ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fusion (Sey1p) for additional roles in nuclear fusion. Here we demonstrate that the ER-localized SNAREs Sec20p, Ufe1p, Use1p, and Bos1p are required for efficient nuclear fusion. In contrast, Sey1p is required indirectly for nuclear fusion; sey1Δ zygotes accumulate ER at the zone of cell fusion, causing a block in nuclear congression. However, double mutants of Sey1p and Sec20p, Ufe1p, or Use1p, but not Bos1p, display extreme ER morphology defects, worse than either single mutant, suggesting that retrograde SNAREs fuse ER in the absence of Sey1p. Together these data demonstrate that SNAREs mediate nuclear fusion, ER fusion after cell fusion is necessary to complete nuclear congression, and there exists a SNARE-mediated, Sey1p-independent ER fusion pathway.     

The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER-ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.     

A Highlights from MBoC Selection: ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative,Sey1p-independent homotypic ER fusion pathway

The peripheral endoplasmic reticulum (ER) network is dynamically maintained by homotypic (ER–ER) fusion. In Saccharomyces cerevisiae, the dynamin-like GTPase Sey1p can mediate ER–ER fusion, but sey1Δ cells have no growth defect and only slightly perturbed ER structure. Recent work suggested that ER-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) mediate a Sey1p-independent ER–ER fusion pathway. However, an alternative explanation—that the observed phenotypes arose from perturbed vesicle trafficking—could not be ruled out. In this study, we used candidate and synthetic genetic array (SGA) approaches to more fully characterize SNARE-mediated ER–ER fusion. We found that Dsl1 complex mutations in sey1Δ cells cause strong synthetic growth and ER structure defects and delayed ER–ER fusion in vivo, additionally implicating the Dsl1 complex in SNARE-mediated ER–ER fusion. In contrast, cytosolic coat protein I (COPI) vesicle coat mutations in sey1Δ cells caused no synthetic defects, excluding perturbed retrograde trafficking as a cause for the previously observed synthetic defects. Finally, deleting the reticulons that help maintain ER architecture in cells disrupted for both ER–ER fusion pathways caused almost complete inviability. We conclude that the ER SNAREs and the Dsl1 complex directly mediate Sey1p-independent ER–ER fusion and that, in the absence of both pathways, cell viability depends upon membrane curvature–promoting reticulons.     

The Dsl1 protein tethering complex is a resident endoplasmic reticulum complex, which interacts with five soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs): implications for fusion and fusion regulation

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.     

Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.     

Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.     

ER network formation requires a balance of the dynamin-like GTPase Sey1p and the Lunapark family member Lnp1p

Although studies on endoplasmic reticulum (ER) structure and dynamics have focused on the ER tubule-forming proteins (reticulons and DP1/Yop1p) and the tubule fusion protein atlastin, nothing is known about the proteins and processes that act to counterbalance this machinery. Here we show that Lnp1p, a member of the conserved Lunapark family, plays a role in ER network formation. Lnp1p binds to the reticulons and Yop1p and resides at ER tubule junctions in both yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, the interaction of Lnp1p with the reticulon protein, Rtn1p, and the localization of Lnp1p to ER junctions are regulated by Sey1p, the yeast orthologue of atlastin. We propose that Lnp1p and Sey1p act antagonistically to balance polygonal network formation. In support of this proposal, we show that the collapsed, densely reticulated ER network in lnp1 Δ cells is partially restored when the GTPase activity of Sey1p is abrogated.     

Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.     

Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-D-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.     

Autophagosome biogenesis requires SNAREs

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.     

Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF₄⁻ but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.     

Autophagosome biogenesis requires SNAREs

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.     

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.     

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

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.     

Rer1p,a retrieval receptor for ER membrane proteins,recognizes transmembrane domains in multiple modes

The yeast Golgi membrane protein Rer1p is required for the retrieval of various endoplasmic reticulum (ER) membrane proteins such as Sec12p and Sec71p to the ER. We demonstrate here that the transmembrane domain (TMD) of Sec71p, a type-III membrane protein, contains an ER localization signal, which is required for physical recognition by Rer1p. The Sec71TMD-GFP fusion protein is efficiently retrieved to the ER by Rer1p. The structural feature of this TMD signal turns out to be the spatial location of polar residues flanking the highly hydrophobic core sequence but not the whole length of the TMD. On the Rer1p side, Tyr152 residue in the 4th TMD is important for the recognition of Sec12p but not Sec71p, suggesting that Rer1p interacts with its ligands at least in two modes. Sec71TMD-GFP expressed in the Deltarer1 mutant cells is mislocalized from the ER to the lumen of vacuoles via the multivesicular body (MVB) sorting pathway. In this case, not only the presence of polar residues in the Sec71TMD but also the length of the TMD is critical for the MVB sorting. Thus, the Rer1p-dependent ER retrieval and the MVB sorting in late endosomes both watch polar residues in the TMD but in a different manner.     

A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.     

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity

In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that not only vacuolar R-SNARE Nyv1p but also the non-cognate R-SNAREs, endosomal Snc2p, and endoplasmic reticulum-Golgi Sec22p caused efficient fusion with vacuolar Qabc-SNAREs. In contrast, their fusion is blocked completely by replacing vacuolar Qc-SNARE Vam7p with the non-cognate endosomal Tlg1p and Syn8p, although these endosomal Qc-SNAREs fully retained the ability to form cis-SNARE complexes with vacuolar SNAREs in solution and on membranes. Thus, our current study establishes that an appropriate assembly of Qabc-SNAREs is crucial for regulating fusion specificity, whereas R-SNARE itself has little contribution to specificity.