Role In Membrane Fusion Research Articles

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity.

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low infectious viral particle production in cells and the lack of cytopathic effects, which are typically useful for screening. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of members of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the methionine at BoDV-1-G residue 307 increases the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell‒cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Frequency of Fusion Inhibitor Resistance Mutations Among Therapy-Naïve HIV Patients.

Glycoprotein 41 (gp41) of the human immunodeficiency virus type 1 (HIV-1) protein plays a critical role in membrane fusion. Gp41 binds to proteins in the plasma membrane of CD4+ T cells, particularly the T-cell antigen receptor (TCR). These findings indicate that gp41 is involved in the assembly of HIV-1 at the plasma membrane of T cells and affects the stimulation of the TCR. To control HIV-1, new inhibitors were introduced to target the gp41 protein. However, mutations in this region might reduce their efficacy. The Gp41 region was amplified from the sera of 30 patients using nested polymerase chain reaction. The sequences were analyzed by bioinformatics tools to identify mutations and gp41 structural features. Subtyping and the interaction between fusion inhibitors and gp41 proteins were also examined. As the first report from Iran, docking analysis between fusion inhibitors and Iranian gp41 proteins showed that mutations in gp41 could not reduce the efficacy of the fusion inhibitors. Most of the patients were infected with CRF35-AD. Several post-modification positions, including glycosylation and phosphorylation sites, were identified in the gp41 protein. Our findings revealed no known multinational drug resistance to gp41 inhibitors; thus, fusion inhibitors can effectively inhibit HIV in Iranian patients. In addition, the present study introduced a new gp41 region (36-44 aa), which considerably influences the interactions between gp41 inhibitors and the gp41 protein. This region may play a pivotal role in suppressing gp41 inhibitors in CFR35-AD. Furthermore, gp41 can be considered a good target for subtyping analysis via the phylogenetic method.

The underlying mechanical properties of membranes tune their ability to fuse

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Hydrophobic Matching Dictates over the Linear Rule of Mixtures in Binary Lipid Membranes.

Biological membranes feature heterogeneous mixtures of lipids with different head and tail characteristics. Their biophysical properties are dictated by the intimate interaction among different constituent lipids. Previous studies suggest that the membrane area-per-lipid (APL) deviates from the linear rule of mixtures (LRM) for binary lipid membranes, but the underlying mechanism remains elusive. Our molecular dynamics (MD) simulations of binary lipid membranes consisting of lipids with different tail characteristics reveal a competitive mechanism whereby lipids tend to deform each other to minimize the hydrophobic mismatch between their tails. Depending on the relative tail lengths and saturation levels, this may result in an either positive or negative deviation of APL from the LRM. As lipid packing plays an essential role in membrane fusion and peptide-membrane binding, our findings may help guide the selection of lipids for the effective rational design of nanoliposomes and membrane-targeting peptides.

Differential Behavior of Eicosapentaenoic and Docosahexaenoic Acids on the Organization, Dynamics, and Fusion of Homogeneous and Heterogeneous Membranes.

Membrane fusion is a common course in innumerable biological processes that helps in the survival of eukaryotes. Enveloped viruses utilize this process to enter the host cells. Generally, the membrane lipid compositions play an important role in membrane fusion by modulating the membrane's physical properties and the behavior of membrane proteins in the cellular milieu. In this work, we have demonstrated the role of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, on the organization, dynamics, and fusion of homogeneous and heterogeneous membranes. We have exploited arrays of steady-state and time-resolved fluorescence spectroscopic methods and polyethylene glycol-induced membrane fusion assay to elucidate the behavior of EPA and DHA on dioleoyl phosphatidylcholine (DOPC)/cholesterol (CH) homogeneous and DOPC/sphingomyelin/CH heterogeneous membranes. Our results suggest that EPA and DHA display differential effects on two different membranes. The effects of PUFAs in homogeneous membranes are majorly attributed to their flexible chain dynamics, whereas the ability of PUFA-induced cholesterol transfer from the lo to the ld phase rules their behavior in heterogeneous membranes. Overall, our results provide detailed information on the effect of PUFAs on homogeneous and heterogeneous membranes.

Membrane tethers at a glance.

Cargo delivery from one compartment to the next relies on the fusion of vesicles with different cellular organelles in a process that requires the concerted action of tethering factors. Although all tethers act to bridge vesicle membranes to mediate fusion, they form very diverse groups as they differ in composition, and in their overall architecture and size, as well as their protein interactome. However, their conserved function relies on a common design. Recent data on class C Vps complexes indicates that tethers play a significant role in membrane fusion beyond vesicle capturing. Furthermore, these studies provide additional mechanistic insights into membrane fusion events and reveal that tethers should be considered as key players of the fusion machinery. Moreover, the discovery of the novel tether FERARI complex has changed our understanding of cargo transport in the endosomal system as it has been shown to mediate 'kiss-and-run' vesicle-target membrane interactions. In this Cell Science at a Glance and the accompanying poster, we compare the structure of the coiled-coil and the multisubunit CATCHR and class C Vps tether families on the basis of their functional analogy. We discuss the mechanism of membrane fusion, and summarize how tethers capture vesicles, mediate membrane fusion at different cellular compartments and regulate cargo traffic.

Synaptotagmin is not only a calcium sensor, but also exerts entropic forces that drive membrane fusion.

Synaptic neurotransmitter (NT) release is accomplished by a cellular machinery that senses presynaptic Ca-entry following an action potential, and responds by fusing the vesicle and plasma membranes for NT release through the fusion pore. In a long-standing view, a clear division of labor exists between Synaptotagmin 1 (Syt1), the Ca sensor for synchronous release, and the SNAREs which catalyze membrane fusion. Here we developed molecular dynamics simulations suggesting that, surprisingly, Syt1 also plays a major role in membrane fusion. Our findings are strongly supported by experiments. A picture of the release machinery emerges in which both SNAREs and Syt1 exert entropic forces that drive fusion.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

Identifying SNARE Proteins Using an Alignment-Free Method Based on Multiscan Convolutional Neural Network and PSSM Profiles

Background: SNARE proteins play a vital role in membrane fusion and cellular physiology and pathological processes. Many potential therapeutics for mental diseases or even cancer based on SNAREs are also developed. Therefore, there is a dire need to predict the SNAREs for further manipulation of these essential proteins, which demands new and efficient approaches. Methods: Some computational frameworks were proposed to tackle the hurdles of biological methods, which take plenty of time and budget to conduct the identification of SNAREs. However, the performances of existing frameworks were insufficiently satisfied, as they failed to retain the SNARE sequence order and capture the mass hidden features from SNAREs. This paper proposed a novel model constructed on the multiscan convolutional neural network (CNN) and position-specific scoring matrix (PSSM) profiles to address these limitations. We employed and trained our model on the benchmark dataset with fivefold cross-validation and two different independent datasets. Results: Overall, the multiscan CNN was cross-validated on the training set and excelled in the SNARE classification reaching 0.963 in AUC and 0.955 in AUPRC. On top of that, with the sensitivity, specificity, accuracy, and MCC of 0.842, 0.968, 0.955, and 0.767, respectively, our proposed framework outperformed previous models in the SNARE recognition task. Conclusions: It is truly believed that our model can contribute to the discrimination of SNARE proteins and general proteins.

Human sperm TMEM95 binds eggs and facilitates membrane fusion

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

Adolescent Verbal Memory as a Psychosis Endophenotype: A Genome-Wide Association Study in an Ancestrally Diverse Sample.

Verbal memory impairment is one of the most prominent cognitive deficits in psychosis. However, few studies have investigated the genetic basis of verbal memory in a neurodevelopmental context, and most genome-wide association studies (GWASs) have been conducted in European-ancestry populations. We conducted a GWAS on verbal memory in a maximum of 11,017 participants aged 8.9 to 11.1 years in the Adolescent Brain Cognitive Development Study®, recruited from a diverse population in the United States. Verbal memory was assessed by the Rey Auditory Verbal Learning Test, which included three measures of verbal memory: immediate recall, short-delay recall, and long-delay recall. We adopted a mixed-model approach to perform a joint GWAS of all participants, adjusting for ancestral background and familial relatedness. The inclusion of participants from all ancestries increased the power of the GWAS. Two novel genome-wide significant associations were found for short-delay and long-delay recall verbal memory. In particular, one locus (rs9896243) associated with long-delay recall was mapped to the NSF (N-Ethylmaleimide Sensitive Factor, Vesicle Fusing ATPase) gene, indicating the role of membrane fusion in adolescent verbal memory. Based on the GWAS in the European subset, we estimated the SNP-heritability to be 15% to 29% for the three verbal memory traits. We found that verbal memory was genetically correlated with schizophrenia, providing further evidence supporting verbal memory as an endophenotype for psychosis.

Molecular mechanisms for understanding the association between TMPRSS2 and beta coronaviruses SARS-CoV-2, SARS-CoV and MERS-CoV infection: scoping review.

The aim of this scoping review was to identify knowledge gaps and to describe the current state of the research on the association between TMPRSS2 and the essential beta coronaviruses (Beta-CoVs) infection and the molecular mechanisms for this association. We searched MEDLINE (OVID), EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL). We included 13 studies. Evidence shows an essential role of TMPRSS2 in Spike protein activation, entry, and spread into host cells. Co-expression of TMPRSS2 with cell surface receptors (ACE2 or DPP4) increased virus entry. This serine protease is involved in the formation of large syncytia between infected cells. TMPRSS2 cleaved the Spike protein of SARS-CoV, SARS-CoV-2, and MERS-CoV, and increased virus propagation. Accumulating evidence suggests that TMPRSS2 is an essential protease for virus replication. We highlighted its critical molecular role in membrane fusion and the impact in viral mRNA replication, then promoting/driving pathogenesis and resistance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00203-021-02727-3.

Molecular machinery turns full circle.

Two proteins called Sec17 and Sec18 may have a larger role in membrane fusion than is commonly assumed in textbook models.

Identification and transcriptional analysis of SNARE vesicle fusion regulators in tomato (Solanum lycopersicum) during plant development and a comparative analysis of the response to salt stress with wild relatives

Intracellular vesicular trafficking ensures the exchange of lipids and proteins between the membranous compartments. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) play a central role in membrane fusion and they are key factors for vesicular trafficking in plants, including crops economically important such as tomato (Solanum lycopersicum). Taking advantage of the complete genome sequence available of S. lycopersicum, we identified 63 genes that encode putative SNARE proteins. Then, phylogenetic analysis allowed the classification of SNAREs in five main groups and recognizing their possible functions. A structure analysis of the genes, their syntenic relationships and their location in the chromosomes were also carried out for their characterization. In addition, the expression profiles of SNARE genes in different tissues were investigated using microarray-based analysis. The results indicated that specific SNAREs had a higher induction in leaf, root, flower and mature green fruit. S. lycopersicum is characterized for being a crop sensitive to saline stress unlike its wild relatives, such as Solanum pennellii, Solanum pimpinellifolium, Solanum habrochaites or Solanum chilense, which are tolerant. In this context, we analyzed different microarrays and evaluated and validated the transcript levels through qRT-PCR experiments. The results showed that SlGOS12.2, SlVAMP727 and SlSYP51.2 could have a positive relationship with salt stress and probably an important role in their tolerance. All these data increase our knowledge and can also be utilized to identify potential molecular targets for conferring tolerance to various stresses in tomato.

A genetic model of CEDNIK syndrome in zebrafish highlights the role of the SNARE protein Snap29 in neuromotor and epidermal development

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease.

Influenza hemagglutinin membrane anchor

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA-detergent micelles with full-length HA-Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.

The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis.

Synaptotagmin (Syt) proteins comprise a 17-member family, many of which trigger exocytosis in response to calcium. Historically, most studies have focused on the isoform Syt-1, which serves as the primary calcium sensor in synchronous neurotransmitter release. Recently, Syt-7 has become a topic of broad interest because of its extreme calcium sensitivity and diversity of roles in a wide range of cell types. Here, we review the known and emerging roles of Syt-7 in various contexts and stress the importance of its actions. Unique functions of Syt-7 are discussed in light of recent imaging, electrophysiological, and computational studies. Particular emphasis is placed on Syt-7-dependent regulation of synaptic transmission and neuroendocrine cell secretion. Finally, based on biochemical and structural data, we propose a mechanism to link Syt-7's role in membrane fusion with its role in subsequent fusion pore expansion via strong calcium-dependent phospholipid binding.

The Role of Fusion Protein F in the Virus Entry and Cell‐to‐Cell Spread for Nipah Virus

Viruses are obligate parasites, where they require host cells to replicate and produce their progeny. Membrane and non‐segmented single‐stranded negative sense RNA genome are important signatures of paramyxoviruses. The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, respiratory syncytial virus, and Nipah virus (NiV) ‐ a deadly paramyxovirus with up to 75% mortality rate, with neither vaccine nor therapeutics available. Membrane fusion is necessary for entry of the membraned viruses like paramyxoviruses into their host cells (viral‐cell fusion), and for cell‐cell virus spread via syncytia formation (cell‐cell fusion). For most paramyxoviruses membrane fusion requires two viral glycoproteins – the attachment glycoprotein (HN/H/G) and fusion (F) glycoprotein. Structurally, type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses. For virus entry into host cells during infection or cell‐cell spread, NiV has a unique pair of G and F proteins as its specific attachment glycoprotein and fusion glycoprotein respectively. NiV G protein made up of C‐terminal end globular head, stalk region, transmembrane (TM) domain and N‐terminal cytoplasmic tail. In contrast, NiV F protein consist of N‐terminal tail on the surface, hydrophobic fusion peptide (FP), single‐pass transmembrane (TM) domain, and C‐terminal cytoplasmic tail. It also has two heptad repeat domains (HR1/HRA [closer to FP] and HR2/HRB [closer to TM]). Both FP and HR domains play key roles in membrane fusion. NiV F proteins are synthesized as biologically inactive F0 precursors. To be active, F0 proteins need to be cleaved into metastable state of F1 and F2 fragments, covalently linked by disulphide bonds. Unlike other paramyxoviruses, where the cleaving of F0 into F1 + F2 is mediated by trypsin or trypsin‐like proteases, NiV F is cleaved by endocytosis and pH dependent process by endosomal proteases (cathepsin L/B) which exposes the FP as new N‐terminal of the F1 fragment. On the surface of NiV membrane, metastable structure of F1 + F2 makes the FP ‘hidden’ or ‘protected’ by protein‐protein interaction with G protein. Upon receptor (Ephrin B2/B3) binding, the NiV G protein alters its structure which then nudges and triggers the adjacent F protein to undergo conformational changes that causes the FP to stab into the host cell membrane, thus bridging between virus membrane and host cell membrane. On further conformational changes of F1+F2, the HRA/HR1 and HRB/HR2 coalesce or conjoin which then forms ‘fusion pores' allowing the NiV genome to enter the host cell cytoplasm and begin replication steps. When many similar NiV F and G mediated membrane fusion steps occur between a NiV‐infected cell with the non‐infected neighboring cells, it produces a multi‐nucleated giant syncytia formation, i.e. to facilitate cell‐cell virus spread. The Walton High School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function of NiV F and G proteins in membrane fusion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Yvc1 acts as a Novel Suppressor of Vacuolar Membrane Fusion

Yeast vacuoles maintain homeostasis in part by balancing membrane fission and fusion; however the regulation of these events is not fully understood. Phosphoinositides (PIs), and the proteins they interact with, are crucial regulators of both fusion and fission. Our work has shown phosphoinositides are essential for membrane fusion, specifically phosphoinositide 3‐phosphate (PI3P), PI4P, and PI(4,5)P2. Comparatively the phosphoinositide PI(3,5)P2 is relatively scarce and uncharacterized in membrane fusion, however others have demonstrated its requirement in osmotic shock induced membrane fission. Creation of PI(3,5)P2 during osmotic shock is coupled with a release of Ca2+ from the vacuole into the cytosol. This Ca2+ release is thought to be caused by a direct interaction of PI(3,5)P2 with the yeast TRP Ca2+ channel (Yvc1). Since Ca2+ release also plays a role in membrane fusion, we sought to determine whether Yvc1 or its activation by PI(3,5)P2 is involved in membrane fusion as well as fission. Utilizing isolated yeast vacuoles we found that the absence of Yvc1 caused a significant increase in membrane fusion. Our data suggest that the augmented fusion was due to elevated levels of PI3P on the vacuole leading to subsequent increased recruitment of fusion machinery. Specifically, we saw enrichment of the soluble SNARE Vam7, subunits of the HOPS tethering complex, as well as the Rab GTPase Ypt7 and its heterodimeric guanine exchange factor Mon1‐Ccz1. These proteins act as components of the tethering machinery and we found that their enrichment in yvc1D vacuoles led to enhanced trans‐SNARE pairing. The increase in SNARE complexes was not due to enhanced rates of priming, suggesting that the change was due to increased vacuole tethering linked to the increase in Mon1‐Ccz1, Ypt7 and HOPS. Taken together our data suggests Yvc1 acts as a novel suppressor of vacuole membrane fusion through its effects on phosphoinositide levels and recruitment of tethering factors.Support or Funding InformationNational Institutes of Health (R01‐GM101132) to RAFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.