only represents a specialized task of the complex or subcomplex. To distinguish between these two possibilities, the localization of Sec5, Sec6, and Sec8 in developing neuropil was examined as well as in sec15 mutant clones (Mehta, 2005).

In the developing lamina of the late third instar larva, Sec5 and Sec15 colocalize. Both are highly enriched in the developing neuropil, whereas immunoreactivity in the functional larval central brain is much lower. In the adult lamina, Sec5 and Sec15 are coexpressed in cartridges. Sec5 colocalizes with Sec15 to a larger extent than any of the other markers tested, including plasmalemmal, synaptic vesicle, or active zone markers. However, the colocalization of Sec15 and Sec5 is not perfect, leaving subdomains marked only by anti-Sec15 or anti-Sec5. These data suggest that Sec5 and Sec15 may have common as well as separate functions (Mehta, 2005).

The expression patterns of two other presumed core members of the exocyst, Sec6 and Sec8, were examined using two newly generated polyclonal antibodies. In adult lamina cartridges, Sec6 immunoreactivity exhibits a very specific pattern that exactly matches the localization of the postsynaptic lamina monopolar cells. In contrast, the antibody against Sec8 exhibits a punctate staining pattern throughout the cartridges that is similar to Sec15. Likewise, Sec6 and Sec8 antibodies have both specific but different staining patterns in the developing brain: Sec6 strongly colocalizes with Sec5 in developing neuropil, whereas Sec8 is enriched in cell bodies but is almost completely excluded from the developing neuropil. Finally, stainings were performed to examine the localization of Sec15, Sec6, and Sec8 at the third instar larval neuromuscular junction. Sec15 is present in both boutons and muscle cells, but seems enriched at boutons. In contrast, Sec 6 is highly enriched at the Z bands of muscle cells and very weakly present in boutons, while Sec8 is not present in muscle cells or neurons, but is in a highly punctate distribution in unidentified processes that may be glial projections. These data are not consistent with a single functional Sec6/8 complex (Mehta, 2005).

To test whether Sec15 at the photoreceptor terminals affects Sec5, Sec6, and Sec8, the presence of these proteins was investigated in sec15 mutant clones. Sec5 immunoreactivity in sec15 mutant clones of photoreceptor terminals in the lamina is markedly reduced and possibly absent in the terminals. Likewise, Sec8 immunoreactivity is reduced in sec15 mutant clones of photoreceptor terminals. However, the levels of Sec6 appear to be unaffected by mutations in sec15. This is likely because Sec6 is enriched in postsynaptic cells in the lamina and because it seems to be absent presynaptically. In addition, the specific developmental and adult staining patterns of Sec8, as well as its downregulation in sec15 mutant clones, suggest common and separable functions compared to sec5, sec6, and sec15 at different points in development. Since loss of sec5 in photoreceptors causes cell lethality, the loss or downregulation of Sec5 in sec15 mutant terminals is unlikely to reflect a global loss of the protein. These data rather suggest that Sec15 is required for localization of Sec5 and Sec8, but not Sec6 to the presynaptic photoreceptor terminal. These data suggest that Sec15 may recruit or stabilize a complex that includes some but not all exocyst members in photoreceptor terminals in a spatiotemporally regulated manner (Mehta, 2005).

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate E-Cadherin trafficking from recycling endosomes to the plasma membrane

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 (Lock, 2005). 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 (Lock, 2005; Miranda, 2001). 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 (Yeaman, 2004). 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 (Lock, 2005). Furthermore, the overexpression of a dominant-negative form of Rab11 impairs the delivery of E-Cadherin to the lateral membrane (Lock, 2005). Consistent with the exocyst regulating trafficking from the REs, exocyst components also localize on the REs, and Sec15 is an effector of Rab11 (Folsch, 2003: Prigent, 2003 ; Zhang, 2004). Finally, E-Cadherin and catenins are associated with exocyst components (Yeaman, 2004; Langevin, 2005).

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).

EVOLUTIONARY HOMOLOGS

Identification and characterization of Sec15 in yeast

SEC15 encodes a 116-kD protein that is essential for vesicular traffic from the Golgi apparatus to the cell surface in yeast. Although the sequence predicts a largely hydrophilic protein, a portion (23%) of Sec15p is found in association with the plasma membrane. The remainder is not associated with a membrane but is found in a 19.5S particle which is not dissociated by 0.5 M NaCl. Sec15p may attach directly to the plasma membrane since it is not found on the Golgi apparatus nor on the secretory vesicle precursors to the plasma membrane. Loss of function of most of the late-acting sec gene products does not alter the distribution of Sec15p. However, the sec8-9 mutation and to a lesser extent the sec10-2 mutation result in a shift of Sec15p to the plasma membrane, suggesting a role for these gene products in the regulation of the Sec15p membrane attachment/detachment processes. Depletion of Sec15p by repression of synthesis indicates that the plasma membrane bound pool is the most stable (Bowser, 1991).

The SEC8 and SEC15 genes are essential for exocytosis in the yeast Saccharomyces cerevisiae and exhibit strong genetic interactions with SEC4, a gene of the ras superfamily. The SEC8 gene encodes a hydrophilic protein of 122 kD, while the temperature-sensitive sec8-9 allele encodes a protein prematurely truncated at 82 kD by an opal stop codon. The Sec8p sequence contains a 202 amino acid region that is 25% identical to the leucine rich domain of yeast adenylate cyclase that has been implicated in ras responsiveness. Fractionation, stability, and cross-linking studies indicate that Sec8p is a component of a 19.5S particle that also contains Sec15p. This particle is found both in the cytosol and peripherally associated with the plasma membrane, but it is not associated with secretory vesicles. Gel filtration studies suggest that a portion of Sec4p is in association with the Sec8p/Sec15p particle. It is proposed that this particle may function as a downstream effector of Sec4p, serving to direct the fusion of secretory vesicles with the plasma membrane (Bowser, 1992)

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides that range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis (TerBush, 1995).

Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. The exocyst complex plays a key role in vesicle targeting. Sec15p, an exocyst component, can associate with secretory vesicles and interact specifically with the rab GTPase, Sec4p, in its GTP-bound form. A chain of protein-protein interactions leads from Sec4p and Sec15p on the vesicle, through various subunits of the exocyst, to Sec3p, which marks the sites of exocytosis on the plasma membrane. Sec4p may control the assembly of the exocyst. The exocyst may therefore function as a rab effector system for targeted secretion (Guo, 1999).

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. SEB1/SBH1 encodes the beta subunit of the Sec61p ER translocation complex, and acts as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. The functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery has been demonstrated. Overexpression of SEB1 suppresses the growth defect in all exocyst sec mutants. In addition, overexpression of SEC61 or SSS1 encoding the other two components of the Sec61p complex suppresses the growth defects of several exocyst mutants. Seb1p coimmunoprecipitates from yeast cell lysates with Sec15p and Sec8p, components of the exocyst complex, and with Sec4p, a secretory vesicle associated Rab GTPase that binds to Sec15p and is essential for exocytosis. The interaction between Seb1p and Sec15p was abolished in sec15-1 mutant and was restored upon SEB1 overexpression. Furthermore, in wild type cells overexpression of SEB1 as well as SEC4 result in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst (Toikkanen, 2003).

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. Photobleaching recovery experiments were used to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis (Boyd, 2004).

Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. This study examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, it was found that all had an altered pattern of ASH1 mRNA localization. ASH1 is a specific repressor of transcription that localizes asymmetrically to the daughter cell nucleus through the localization of ASH1 mRNA to the distal tip of the daughter cell. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. These novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization (Aronov, 2004).

Polarized exocytosis is important for morphogenesis and cell growth. The exocyst is a multiprotein complex implicated in tethering secretory vesicles at specific sites of the plasma membrane for exocytosis. In the budding yeast, the exocyst is localized to sites of bud emergence or the tips of small daughter cells, where it mediates secretion and cell surface expansion. To understand how exocytosis is spatially controlled, the localization of Sec15p, a member of the exocyst complex and downstream effector of the rab protein Sec4p, was systematically analyzed in various mutants. The polarized localization of Sec15p relies on functional upstream membrane traffic, activated rab protein Sec4p, and its guanine exchange factor Sec2p. The initial targeting of both Sec4p and Sec15p to the bud tip depends on polarized actin cable. However, different recycling mechanisms for rab and Sec15p may account for the different kinetics of polarization for these two proteins. Sec3p and Sec15p, though both members of the exocyst complex, rely on distinctive targeting mechanisms for their localization. The assembly of the exocyst may integrate various cellular signals to ensure that exocytosis is tightly controlled. Key regulators of cell polarity such as Cdc42p are important for the recruitment of the exocyst to the budding site. Conversely, it was found that the proper localization of these cell polarity regulators themselves also requires a functional exocytosis pathway. Bem1p, a protein essential for the recruitment of signaling molecules for the establishment of cell polarity, interacts with the exocyst complex. It is proposed that a cyclical regulatory network contributes to the establishment and maintenance of polarized cell growth in yeast (Zajac, 2005).

Spatial regulation of the secretory machinery is essential for the formation of a new bud in Saccharomyces cerevisiae. Yet, the mechanisms underlying cross-talk between the secretory and the cell-polarity-establishment machineries have not been fully elucidated. This study reports that Sec15p, a subunit of the exocyst complex, might provide one line of communication. Not only is Sec15p an effector of the rab protein Sec4p, the master regulator of post-Golgi trafficking, but it also interacts with components of the polarity-establishment machinery. A direct physical interaction has been demonstrated between Sec15p and Bem1p, a protein involved in the Cdc42p-mediated polarity-establishment pathway, confirming a prior two-hybrid study. When this interaction is compromised, as in the case of cells lacking the N-terminal 138 residues of Bem1p, including the first Src-homology 3 (SH3) domain, the localization of green fluorescent protein (GFP)-tagged Sec15 is affected, especially in the early stage of bud growth. In addition, Sec15-1p, which is defective in Bem1p binding, mislocalizes along with Sec8p, another exocyst subunit. Overall, this evidence suggests that the interaction of Sec15p with Bem1p is important for Sec15p localization at the early stage of bud growth and, through this interaction, Sec15p might play a crucial role in integrating the signals between Sec4p and the components of the early-polarity-establishment machinery. This, in turn, helps to coordinate the secretory pathway and polarized bud growth (France, 2006).

Isolation and characterization of mammalian Sec15 homologs

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. cDNAs have been isolated encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis (Kee, 1997).

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. The exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, it was 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, an in vitro exocyst reconstitution assay was established and exocyst effect on microtubule dynamics was studied. The exocyst complex reconstituted from eight recombinant exocyst subunits inhibits tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminishes its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself is also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion does not lose its activity completely. Overexpression of exo70 in NRK cells results in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions is greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that does not show tubulin polymerization inhibition activity, does 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 (Wang, 2004).

Hemoglobin deficit (hbd) mice carry a spontaneous mutation that impairs erythroid iron assimilation but does not cause other defects. Normal delivery of iron to developing erythroid precursors is highly dependent on the transferrin cycle. Through genetic mapping and complementation experiments, the hbd mutation has been shown to be an in-frame deletion of a conserved exon of the mouse gene Sec15l1, encoding one of two Sec15 proteins implicated in the mammalian exocyst complex. Sec15l1 is linked to the transferrin cycle through its interaction with Rab11, a GTPase involved in vesicular trafficking. It is proposed that inactivation of Sec15l1 alters recycling of transferrin cycle endosomes and increases the release of transferrin receptor exocytic vesicles. This in turn decreases erythroid iron uptake. Determining the molecular basis of the hbd phenotype provides new insight into the intricate mechanisms necessary for normal erythroid iron uptake and the function of a mammalian exocyst protein (Lim, 2005).

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. A positional cloning project was undertaken to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. Reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. A strong candidate gene for hbd has been identified, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals (White, 2005).

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, the localization and function of the Sec15 exocyst subunit was examined in mammalian cells. Overexpressed Sec15 associates with clusters of tubular/vesicular elements that are concentrated in the perinuclear region. The tubular/vesicular clusters are dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and are accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalizes selectively with the recycling endosome marker Rab11 and exhibits a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells (Zhang, 2004).

REFERENCES

Search PubMed for articles about Drosophila Secretory 15

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(12): 906-21. 12453153

Ang, A. L., et al. (2004). Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 167(3): 531-43. 15534004

Aronov, S. and Gerst, J. E. (2004). Involvement of the late secretory pathway in actin regulation and mRNA transport in yeast. J. Biol. Chem. 279(35): 36962-71. 15192110

Bennett, M. K. and Scheller, R. H. (1993). The molecular machinery for secretion is conserved from yeast to neurons. Proc. Natl. Acad. Sci. 90(7): 2559-63. 8096639

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. 15897260

Bowser, R., and Novick, P (1991). Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle. J. Cell Biol. 112: 1117-1131. 1900300

Bowser, R., Muller, H., Govindan, B. and Novick P. (1992). Sec8p and Sec15p are components of a plasma membrane-associated 19.5S particle that may function downstream of Sec4p to control exocytosis. J. Cell Biol. 118(5): 1041-56. 1512289

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. 15583031

Chabu, C. and Xu, T. (2014). Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth. Development 141(24):4729-39. PubMed ID: 25411211

Folsch, H., Pypaert, M., Maday, S., Pelletier, L. and Mellman, I. (2003). The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. J. Cell Biol. 163(2): 351-62. 14581457

France, Y. E., Boyd, C., Coleman, J. and Novick, P. J. (2006). The polarity-establishment component Bem1p interacts with the exocyst complex through the Sec15p subunit. J. Cell Sci. 119(Pt 5): 876-88. 16478783

Grindstaff, K. K., et al. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 93(5): 731-40. 9630218

Guo, W., Roth, D., Walch-Salimena, C. and Novick, P. (1999). The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J. 18: 1071-1080. 10022848

Hsu, S. C, Hazuka, C. D., Foletti, D. L. and Scheller, R. H. (1999). Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex. Trends Cell Biol. 9(4): 150-3. 10203793

Inoue, M., Chang, L., Hwang, J., Chiang, S. H. and Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature 422(6932): 629-33. 12687004

Izaddoost, S., Nam, S. C., Bhat, M. A., Bellen, H. J. and Choi, K. W. (2002). Drosophila Crumbs is a positional cue in photoreceptor adherens junctions and rhabdomeres. Nature 416(6877): 178-83. 11850624

Kee, Y., Yoo, J. S., Hazuka, C. D., Peterson, K. E., Hsu, S. C. and Scheller, R. H. (1997). Subunit structure of the mammalian exocyst complex. Proc. Natl. Acad. Sci. 94(26): 14438-43. 9405631

Jafar-Nejad, H., et al. (2005). Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors. Dev. Cell 9(3): 351-63. 16137928

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): 355-76. 16224820

Lim, J. E., et al. (2005). A mutation in Sec15l1 causes anemia in hemoglobin deficit (hbd) mice. Nat Genet. 37(11): 1270-3. 16227995

Lock, J. G. and Stow, J. L. (2005). Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Mol. Biol. Cell. 16(4): 1744-55. 15689490

Mehta, S. Q., et al. (2005). Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 46(2): 219-32. 15848801

Miranda, K. C., et al. (2001). A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells. J. Biol. Chem. 276(25): 22565-72. 11312273

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. 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. 14681190

Murthy, M., et al. (2005). Sec6 mutations and the Drosophila exocyst complex. J. Cell Sci. 118: 1139-1150. 15728258

Nelson, W.J. (2003). Adaptation of core mechanisms to generate cell polarity. Nature 422: 766-774. 12700771

Novick, P., Field, C. and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21: 205-215. 6996832

Prigent, M., et al. (2003). ARF6 controls post-endocytic recycling through its downstream exocyst complex effector. J. Cell Biol. 163: 1111-1121. 14662749

Sans, N., et al. (2001). NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex, Nat. Cell Biol. 5: 520-530. 12738960

Schekman, R. and Novick, P. (2004). 23 genes, 23 years later. Cell 116: S13-S15. 15055575

TerBush, D. R. and Novick, P. (1995). Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J. Cell Biol. 130(2): 299-312. 7615633

Toikkanen, J. H., Miller, K. J, Soderlund, H., Jantti, J. and Keranen, S. (2003). The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae. J. Biol. Chem. 278(23): 20946-53. 12665530

Vik-Mo, E. O., et al. (2003). Sec6 is localized to the plasma membrane of mature synaptic terminals and is transported with Secretogranin II-containing vesicles. Neuroscience 119: 73-85. 12763070

Wang, S., Liu, Y., Adamson, C. L., Valdez, G., Guo, W. and Hsu, S. C. (2004). The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization. J. Biol. Chem. 279(34): 35958-66. 15205466

White, R. A, et al. (2005). Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking. Genomics 86(6): 668-73. 16289749

Whyte, J. R. and Munro, S. (2001). The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic. Dev. Cell 1: 527-537. 11703943

Whyte, J. R. and Munro, S. (2002). Vesicle tethering complexes in membrane traffic, J. Cell Sci. 115: 2627-37. 12077354

Wu, S., et al. (2005). Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat. Struct. Mol. Biol. 12(10): 879-85. 16155582

Yeaman, C., Grindstaff, K. K. and Nelson, W. J. (2004). Mechanism of recruiting Sec6/8 (exocyst) complex to the apical junctional complex during polarization of epithelial cells. J. Cell Sci. 117: 559-570. 14709721

Zajac, A., Sun, X., Zhang, J. and Guo, W. (2005). Cyclical regulation of the exocyst and cell polarity determinants for polarized cell growth. Mol. Biol. Cell 16(3): 1500-12. 15647373

Zhang, X. M., Ellis, S., Sriratana, A., Mitchell, C. A. and Rowe, T. (2004). Sec15 is an effector for the Rab11 GTPase in mammalian cells. J. Biol. Chem. 279(41): 43027-34. 15292201

date revised: 12 January 2018

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