Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.     

The structure of the exocyst subunit Sec6p defines a conserved architecture with diverse roles

The exocyst is a conserved protein complex essential for trafficking secretory vesicles to the plasma membrane. The structure of the C-terminal domain of the exocyst subunit Sec6p reveals multiple helical bundles, which are structurally and topologically similar to Exo70p and the C-terminal domains of Exo84p and Sec15, despite <10% sequence identity. The helical bundles appear to be evolutionarily related molecular scaffolds that have diverged to create functionally distinct exocyst proteins.     

The rab exchange factor Sec2p reversibly associates with the exocyst

Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.     

The synaptobrevin homologue Snc2p recruits the exocyst to secretory vesicles by binding to Sec6p

A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.     

The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.     

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

The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.     

The role of Sec3p in secretory vesicle targeting and exocyst complex assembly

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.     

CK2 phosphorylation of human Sec63 regulates its interaction with Sec62

Background

Protein kinase CK2 is a pleiotropic enzyme which is ubiquitously expressed in eukaryotic cells. Several years ago CK2 was found to be associated with the mammalian endoplasmic reticulum. So far nothing is known about the function of CK2 at the ER.

Methods

CK2 phosphorylation sites in the polypeptide chain of Sec63 were mapped using deletion mutants and a peptide library. Binding of Sec63 to CK2 and to Sec62 was analyzed by pull-down assays and by co-immunoprecipitation

Results

Sec63 was identified as a novel substrate and binding partner of protein kinase CK2.We identified serine 574, serine 576 and serine 748 as CK2 phosphorylation sites. Phosphorylation of Sec63 by CK2 enhanced its binding to Sec62.

Conclusions

Protein kinase CK2 phosphorylation of Sec63 leads to an enhanced binding of Sec63 to Sec62. This complex formation is a prerequisite for a functional ER protein translocon.

General significance

Thus, our present data indicate a regulatory role of CK2 in the ER protein translocation.     

The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae

The exocyst is a conserved protein complex proposed to mediate vesicle tethering at the plasma membrane. Previously, we identified SEB1/SBH1, encoding the beta subunit of the Sec61p ER translocation complex, as a multicopy suppressor of the sec15-1 mutant, defective for one subunit of the exocyst complex. Here we show the functional and physical interaction between components of endoplasmic reticulum translocon and the exocytosis machinery. We show that 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 suppressed the growth defects of several exocyst mutants. Seb1p was coimmunoprecipitated 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 resulted in increased production of secreted proteins. These findings propose a novel functional and physical link between the endoplasmic reticulum translocation complex and the exocyst.     

Functional specialization within a vesicle tethering complex: bypass of a subset of exocyst deletion mutants by Sec1p or Sec4p

The exocyst is an octameric protein complex required to tether secretory vesicles to exocytic sites and to retain ER tubules at the apical tip of budded cells. Unlike the other five exocyst genes, SEC3, SEC5, and EXO70 are not essential for growth or secretion when either the upstream activator rab, Sec4p, or the downstream SNARE-binding component, Sec1p, are overproduced. Analysis of the suppressed sec3Delta, sec5Delta, and exo70Delta strains demonstrates that the corresponding proteins confer differential effects on vesicle targeting and ER inheritance. Sec3p and Sec5p are more critical than Exo70p for ER inheritance. Although nonessential under these conditions, Sec3p, Sec5p, and Exo70p are still important for tethering, as in their absence the exocyst is only partially assembled. Sec1p overproduction results in increased SNARE complex levels, indicating a role in assembly or stabilization of SNARE complexes. Furthermore, a fraction of Sec1p can be coprecipitated with the exoycst. Our results suggest that Sec1p couples exocyst-mediated vesicle tethering with SNARE-mediated docking and fusion.     

Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p.

Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores. These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD). In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p). In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived. Its proteolysis is mediated specifically by the unique Ubc6p tail region. Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine. However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p. In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p. Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates. These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p.     

Identification of Sec36p,Sec37p,and Sec38p: components of yeast complex that contains Sec34p and Sec35p

The Saccharomyces cerevisiae proteins Sec34p and Sec35p are components of a large cytosolic complex involved in protein transport through the secretory pathway. Characterization of a new secretion mutant led us to identify SEC36, which encodes a new component of this complex. Sec36p binds to Sec34p and Sec35p, and mutation of SEC36 disrupts the complex, as determined by gel filtration. Missense mutations of SEC36 are lethal with mutations in COPI subunits, indicating a functional connection between the Sec34p/sec35p complex and the COPI vesicle coat. Affinity purification of proteins that bind to Sec35p-myc allowed identification of two additional proteins in the complex. We call these two conserved proteins Sec37p and Sec38p. Disruption of either SEC37 or SEC38 affects the size of the complex that contains Sec34p and Sec35p. We also examined COD4, COD5, and DOR1, three genes recently reported to encode proteins that bind to Sec35p. Each of the eight genes that encode components of the Sec34p/sec35p complex was tested for its contribution to cell growth, protein transport, and the integrity of the complex. These tests indicate two general types of subunits: Sec34p, Sec35p, Sec36p, and Sec38p seem to form the essential core of a complex to which Sec37p, Cod4p, Cod5p, and Dor1p seem to be peripherally attached.     

Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p

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. We have used photobleaching recovery experiments 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.     

The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis

Polarized secretion requires proper targeting of secretory vesicles to specific sites on the plasma membrane. Here we report that 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.     

The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p

The Sec61 protein translocation complex in the endoplasmic reticulum (ER) membrane is composed of three subunits. The alpha-subunit, called Sec61p in yeast, is a multispanning membrane protein that forms the protein conducting channel. The functions of the smaller, carboxyl-terminally tail-anchored beta subunit Sbh1p, its close homologue Sbh2p, and the gamma subunit Sss1p are not well understood. Here we show that co-translational protein translocation into the ER is reduced in sbh1Delta sbh2Delta cells, whereas there is a limited reduction of post-translational translocation and no effect on export of a mutant form of alpha-factor precursor for ER-associated degradation in the cytosol. The translocation defect and the temperature-sensitive growth phenotype of sbh1Delta sbh2Delta cells were rescued by expression of the transmembrane domain of Sbh1p alone, and the Sbh1p transmembrane domain was sufficient for coimmunoprecipitation with Sec61p and Sss1p. Furthermore, we show that Sbh1p co-precipitates with the ER transmembrane protein Rtn1p. Sbh1p-Rtn1p complexes do not appear to contain Sss1p and Sec61p. Our results define the transmembrane domain as the minimal functional domain of the Sec61beta homologue Sbh1p in ER translocation, identify a novel interaction partner for Shb1p, and imply that Sbh1p has additional functions that are not directly linked to protein translocation in association with the Sec61 complex.     

Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.     

Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein are required for the stability of the Q/t-SNARE complex at the endoplasmic reticulum in yeast

The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi-ER retrograde transport. Size exclusion chromatography and affinity purification approaches confirmed that Dsl3p is associated with subunits of the "Dsl1p complex." The complex also includes the Q/t-SNARE proteins, Use1p, Sec20p, and Ufe1p, integral membrane proteins that constitute the trimeric acceptor for R/v-SNAREs on Golgi-derived vesicles at the ER. Using mutants, we performed a detailed analysis of interactions between subunits of the Dsl1p complex and the ER-localized SNARE proteins. This analysis showed that both Dsl1p and Dsl3p are required for the stable interaction of the SNARE Use1p with a central subcomplex consisting of Tip20p and the SNARE proteins Ufe1p and Sec20p.     

Secretion of the mammalian Sec14p-like phosphoinositide-binding p45 protein

Protein-lipid interactions are important for protein targeting, signal transduction, lipid transport, and the maintenance of cellular compartments and membranes. Specific lipid-binding protein domains, such as PH, FYVE, PX, PHD, C2 and SEC14 homology domains, mediate interactions between proteins and specific phospholipids. We recently cloned a 45-kDa protein from rat olfactory epithelium, which is homologous to the yeast Sec14p phosphatidylinositol (PtdIns) transfer protein and we report here that this protein binds to PtdIns(3,4,5)P3 and far weaker to less phosphorylated derivatives of PtdIns. Expression of the p45 protein in COS-1 cells resulted in accumulation of the protein in secretory vesicles and in the extracellular space. The secreted material contained PtdIns(3,4,5)P3. Our findings are the first report of a Sec14p-like protein involved in transport out of a cell and, to the best of our knowledge, inositol-containing phospholipids have not previously been detected in the extracellular space. Our findings suggest that p45 and phosphoinositides may participate in the formation of the protective mucus on nasal epithelium.     

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.     

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.