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SNARE domain alone stimulates vacuole fusion. A, vacuole fusion reactions were incubated with indicated concentrations of GST-SNARE domain from Vam7 (SD). B, vacuoles were incubated with 1 M SD in the absence of ATP to block priming. SD-supplemented reactions were incubated with known inhibitors of vacuole fusion. Fusion inhibitors were 353 nM -Vam3p, 32 nM -Vps33p, 133 nM -Ypt7p, 1 M FVYE, 1 M PX, 1 M MTM, 2 M SigD, 30 M ENTH, 10 M MED, 12 M FAPP-PH, 10 M C1b, 17 M filipin, or 2.7 M Plc1p. Data represent mean fusion S.E. (n 3).
Source publication
Regulated membrane fusion requires organelle tethering, enrichment of selected proteins and lipids at the fusion site, bilayer
distortion, and lipid rearrangement. Yeast vacuole homotypic fusion requires regulatory lipids (ergosterol, diacylglycerol,
and phosphoinositides), the Rab family GTPase Ypt7p, the multisubunit Ypt7p-effector complex HOPS (...
Contexts in source publication
Context 1
... through its affinities for PI(3)P (10) and HOPS (5) and may be primarily required for membrane targeting. To test whether the PX domain has additional essential functions, we purified the SD of Vam7p and assayed its ability to support fusion. High concentrations of the SD of Vam7p (1-10 M) support vacuole fusion, but only in the absence of ATP (Fig. 4A). These concen- trations are 100-fold higher than the Vam7 Y42A needed for fusion under the same assay conditions ( Fig. 2A), suggesting that the affinities of the PX domain of Vam7p for HOPS and PI(3)P are important to target Vam7p for its function. To test whether this fusion supported by the SD domain was on the physiological ...
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... Vam7p for its function. To test whether this fusion supported by the SD domain was on the physiological pathway, we used a panel of well studied fusion inhibitors. Fusion was sensitive to most standard inhibitors, such as antibodies to the SNARE Vam3p, Vps33p (the SM sub- unit of HOPS), or the Rab Ypt7p, or to many (but not all) lipid ligands (Fig. 4B). In the presence of ATP, where SD supports very little bypass fusion (Fig. 4A), we observed a striking enhancement of SD-mediated fusion by the PLC inhibitor U73122 (Fig. 5A). The specificity of this stimulation is shown by its sensitivity to the addition of recombinant yeast Plc1p, by the fact that stimulation was not seen with the ...
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... was on the physiological pathway, we used a panel of well studied fusion inhibitors. Fusion was sensitive to most standard inhibitors, such as antibodies to the SNARE Vam3p, Vps33p (the SM sub- unit of HOPS), or the Rab Ypt7p, or to many (but not all) lipid ligands (Fig. 4B). In the presence of ATP, where SD supports very little bypass fusion (Fig. 4A), we observed a striking enhancement of SD-mediated fusion by the PLC inhibitor U73122 (Fig. 5A). The specificity of this stimulation is shown by its sensitivity to the addition of recombinant yeast Plc1p, by the fact that stimulation was not seen with the analog U73343 (which does not inhibit phospholipase C (26)) and by its sensi- ...
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... U73343 (which does not inhibit phospholipase C (26)) and by its sensi- tivity to affinity-purified antibody to Vam3p (Fig. 5A) and other reaction pathway inhibitors (data not shown). U73122 only stimulated fusion over a narrow range of SD concentrations (Fig. 5B). In the absence of ATP, where little fusion was seen with M or lower levels of SD (Fig. 4A), U73122 allowed even 0.1 M SD to strongly stimulate fusion (Fig. 5C). In the absence of ATP or U73122, the substantial fusion supported by 1-10 M SD is strongly inhibited by U73433. Even though U73433 does not inhibit PLC activity (26), it might bind to PLC in a manner that interferes with its localization or other proper regulation. ...
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Although diacylglycerol (DAG) can trigger liposome fusion, biological membrane fusion requires Rab and SNARE proteins. We
have investigated whether DAG and phosphoinositide-specific phospholipase C (PLC) have a role in the Rab- and SNARE-dependent
homo-typic vacuole fusion in Saccharomyces cerevisiae. Vacuole fusion was blocked when DAG was sequest...
Citations
... R AGTT GAGC TCGT CGAC TATG GCTT TGAC AACT GCAGGA. GST-PX was prepared as described (Fratti and Wickner, 2007). Proteoliposome preparation and fusion assays were as described in Song et al., 2021. ...
Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans -complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.
... Proteins HOPS (Zick and Wickner, 2013), the vacuolar SNAREs (Mima et al., 2008;Zucchi andZick, 2011), soluble R andQa (Thorngren et al., 2004;Song and Wickner, 2017), the MBP-tagged SNARE domain of Qb , Ypt7-TM bearing an apolar membrane anchor of the same sequence as Qb , prenyl Ypt7 (Zick and Wickner, 2013), Sec17 (Schwartz and Merz, 2009), Sec18 (Mayer et al., 1996), and GST-PX (Fratti and Wickner, 2007) were purified as described. ...
Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the 8 major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.
... Reagents were purchased, and proteins purified, as described in Song GST-PX was prepared as described (Fratti and Wickner, 2007 zippering. Proteoliposomes were prepared with vacuolar lipids, membrane-anchored Ypt7, and 281 either R or the 3 Q-SNAREs with molar ratios of 1Ypt7:8,000 lipids and 1 of each SNARE/16,000 282 lipids. ...
Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that SNARE zippering-arrested fusion intermediates that are formed without HOPS support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.
... Additionally, PI3P is required for fusion of vesicles with the vacuole, and homotypic vacuole fusion. This is due in part to the direct binding of the SNARE Vam7 (Cheever et al., 2001;Fratti and Wickner, 2007) and the heterodimeric GEF subunits Mon1 and Ccz1 to PI3P. Moreover, Vps34-dependent generation of PI3P is required for the vacuole association of many additional proteins required for vacuole fusion (Lawrence et al., 2014). ...
Phosphoinositide signaling lipids are essential for several cellular processes. The requirement for a phosphoinositide is conventionally studied by depleting the corresponding lipid kinase. However, there are very few reports on the impact of elevating phosphoinositides. That phosphoinositides are dynamically elevated in response to stimuli suggests that, in addition to being required, phosphoinositides drive downstream pathways. To test this hypothesis, we elevated the levels of phosphatidylinositol-3-phosphate (PI3P) by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 drives certain pathways, including PI(3,5)P 2 synthesis and retrograde transport from the vacuole. This demonstrates that PI3P is rate limiting in some pathways. Interestingly, hyperactive Vps34 does not affect ESCRT function. Thus, elevating PI3P does not always increase the rate of PI3P-dependent pathways. Elevating PI3P can also delay a pathway. Elevating PI3P slowed late steps in autophagy, in part by delaying the disassembly of autophagy proteins from mature autophagosomes as well as delaying fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as assessed by changes in vacuole morphology. These studies suggest that stimulus-induced elevation of phosphoinositides provides a way for these stimuli to selectively regulate downstream processes.
... Fluorescence was scanned across a temperature gradient of 20 to 95°C and the first derivative of the fluorescence data was used to determine the Tm for each condition. DSF has the ability to show multiple melting transitions (40,41). Our data show that mSec18 has three melting transitions. ...
... In addition to mediating ApoB-Lp secretion, Sortilin has a significant role in ApoB degradation by the lysosome 41 . Our previous work showed that blocking autophagy prevents insulin signaling from suppressing ApoB secretion. ...
... 494 nm, em. 515 nm).Vacuole Isolation and in vitro vacuole fusion assay -Vacuoles were isolated from yeast strains by density gradient floatation as previously described(41). Fusion reactions(30 µL) contained 3 µg each of vacuoles from BJ3505 (pep4∆ PHO8) and DK6281 (PEP4 pho8∆), fusion assay buffer (125 mM KCl, 5 mM MgCl2, 20 mM PIPES-KOH pH 6.8, 200 mM sorbitol), ATP regenerating system (1 mM ATP, 29 mM creatine phosphate, 0.1 mg/ml creatine kinase), 10 µM CoA, and 283 nM Pbi2p. ...
The segregation and upkeep of distinct compartmental organization within the cell requires both organelle specific SNAREs as well as sorting receptors to allow for continual rounds of productive fusion bringing in appropriate cargo into designated organelles. Generally, sorting receptors function by providing unique signal sequences in the c-terminal tail region of the receptor that bind to specific sorting proteins allowing cargo to be transmitted to appropriate organelles. However, the yeast Vps10 vacuolar sorting protein functions by an as yet undefined signal sequence segregating proteins to either the secretory pathway or to the vacuole. Additionally, we have found that the mammalian Vps-10 orthologue, sortilin binds to PI(3,4,5)tri-phosphate (PIP3) and this may have relevance to the eventual sorting functions in this pathway. Furthermore, this pathway is defined by its requirement for the protein Vps-34, the only class III phosphatidyl-inositol-3-kinase that makes PI3P in both yeast and mammalian cells. The relationship between Vps-34 or PI3P production and phosphatidic acid (PA) levels is a fairly unexplored subject. However, our group identified the PA phosphatase Pah1 as being the sole PA phosphatase, which when knocked out showed any effect on yeast vacuole fusion. Furthermore, in Pah1 knockouts, there is no Vps34 at the yeast vacuole and levels of PI3P at the yeast vacuole are almost completely abolished. We have shown that PA levels at the vacuole affect fusion via regulation at the priming step of fusion. Priming is the unraveling of cis-SNAREs resulting from trans-SNARE complexes remaining from a prior fusion event. The unique job of priming all cellular SNAREs is the responsibility of AAA+ ATPase NSF or Sec18, which transmits the energy released from ATP hydrolysis to mechanically unwind SNAREs. In order to investigate Vps10 dependent sorting and SNARE priming, small molecules were developed to probe the lipid binding pockets of both yeast Vps-10 and Sec18. These molecules have been shown to affect levels of priming as well as targets of sorting protein Vps-10. It is proposed that computational drug discovery in companion with molecular dynamics (MD) simulations can be coupled to biochemical techniques to further elucidate mechanisms of protein fusion and sorting involving specific lipid-protein interactions to further understand the mechanisms by which pathways such as the Vps-10 protein sorting pathway and the yeast fusion priming pathway operate. iii ACKNOWLEDGEMENTS
... Membrane scaffold protein 1D1 (MSP1D1-His) was prepared as described previously (39). GST-Vam7 Y42A was expressed and purified as described previously (40). ...
The homeostasis of most organelles requires membrane fusion mediated by soluble
N
-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
... For this, we used a SNARE complex formation assay using exogenous GST-Vam7 in reactions treated with anti-Sec17 IgG to block priming. [31][32][33][34] Thus, only the formation of new SNARE complexes will be measured in the presence of Cu 2+ . After the addition of anti-Sec17 we further treated individual reactions with GDI to block tethering or Cu 2+ . ...
... Specific proteins were detected by Western blotting. As previously seen, GST-Vam7 formed complexes with its cognate SNAREs Vam3 and Nyv1 when incubated at 27°C, but not when incubation on ice prevented complex formation as seen previously 31,32 (Fig. 4A-B). As a negative control GDI inhibited SNARE complex formation. ...
... Anti-Sec17 IgG 26 , and Pbi2 (Protease B inhibitor) 57 were prepared as described previously. Recombinant GST-Vam7 and GDI were prepared as previously described 31,32,58 NEM (N-ethylmaleimide), acridine orange and FCCP were from Sigma. Quinacrine was from Cayman Biochemical. ...
The accumulation of Copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+, and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V‐ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu2+ is responsible for the inhibition of vacuole fusion and V‐ATPase function. This notion is supported by the differential effects of chelators. The Cu2+‐specific chelator TETA rescued fusion, whereas the Cu+‐specific chelator BCS had no effect on the inhibited fusion. This article is protected by copyright. All rights reserved. In this study we found that Cu2+ inhibits in vitro vacuole homotypic fusion. The inhibition was not due to generating reactive oxygen radicals and membrane damage. Rather Cu2+ blocked fusion through reducing trans‐SNARE complex formation and by blocking the V‐ATPase function and vacuole acidification
... Next, we examined if SNARE complex formation was affected by Cu 2+ . For this, we used a SNARE complex formation assay using exogenous GST-Vam7 in reactions treated with anti-Sec17 IgG to block priming (23)(24)(25)(26). Thus, only the formation of new SNARE complexes will be measured in the presence of Cu 2+ . ...
... Anti-Sec17 IgG (18), and Pbi2 (Protease B inhibitor) (46) were prepared as described previously. Recombinant GST-Vam7 and GDI were prepared as previously described (23,24,47) NEM (N-ethylmaleimide), acridine orange and FCCP were from Sigma. Quinacrine was from Cayman Biochemical. ...
The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg + , Cd 2+ , and Cu 2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu 2+ to Cu +. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl 2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H + pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu 2+ is responsible for the inhibition of vacu-ole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu 2+-specific chelator TETA rescued fusion, whereas the Cu +-specific chelator BCS had no effect on the inhibited fusion.
... Pho04 and Pho15 hold the SNARE-associated domain DedA. SNARE-associated proteins are classified as structural proteins that function as a protein-protein interaction module (31). To our knowledge, no proteins with SNARE domains have been previously discovered to possess phosphatase activity. ...
Phosphorus (P) is a key element involved in numerous cellular processes and essential to meet global food demand. Phosphatases play a major role in cell metabolism and contribute to control the release of P from phosphorylated organic compounds, including phytate. Apart from the relationship with pathogenesis and the enormous economic relevance, phosphatases/phytases are also important for reduction of phosphorus pollution. Almost all known functional phosphatases/phytases are derived from cultured individual microorganisms. We demonstrate here for the first time the potential of functional metagenomics to exploit the phosphatase/phytase pools hidden in environmental soil samples. The recovered diversity of phosphatases/phytases comprises new types and proteins exhibiting largely unknown characteristics, demonstrating the potential of the screening method for retrieving novel target enzymes. The insights gained into the unknown diversity of genes involved in the P cycle highlight the power of function-based metagenomic screening strategies to study Earth’s phosphatase pools.
... Membrane scaffold protein 1D1 (MSP1D1-His) was prepared as described (62). GST-Vam7 and Sec17 were expressed and purified as shown previously (32, 63,64). Purification of GST-Nyv1(DTM), was performed as previously described with minor changes (65). ...
Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes. We now report that PA also induces conformational changes in Sec18 protomers and that hexameric Sec18 cannot bind PA membranes. Molecular dynamics (MD) analyses revealed that the D1 and D2 domains of Sec18 contain PA-binding sites and that the residues needed for PA binding are masked in hexameric Sec18. Importantly, these simulations also disclosed that a major conformational change occurs in the linker region between the D1 and D2 domains, which is distinct from the conformational changes that occur in hexameric Sec18 during SNARE priming. Together, these findings indicate that PA regulates Sec18 function by altering its architecture and stabilizing membrane-bound Sec18 protomers. © 2019 Starr et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
