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Vacuole fusion is driven by SNARE proteins that require activation-or priming-by the AAA+ protein Sec18 (NSF) before they can bring membranes together and trigger the merger of two bilayers into a continuous membrane. Sec18 resides on vacuoles prior to engaging inactive cis-SNARE complexes through its interaction with the regulatory lipid phosphatidic acid (PA). Binding PA causes Sec18 to undergo large conformational changes that keeps it bound to the membrane, thus precluding its interactions with SNAREs. Such conformational changes can be measured by various biochemical and biophysical assays. The conversion of PA to diacylglycerol by the PA phosphatase Pah1 releases Sec18 from the membrane-bound pool and promotes its transfer to SNARE complexes allowing priming to occur. Here we describe four spectroscopy-based methods to distinguish conformational changes from alterations in secondary structure during PA binding. These methods only require purified protein and short chain soluble lipids, making the methods rapid and affordable ways to screen the effects of specific protein-lipid interactions. The assays described in this chapter include 1-anilino-8-naphthalenesulfonate (ANS) spectroscopy and intrinsic tryptophan fluorescence to detect exposure of hydrophobic regions; differential scanning fluorometry to measure changes in protein stability across a temperature gradient; and circular dichroism to examine changes in secondary structure., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies. Unlike surface plasmon resonance (SPR), BLI is an open system that does not require microfluidics, which eliminates issues that result from clogging and changes in viscosity. Importantly, BLI readings can be completed in minutes and can be formatted for high throughput screening. Here we use biotinylated short chain phosphoinositides and phosphatidic acid bound to streptavidin BLI biosensors to measure the binding of the soluble Qc SNARE Vam7 from Saccharomyces cerevisiae. Unlike most SNAREs, Vam7 lacks a transmembrane domain or lipid anchor to associate with membranes. Instead Vam7 associates to yeast vacuolar membranes using its N-terminal PX domain that binds to phosphatidylinositol 3-phosphate (PI3P) and phosphatidic acid (PA). Using full length Vam7, Vam7 Y42A , and PX domain alone, we determined and compared the dissociation constants (K D ) of each to biotinylated PI3P and PA biosensors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds the last two carbons to VLCFAs that are incorporated into sphingolipids. Cells lacking Elo3 have fragmented vacuoles, which is also seen when WT cells are treated with the sphingolipid synthesis inhibitor Aureobasidin-A. Isolated elo3Δ vacuoles show acidification defects and increased membrane fluidity, and this correlates with deficient fusion. Fusion arrest occurs at the tethering stage as elo3Δ vacuoles fail to cluster efficiently in vitro. Unlike HOPS and fusogenic lipids, GFP-Ypt7 does not enrich at elo3Δ vertex microdomains, a hallmark of vacuole docking prior to fusion. Pulldown assays using bacterially expressed GST-Ypt7 showed that HOPS from elo3Δ vacuole extracts failed to bind GST-Ypt7 while HOPS from WT extracts interacted strongly with GST-Ypt7. Treatment of WT vacuoles with the fluidizing anesthetic dibucaine recapitulates the elo3Δ phenotype and shows increased membrane fluidity, mislocalized GFP-Ypt7, inhibited fusion, and attenuated acidification. Together these data suggest that sphingolipids contribute to Rab-mediated tethering and docking required for vacuole fusion., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipid composition impacts fusion through direct protein lipid interactions or through modulating the physical properties of membranes at the site of contact, including the induction of positive curvature by lysophospholipids (LPLs). The degree of positive curvature induced is due to the length and saturation of the single acyl chain in addition to the size of the head group. Here we examined how yeast vacuole fusion and ion transport were differentially affected by changes in lysolipid properties. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons all inhibited fusion with IC 50 values ranging from ∼40-120 µM. The monounsaturation of LPC-18:1 had no effect when compared to its saturated counterpart LPC-18:0. On the other hand, head group size played a more significant role in blocking fusion as lysophosphatidic acid (LPA)-18:1 failed to fully inhibit fusion. We also show that both Ca 2+ uptake and SNARE-dependent Ca 2+ efflux was sensitive to changes in the acyl chain length and saturation of LPCs, while LPA only affected Ca 2+ efflux. Finally, we tested these LPLs on vacuole acidification by the V-ATPase. This showed that LPC-18:0 could fully inhibit acidification whereas other LPCs had moderate effects. Again, LPA had no effect. Together these data suggest that the effects of LPLs were due to a combination of head group size and acyl chain length leading to a range in degree of positive curvature.

Eukaryotic cells are compartmentalized into membrane-bound organelles, allowing each organelle to maintain the specialized conditions needed for their specific functions. One of the features that change between organelles is lumenal pH. In the endocytic and secretory pathways, lumenal pH is controlled by isoforms and concentration of the vacuolar-type H + -ATPase (V-ATPase). In the endolysosomal pathway, copies of complete V-ATPase complexes accumulate as membranes mature from early endosomes to late endosomes and lysosomes. Thus, each compartment becomes more acidic as maturation proceeds. Lysosome acidification is essential for the breakdown of macromolecules delivered from endosomes as well as cargo from different autophagic pathways, and dysregulation of this process is linked to various diseases. Thus, it is important to understand the regulation of the V-ATPase. Here we describe a high-throughput method for screening inhibitors/activators of V-ATPase activity using Acridine Orange (AO) as a fluorescent reporter for acidified yeast vacuolar lysosomes. Through this method, the acidification of purified vacuoles can be measured in real-time in half-volume 96-well plates or a larger 384-well format. This not only reduces the cost of expensive low abundance reagents, but it drastically reduces the time needed to measure individual conditions in large volume cuvettes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Yeast vacuoles are acidified by the v-type H + -ATPase (V-ATPase) that is comprised of the membrane embedded V O complex and the soluble cytoplasmic V 1 complex. The assembly of the V 1 -V O holoenzyme on the vacuole is stabilized in part through interactions between the V O a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ). PI(3,5)P 2 also affects vacuolar Ca 2+ release through the channel Yvc1 and uptake through the Ca 2+ pump Pmc1. Here, we asked if H + and Ca 2+ transport activities were connected through PI(3,5)P 2 . We found that overproduction of PI(3,5)P 2 by the hyperactive fab1 T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1 EEE mutant attenuated the formation of a H + gradient. Separately, we tested the effects of excess Ca 2+ on vacuole acidification. Adding micromolar Ca 2+ blocked vacuole acidification, whereas chelating Ca 2+ accelerated acidification. The effect of adding Ca 2+ on acidification was eliminated when the Ca 2+ /H + antiporter Vcx1 was absent, indicating that the vacuolar H + gradient can collapse during Ca 2+ stress through Vcx1 activity. This, however, was independent of PI(3,5)P 2 , suggesting that PI(3,5)P 2 plays a role in submicromolar Ca 2+ flux but not under Ca 2+ shock. To see if the link between Ca 2+ and H + transport was bidirectional, we examined Ca 2+ transport when vacuole acidification was inhibited. We found that Ca 2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca 2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P 2 ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial fruit blotch (BFB) is a bacterial disease that devastates Cucurbitaceae crops worldwide, causing significant economic losses. Currently, there is no means to treat or control the disease. This study focused on exploring the antibacterial properties of endophytic fungi against Acidovorax citrulli ( Aac ), the causative agent of BFB. Based on disc diffusion, time kill and MIC microdilution broth assays, four endophytes showed promise in controlling Aac . Nonetheless, only one strain, Neocosmospora sp. MFLUCC 17-0253, reduced the severity of disease on watermelon and melon seedlings up to 80%. Structure analysis revealed production of several compounds by the fungus. Three of these secondary metabolites, including mixture of 2-methoxy-6-methyl-7-acetonyl-8-hydroxy-1,4-maphthalenedione and 5,8-dihydroxy-7-acetonyl-1,4-naphthalenedione, anhydrojavanicin, and fusarnaphthoquinones B exhibited antagonistic activity against Aac . The chemical profile data in planta experiment analyzed by LC-Q/TOF-MS suggested successful colonization of endophytic fungi in their host plant and different metabolic profiles between treated and untreated seedling. Biofilm assay also demonstrated that secondary metabolites of Neocosmospora sp. MFLUCC 17-0253 significantly inhibited biofilm development of Aac . To the best of our knowledge, secondary metabolites that provide significant growth inhibition of Aac are reported for the first time. Thus, Neocosmospora sp. MFLUCC 17-0253 possesses high potential as a biocontrol agent for BFB disease.

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties., Competing Interests: The authors declare no competing interest.