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Addition of exogenous PA or NEM causes a severe membrane fusion defect. Vacuoles were harvested from WT BJ3505 and DKY6281 and tested for fusion by luminal mixing and proPho8p maturation. Fusion reactions containing 3 μg of each vacuole type were incubated in the presence of diC8-PA (A) or diC8-DAG (B) at the indicated concentrations. C) Fusion reactions containing 100 μg/mL diC8-PA were incubated in the presence of 188 nm recombinant Sec18p. Fusion reactions containing 3 μg of each vacuole type were incubated in the presence of GST-DEP (D) or NEM (E) at the indicated concentrations. Fusion results were normalized to untreated WT vacuoles at standard conditions. F) Exogenous recombinant Vam7p (200 nm) was used to bypass fusion inhibitors. G) Gain of resistance kinetic assays was performed in the presence of 140 μg/mL α-Sec17p IgG, 2 mm propranolol, 300 μm diC8-PA, 1 mm NEM or PS buffer. Data were fit using first-order exponential decay with weights and errors. (H) Calculated half-times from first-order exponential decay fit. **p < 0.001.
Source publication
Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/NSF and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane assoc...
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... first assayed if the addition of diC8-PA or diC8-DAG showed a signif- icant effect on overall vacuolar fusion activity. Addition of diC8-PA led to potent inhibition of vacuolar con- tent mixing in a dose-dependent manner ( Figure 3A), whereas adding the same concentrations of diC8-DAG did not affect fusion ( Figure 3B). Importantly, inhibition by diC8-PA could be reversed by the addition of recombinant Sec18p suggesting a direct interaction between the protein and lipid ( Figure 3C). ...
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... first assayed if the addition of diC8-PA or diC8-DAG showed a signif- icant effect on overall vacuolar fusion activity. Addition of diC8-PA led to potent inhibition of vacuolar con- tent mixing in a dose-dependent manner ( Figure 3A), whereas adding the same concentrations of diC8-DAG did not affect fusion ( Figure 3B). Importantly, inhibition by diC8-PA could be reversed by the addition of recombinant Sec18p suggesting a direct interaction between the protein and lipid ( Figure 3C). ...
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... of diC8-PA led to potent inhibition of vacuolar con- tent mixing in a dose-dependent manner ( Figure 3A), whereas adding the same concentrations of diC8-DAG did not affect fusion ( Figure 3B). Importantly, inhibition by diC8-PA could be reversed by the addition of recombinant Sec18p suggesting a direct interaction between the protein and lipid ( Figure 3C). We then wanted to test if seques- tering PA with the PA-specific binding domain GST-DEP Fusion results were normalized to untreated WT vacuoles at stan- dard conditions. ...
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... have a measurable effect on fusion activity. Addi- tion of GST-DEP had a minimal effect on fusion except at the highest concentration tested (10 μM), which reduced fusion by approximately 50% ( Figure 3D). Higher concen- trations were not used because of buffer interference with the fusion assay. ...
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... NEM is known to alky- late NSF, we reasoned that a similar modification could take place with Sec18p and perturb priming activity. We found that NEM potently inhibited fusion at ≥1 mM suggesting that NEM might modify and inhibit Sec18p ( Figure 3E). To test whether diC8-PA, diC8-DAG or NEM had priming-specific inhibition, we employed a variant of the content mixing assay that utilizes a Vam7p bypass of priming. ...
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... expected that a priming-specific defect caused by addition of diC8-PA or NEM should also be rescued in this manner. Sur- prisingly, a Vam7p bypass did not rescue fusion activity in the presence of diC8-PA or NEM ( Figure 3F). These data suggest that diC8-PA and NEM could additionally be affecting stages downstream of priming in the fusion cascade. ...
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... compared diC8-PA inhibition kinetics to that of α-Sec17p IgG, propranolol, NEM and GDI through calculated gain of resistance half-times using first-order exponential decay fitting ( Figure 3G) (4,17). Interestingly, diC8-PA showed similar recovery of fusion to that of the tethering stage inhibitor GDI ( Figure 3H). This suggests that PA could be inhibiting fusion during a stage downstream of priming, independent of Sec18p; however, we cannot rule out that diC8-PA may be blocking additional rounds of fusion. ...
Citations
... PA also binds to and regulates proteins/enzymes important to vesicle fission/fusion; moreover, PA can move across the lipid bilayer by both spontaneous flip-flop and in an enzyme-dependent manner (Contreras et al., 2010;Homan & Pownall, 1988;Tanguy et al., 2019;Zhukovsky et al., 2019). This complex behavior of PA accounts for its multiple effects on vesicular trafficking and membrane fusion/fission, depending on cell types and conditions (Nakanishi et al., 2006;Pagliuso et al., 2016;Starr et al., 2016;Valente et al., 2013). In animal cells, PA was reported to enter the nucleus via vesicular trafficking (Henkels et al., 2016). ...
Cytosolic glyceraldehyde‐3‐phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but undergoes stress‐induced nuclear translocation for moonlighting. We previously reported that in response to heat stress, GAPC accumulated in the nucleus to modulate transcription and thermotolerance. Here we show a cellular and molecular mechanism that mediates heat‐induced nuclear translocation of cytosolic GAPC in Arabidopsis thaliana. Heat‐induced GAPC nuclear accumulation and plant heat tolerance were reduced in Arabidopsis phospholipase D (PLD) knockout mutants of pldδ and pldα1pldδ, but not of pldα1. These changes were restored to wild type by genetic complementation with active PLDδ, but not with catalytically inactive PLDδ. GAPC overexpression enhanced the seedling thermotolerance and the expression of heat‐inducible genes, but this effect was abolished in the pldδ background. Heat stress elevated the levels of PLD product phosphatidic acid (PA) in the nucleus in wild type, but not in pldδ plants. Lipid labeling demonstrated the heat‐induced nuclear co‐localization of PA and GAPC, which was impaired by zinc that inhibited the PA‐GAPC interaction and by a membrane trafficking inhibitor brefeldin A (BFA). The GAPC nuclear accumulation and seedling thermotolerance were also decreased by treatments with zinc or BFA. Our data suggest that PLDδ and PA are critical for the heat‐induced nuclear translocation of GAPC. We propose that PLDδ‐produced PA mediates the process via lipid‐protein interaction and that the lipid mediation act as a cellular conduit linking stress perturbations at cell membranes to nuclear functions in plant coping with heat stress.
... Insect: LKB1 [181], TREK-1 [182]. Yeast: Pah1 [183], Opi1p [38,184], Spo20p [38,185,186], Sso1p [187], Sec18p [188,189], Ups1 [190,191], Chm7 [192]. Plant: ABI1 [193], PP2CA [194], TGD2 [195,196], TGD4 [197], AtPDK1 [198], MKK7/9 [199], SnRK2.4 [200], PID [201], AtSphK1 [202], RGS1 [203], PEPC [204], LHY [205], CCA1 [205], Werewolf [206], AHL4 [207], AKT2 [208], MAP65-1 [209], RbohD160 [210], 14-3-3 protein [211], MtDef4 [211], NsD7 [212], SNX [213]. ...
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.
... In another technique, called 'liposome co-precipitation' , proteins are co-precipitated with insoluble lipid vesicles ('liposomes') composed of PA-containing lipid bilayers, and are detected by immunoblotting. It is usually preferred due to the compositional property of liposomes similar to that of cellular membranes, and can be applied to quantify the co-precipitated proteins by densitometric analyses [103,76]. PA-conjugated beads (e.g. ...
... The presence of PA in the outer leaflet of a membrane should inhibit membrane fission, but in some cases rather promotes it, for example on trans-Golgi network by recruiting a fission-inducing protein C-terminal-binding protein1-S/brefeldin A ADP-ribosylation substrate (CtBP1-S/BARS) [120,121]. Likewise, PA is able to both promote and inhibit soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE)-mediated membrane fusion by interacting with different sets of SNARE proteins [58,76,[122][123][124]. PA can also promote mitochondrial fusion by stimulating mitofusin 1-mediated fusion and suppressing fission-inducing dynamin-related protein 1 (Drp1) [66,125,126]. ...
Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking.
... Additionally, it was revealed that the recruited PIP5K regulates the actin cytoskeleton at the IS, facilitating targeted granule secretion by the CTL. PA-regulated exocytosis was previously described in a variety of cell types including neutrophils and neurons [70,71], and it was mentioned to be facilitated by PA's conical shape, which induces negative membrane curvatures, as well as its participation in the modulation of SNARE complexes [72][73][74]. These data suggest that PA, as DAG, may be influencing membrane IS structure and function. ...
Recognition of antigens displayed on the surface of an antigen-presenting cell (APC) by T-cell receptors (TCR) of a T lymphocyte leads to the formation of a specialized contact between both cells named the immune synapse (IS). This highly organized structure ensures cell–cell communication and sustained T-cell activation. An essential lipid regulating T-cell activation is diacylglycerol (DAG), which accumulates at the cell–cell interface and mediates recruitment and activation of proteins involved in signaling and polarization. Formation of the IS requires rearrangement of the cytoskeleton, translocation of the microtubule-organizing center (MTOC) and vesicular compartments, and reorganization of signaling and adhesion molecules within the cell–cell junction. Among the multiple players involved in this polarized intracellular trafficking, we find sorting nexin 27 (SNX27). This protein translocates to the T cell–APC interface upon TCR activation, and it is suggested to facilitate the transport of cargoes toward this structure. Furthermore, its interaction with diacylglycerol kinase ζ (DGKζ), a negative regulator of DAG, sustains the precise modulation of this lipid and, thus, facilitates IS organization and signaling. Here, we review the role of SNX27, DAG metabolism, and their interplay in the control of T-cell activation and establishment of the IS.
... 6. His 6 -Sec18: His 6 -Sec18 can be expressed as a fusion protein in E. coli and purified using Ni-NTA and size-exclusion chromatography [6]. Hexamer and monomer pools are separated by size-exclusion chromatography. ...
The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of Microscale thermophoresis (MST) MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins, however, many analytes, including pharmaceuticals fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes. Available at: jove.com/video/60607
... 16,26 Yet, inhibiting PA phosphatase activity to produce diacylglycerol (DAG), resulting in elevating PA concentrations, blocks vacuole fusion at the priming stage through sequestering Sec18 from cis-SNARE complexes. 20,48 Likewise, low levels of phospholipase C (PLC) stimulates vacuole fusion through converting PI(4,5)P 2 to DAG, while high levels of PLC potently inhibits fusion. 49 Finally, while PI(4,5)P 2 is well known to stimulate vacuole fusion, an excess of the lipid can also inhibit the pathway. ...
The transport of Ca²⁺ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca²⁺ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P2 pools. Ca²⁺ is also released during vacuole fusion upon trans‐SNARE complex assembly, however its role remains unclear. The effect of PI(3,5)P2 on Ca²⁺ flux during fusion was independent of Yvc1. Here we show that while low levels of PI(3,5)P2 were required for Ca²⁺ uptake into the vacuole, increased concentrations abolished Ca²⁺ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P2 or increased endogenous production of by the hyperactive fab1T2250A mutant. In contrast, the lack of PI(3,5)P2 on vacuoles from the kinase dead fab1EEE mutant showed delayed and decreased Ca²⁺ uptake. The effects of PI(3,5)P2 were linked to the Ca²⁺ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P2. Experiments with Verapamil inhibited Ca²⁺ uptake when added at the start of the assay, while adding it after Ca²⁺ had been taken up resulted in the rapid expulsion of Ca²⁺. Vacuoles lacking both Pmc1 and the H⁺/Ca²⁺ exchanger Vcx1 lacked the ability to take up Ca²⁺ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P2 can modulate which path predominates.
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... In the case of Saccharomyces cerevisiae, the spatiotemporal localization and activity of many proteins and lipids that participate in homotypic vacuole fusion are well understood. Priming of the membranes is dependent on the conversion of phosphatidic acid (PA) to diacylglycerol (DAG) by Pah1p in the membrane, and the action of the chaperone proteins Sec17p (α-SNAP) and the AAA+ ATPase Sec18p (NSF), which releases SNAREs (soluble NSF attachment protein receptors) (Vam3p, Nyv1p, Vti1p, and Vam7p) from inactive cis-bundles (Mayer et al., 1996;Mayer and Wickner, 1997;Starr et al., 2016). This is followed by tethering of the vacuoles through interactions between a Rab GTPase (Ypt7p), and the heterohexameric HOPS (homotypic fusion and protein sorting) complex (Miner et al., 2016;Seals et al., 2000;Stroupe et al., 2006). ...
... In some cases, these regulatory lipids have been found to colocalize with fusion machinery proteins in a region termed the "vertex domain", the ring-like area surrounding two membranes in contact during the docking stage (Fratti et al., 2004;Wang et al., 2002). The regulatory glycerophospholipids diacylglycerol (DAG), phosphatidic acid (PA) and a number of phosphatidylinositol phosphates (PtdInsP) have been shown to influence the recruitment and activity of Rab and Rho GTPases, chaperone proteins, HOPS, SNAREs, and actin (Karunakaran et al., 2012;Karunakaran and Fratti, 2013;Miner et al., 2016;Miner et al., 2017;Starr et al., 2016). ...
The role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolated from these cells displayed reduced levels of in vitro fusion, which we traced to a block in tethering and docking. We found that C26 VLCFA deficient yeast mislocalize fusion markers, and the small GTPases Rho1p and Ypt7p fail to selectively concentrate at the boundary and vertex domains of vacuoles isolated from these yeasts. Surprisingly, we only observed mild changes to the localization of other regulatory lipids, but membrane fluidity and solubility was significantly altered. Taken together, these results suggest that sphingolipids containing C26 VLCFAs act as regulatory lipids in the homotypic vacuolar fusion cascade by assembling membrane microdomains that promote the protein and lipid machinery required for the tethering and docking of vacuoles.
Summary
Many sphingolipids contain very-long chain fatty-acids with 26 carbons. The deletion of Elo3, the elongase that adds the final two carbons results in pleiotropic effects that negatively alter membrane fusion at the tethering and docking stages.
... We previously demonstrated the importance of phosphatidic acid (PA) in regulating Sec18/NSF priming activity (10)(11)(12). Deletion of the PA phosphatase PAH1, the yeast orthologue of mammalian lipin1, leads to elevated concentrations of PA on the vacuole that we hypothesized sequesters Sec18 away from cis-SNAREs (10). Even when Pah1 is present, released Sec18 can be inhibited by adding soluble dioctanoyl PA (C8-PA). ...
... To examine if IPA blocked other PA binding proteins we used the DEP domain from the murine protein Dvl2 (25). Previously we used DEP to bind PA liposomes and vacuolar PA to displace Sec18 from membranes (11). Here we tested PA liposome binding DEP in the presence of 100 µM IPA. ...
... Nevertheless, this is the first demonstration of a specific Sec18 ligand to inhibit membrane fusion. In comparison, inhibiting fusion with NEM requires millimolar concentrations (11). ...
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.
... After the fusion step, the SNAREs are held together in a cis-conformation by Sec17. Sec18, one of the most important players of priming, detaches itself from the vacuolar membrane and transfers itself on to Sec17 where Sec18 works to sequester Sec17 from the SNARE bundle, thereby releasing the SNAREs for the next fusion activity (Ungermann et al. 1998;Starr et al. 2016). ...
... Taking a closer look, evidence suggests that ATP bound Sec18 resides on the vacuolar membrane by attaching itself to the phosphomonoester head group of phosphatidic acid (PA) where it also serves as a membrane receptor for LMA1 (Xu et al. 1998;Starr et al. 2016). Meanwhile, Sec17 resides on the cis-SNARE complex. ...
... Next, in the budding yeast it was shown that the PA phosphatase, Pah1, helps in the conversion of PA to diacylglycerol (figure 4). When this conversion occurs, Sec18 is released from the vacuolar membrane and is able to indirectly bind the cis-SNARE complex by using its N-terminal domain to bind Sec17/a-SNAP (Hohl et al. 1998;Starr et al. 2016). In mammalian cells, this interaction is mediated via N-(1-160) and C-(160-295) amino acid residues on a-SNAP that interact with NSF. ...
Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part of the reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellular trafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when a cargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypic fusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole to vacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when an endosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components – Rabs, Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs – that have been known to function in both of these types of fusion. We hope to develop a model of how all of these constituents function together to achieve membrane fusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degree of importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypic or homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson’s and Alzheimer’s disease. Although much research has significantly expanded our knowledge of fusion, there is still much more to be learned.
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... Vacuoles were incubated with reaction buffer, 100 µM Cu 2+ or 1 mM N-ethylmaleimide (NEM), an inhibitor of Sec18/NSF function. 27,28 Individual reactions were incubated at increasing time increments after which they were centrifuged to separate the membrane bound (pellet) and solubilized (supernatant) fractions of Sec17. These experiments showed that Cu 2+ did not negatively affect SNARE priming, as similar levels of Sec17 were released relative to the buffer control (Fig. 3A-B). ...
... To further resolve the stage of the fusion pathway that was inhibited by Cu 2+ we performed a temporal gain of resistance assay. [28][29][30] Individual fusion reactions were treated with inhibitors at different time-points throughout the incubation period. Vacuole fusion gains resistance to an inhibitor once the target of the reagent has had completed its function. ...
... In comparison, the inhibition of SNARE pairing has been shown many times to occur much earlier during the docking stage the pathway with a halftime of 20 min. 28,44,45 While kinetically separate, the effects of Cu 2+ on these functions could have a common origin such as the reduction of membrane fluidity. The mean by which Cu 2+ and not Zn 2+ or other metals (except for Ag 2+ ) alter membrane fluidity is due coppercopper interactions when bound to anionic phospholipids. ...
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
