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).
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).
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