Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae

Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.     

Binding interactions control SNARE specificity in vivo

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.     

In vitro fusion catalyzed by the sporulation-specific t-SNARE light-chain Spo20p is stimulated by phosphatidic acid

Sec9p and Spo20p are two SNAP25 family SNARE proteins specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate intracellular trafficking between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires enzymatically active Spo14p, which is a phosphatidylcholine (PC)-specific phospholipase D that hydrolyzes PC to generate phosphatidic acid (PA). Phosphatidic acid is required to localize Spo20p properly during sporulation; however, it seems to have additional roles that are not fully understood. Here we compared the fusion mediated by all combinations of the Sec9p or Spo20p C-terminal domains with Sso1p/Sso2p and Snc1p/Snc2p. Our results show that Spo20p forms a less efficient SNARE complex than Sec9p. The combination of Sso2p/Spo20c is the least fusogenic t-SNARE complex. Incorporation of PA in the lipid bilayer stimulates SNARE-mediated membrane fusion by all t-SNARE complexes, likely by decreasing the energetic barrier during membrane merger. This effect may allow the weak SNARE complex containing Spo20p to function during sporulation. In addition, PA can directly interact with the juxtamembrane region of Sso1p, which contributes to the stimulatory effects of PA on membrane fusion. Our results suggest that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions may be coordinately regulated to control the rate and specificity of membrane fusion.     

Genetic evidence of a role for membrane lipid composition in the regulation of soluble NEM-sensitive factor receptor function in Saccharomyces cerevisiae

SEC9 and SPO20 encode SNARE proteins related to the mammalian SNAP-25 family. Sec9p associates with the SNAREs Sso1/2p and Snc1/2p to promote the fusion of vesicles with the plasma membrane. Spo20p functions with the same two partner SNAREs to mediate the fusion of vesicles with the prospore membrane during sporogenesis. A chimeric molecule, in which the helices of Sec9p that bind to Sso1/2p and Snc1/2p are replaced with the homologous regions of Spo20p, will not support vesicle fusion in vegetative cells. The phosphatidylinositol-4-phosphate-5-kinase MSS4 was isolated as a high-copy suppressor that permits this chimera to rescue the temperature-sensitive growth of a sec9-4 mutant. Suppression by MSS4 is specific to molecules that contain the Spo20p helical domains. This suppression requires an intact copy of SPO14, encoding phospholipase D. Overexpression of MSS4 leads to a recruitment of the Spo14 protein to the plasma membrane and this may be the basis for MSS4 action. Consistent with this, deletion of KES1, a gene that behaves as a negative regulator of SPO14, also promotes the function of SPO20 in vegetative cells. These results indicate that elevated levels of phosphatidic acid in the membrane may be required specifically for the function of SNARE complexes containing Spo20p.     

Molecular interactions position Mso1p, a novel PTB domain homologue, in the interface of the exocyst complex and the exocytic SNARE machinery in yeast

In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p-Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

SPO73 and SPO71 Function Cooperatively in Prospore Membrane Elongation During Sporulation in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, cells undergoing sporulation form prospore membranes to surround their meiotic nuclei. The prospore membranes ultimately become the plasma membranes of the new cells. The putative phospholipase Spo1 and the tandem Pleckstrin Homology domain protein Spo71 have previously been shown to be required for prospore membrane development, along with the constitutively expressed Vps13 involved in vacuolar sorting. Here, we utilize genetic analysis, and find that SPO73 is required for proper prospore membrane shape and, like SPO71, is necessary for prospore membrane elongation. Additionally, similar to SPO71, loss of SPO73 partially suppresses spo1Δ. Spo73 localizes to prospore membranes and complexes with Spo71. We also find that phosphatidylserine localizes to the prospore membrane. Our results suggest a model where SPO71 and SPO73 act in opposition to SPO1 to form and elongate prospore membranes, while VPS13 plays a distinct role in prospore membrane development.     

Ady4p and Spo74p are components of the meiotic spindle pole body that promote growth of the prospore membrane in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membrane-organizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74Δ/spo74Δ cells and occur aberrantly in ady4Δ/ady4Δ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.     

A novel complex of membrane proteins required for formation of a spherical nucleus.

Two membrane proteins were identified through their genetic interaction with the nucleoporin Nup84p and shown to participate in nuclear envelope morphogenesis in yeast. One component is a known sporulation factor Spo7p, and the other, Nem1p, a novel protein whose C-terminal domain is conserved during eukaryotic evolution. Spo7p and Nem1p localize to the nuclear/ER membrane and behave biochemically as integral membrane proteins. Nem1p binds to Spo7p via its conserved C-terminal domain. Although cells without Spo7p or Nem1p are viable, they exhibit a drastically altered nuclear morphology with long, pore-containing double nuclear membrane extensions. These protrusions emanate from a core nucleus which contains the DNA, and penetrate deeply into the cytoplasm. Interestingly, not only Spo7(-) and Nem1(-), but also several nucleoporin mutants are defective in sporulation. Thus, Spo7p and Nem1p, which exhibit a strong genetic link to nucleoporins of the Nup84p complex, fulfil an essential role in formation of a spherical nucleus and meiotic division.     

The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo

Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.     

The Arf-GTPase-activating protein Gcs1p is essential for sporulation and regulates the phospholipase D Spo14p

SPO14, encoding the major Saccharomyces cerevisiae phospholipase D (PLD), is essential for sporulation and mediates synthesis of the new membrane that encompasses the haploid nuclei that arise through meiotic divisions. PLD catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid (PA) and choline. PA stimulates Arf-GTPase-activating proteins (Arf-GAPs), which are involved in membrane trafficking events and actin cytoskeletal function. To determine if Spo14p-generated PA mediates its biological response through Arf-GAPs, we analyzed the sporulation efficiencies of cells deleted for each of the five known and potential yeast Arf-GAPs. Only gcs1delta mutants display a sporulation defect similar to that of spo14 mutants: cells deleted for GCS1 initiate the sporulation program but are defective in synthesis of the prospore membrane. Endosome-to-vacuole transport is also impaired in gcs1delta cells during sporulation. Furthermore, Arf-GAP catalytic activity, but not the pleckstrin homology domain, is required for both prospore membrane formation and endosome-to-vacuole trafficking. An examination of Gcs1p-green fluorescent protein revealed that it is a soluble protein. Interestingly, cells deleted for GCS1 have reduced levels of Spo14p-generated PA. Taken together, these results indicate that GCS1 is essential for sporulation and suggest that GCS1 positively regulates SPO14.     

Differential regulation of Saccharomyces cerevisiae phospholipase D in sporulation and Sec14-independent secretion

Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation. Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion). In sporulating cells Spo14 is phosphorylated and relocalized within the cell. In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion. Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14(R894G), revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity. Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion. Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation. These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion. Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis.     

Sorting signals within the Saccharomyces cerevisiae sporulation-specific dityrosine transporter, Dtr1p, C terminus promote Golgi-to-prospore membrane transport

During sporulation in Saccharomyces cerevisiae, the dityrosine transporter Dtr1p, which is required for formation of the outermost layer of the spore wall, is specifically expressed and transported to the prospore membrane, a novel double-lipid-bilayer membrane. Dtr1p consists of 572 amino acids with predicted N- and C-terminal cytoplasmic extensions and 12 transmembrane domains. Dtr1p missing the largest internal cytoplasmic loop was trapped in the endoplasmic reticulum in both mitotically dividing cells and cells induced to sporulate. Deletion of the carboxyl 15 amino acids, but not the N-terminal extension of Dtr1p, resulted in a protein that failed to localize to the prospore membrane and was instead observed in cytoplasmic puncta. The puncta colocalized with a cis-Golgi marker, suggesting that Dtr1p missing the last 15 amino acids was trapped in an early Golgi compartment. Deletion of the C-terminal 10 amino acids resulted in a protein that localized to the prospore membrane with a delay and accumulated in cytoplasmic puncta that partially colocalized with a trans-Golgi marker. Both full-length Dtr1p and Dtr1p missing the last 10 amino acids expressed in vegetative cells localized to the plasma membrane and vacuoles, while Dtr1p deleted for the carboxyl-terminal 15 amino acids was observed only at vacuoles, suggesting that transport to the prospore membrane is mediated by distinct signals from those that specify plasma membrane localization. Transfer-of-function experiments revealed that both the carboxyl transmembrane domain and the C-terminal tail are important for Golgi complex-to-prospore membrane transport.     

SPO71 Mediates Prospore Membrane Size and Maturation in Saccharomyces cerevisiae

The mechanisms that control the size and shape of membranes are not well understood, despite the importance of these structures in determining organelle and cell morphology. The prospore membrane, a double lipid bilayer that is synthesized de novo during sporulation in S. cerevisiae, grows to surround the four meiotic products. This membrane determines the shape of the newly formed spores and serves as the template for spore wall deposition. Ultimately, the inner leaflet of the prospore membrane will become the new plasma membrane of the cell upon germination. Here we show that Spo71, a pleckstrin homology domain protein whose expression is induced during sporulation, is critical for the appropriate growth of the prospore membrane. Without SPO71, prospore membranes surround the nuclei but are abnormally small, and spore wall deposition is disrupted. Sporulating spo71Δ cells have prospore membranes that properly localize components to their growing leading edges yet cannot properly localize septin structures. We also found that SPO71 genetically interacts with SPO1, a gene with homology to the phospholipase B gene that has been previously implicated in determining the shape of the prospore membrane. Together, these results show that SPO71 plays a critical role in prospore membrane development.     

SPO21 Is Required for Meiosis-specific Modification of the Spindle Pole Body in Yeast

During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.     

Ady3p links spindle pole body function to spore wall synthesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the de novo synthesis of prospore membranes and spore walls. Ady3p has been identified as an interaction partner for Mpc70p/Spo21p, a meiosis-specific component of the outer plaque of the spindle pole body (SPB) that is required for prospore membrane formation, and for Don1p, which forms a ring-like structure at the leading edge of the prospore membrane during meiosis II. ADY3 expression has been shown to be induced in midsporulation. We report here that Ady3p interacts with additional components of the outer and central plaques of the SPB in the two-hybrid assay. Cells that lack ADY3 display a decrease in sporulation efficiency, and most ady3Delta/ady3Delta asci that do form contain fewer than four spores. The sporulation defect in ady3Delta/ady3Delta cells is due to a failure to synthesize spore wall polymers. Ady3p forms ring-like structures around meiosis II spindles that colocalize with those formed by Don1p, and Don1p rings are absent during meiosis II in ady3Delta/ady3Delta cells. In mpc70Delta/mpc70Delta cells, Ady3p remains associated with SPBs during meiosis II. Our results suggest that Ady3p mediates assembly of the Don1p-containing structure at the leading edge of the prospore membrane via interaction with components of the SPB and that this structure is involved in spore wall formation.     

Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.     

SPO71 Encodes a Developmental Stage-Specific Partner for Vps13 in Saccharomyces cerevisiae

The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.