Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae.

BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production. RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells. CONCLUSION: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae.

BACKGROUND: Target of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear. RESULTS: We demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-responsive genes mainly via the Tap42-Sit4-Rrd1/2 pathway. To confirm TORC1's function in glucose signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination. CONCLUSIONS: Our studies indicate that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

Expression of a Functional IL-2 Receptor in Vascular Smooth Muscle Cells.

Many nonlymphoid cell types express at least two, if not all three, subunits of the IL-2R; although, compared with lymphocytes, relatively little is known about how IL-2 affects the function of nonlymphoid cells. The limited information available suggests that IL-2 has a substantial impact on cells such as gastrointestinal epithelial cells, endothelial cells, and fibroblasts. In a previous report from our laboratory, we noted that IL-2 and IL-2Rß-deficient mice lose smooth muscle cells over time, eventually resulting in aneurysmal aortas and ectatic esophagi. This finding, combined with our work showing that IL-2 surrounds vascular smooth muscle cells by association with perlecan, led us to ask whether vascular smooth muscle cells express an IL-2R. Toward this end, we reported the expression of IL-2Rß on human and murine vascular smooth muscle cells. We now report that vascular smooth muscle cells express all three subunits of the IL-2R, and that expression of IL-2Rα varies with vascular smooth muscle cell phenotype. Furthermore, we show that, through a functional IL-2R, IL-2 initiates signaling pathways and impacts vascular smooth muscle cell function. Finally, we demonstrate that IL-2 expression increases upon initiation of conditions that promote intimal hyperplasia, suggesting a mechanism by which the IL-2/IL-2R system may impact this widespread vascular pathology.

Genomics-driven discovery of a biosynthetic gene cluster required for the synthesis of BII-Rafflesfungin from the fungus Phoma sp. F3723.

BACKGROUND: Phomafungin is a recently reported broad spectrum antifungal compound but its biosynthetic pathway is unknown. We combed publicly available Phoma genomes but failed to find any putative biosynthetic gene cluster that could account for its biosynthesis. RESULTS: Therefore, we sequenced the genome of one of our Phoma strains (F3723) previously identified as having antifungal activity in a high-throughput screen. We found a biosynthetic gene cluster that was predicted to synthesize a cyclic lipodepsipeptide that differs in the amino acid composition compared to Phomafungin. Antifungal activity guided isolation yielded a new compound, BII-Rafflesfungin, the structure of which was determined. CONCLUSIONS: We describe the NRPS-t1PKS cluster 'BIIRfg' compatible with the synthesis of the cyclic lipodepsipeptide BII-Rafflesfungin [HMHDA-L-Ala-L-Glu-L-Asn-L-Ser-L-Ser-D-Ser-D-allo-Thr-Gly]. We report new Stachelhaus codes for Ala, Glu, Asn, Ser, Thr, and Gly. We propose a mechanism for BII-Rafflesfungin biosynthesis, which involves the formation of the lipid part by BIIRfg_PKS followed by activation and transfer of the lipid chain by a predicted AMP-ligase on to the first PCP domain of the BIIRfg_NRPS gene.

A yeast two-hybrid system for the screening and characterization of small-molecule inhibitors of protein-protein interactions identifies a novel putative Mdm2-binding site in p53.

BACKGROUND: Protein-protein interactions (PPIs) are fundamental to the growth and survival of cells and serve as excellent targets to develop inhibitors of biological processes such as host-pathogen interactions and cancer cell proliferation. However, isolation of PPI inhibitors is extremely challenging. While several in vitro assays to screen for PPI inhibitors are available, they are often expensive, cumbersome, and require large amounts of purified protein. In contrast, limited in vivo assays are available to screen for small-molecule inhibitors of PPI. METHODS: We have engineered a yeast strain that is suitable for screening of small-molecule inhibitors of protein-protein interaction using the Yeast 2-hybrid Assay. We have optimised and validated the assay using inhibitors of the p53-Mdm2 interaction and identified a hitherto unreported putative Mdm2-binding domain in p53. RESULTS: We report a significantly improved and thoroughly validated yeast two-hybrid (Y2H) assay that can be used in a high throughput manner to screen for small-molecule PPI inhibitors. Using the p53-Mdm2 interaction to optimize the assay, we show that the p53-Mdm2 inhibitor nutlin-3 is a substrate for the yeast ATP-binding cassette (ABC) transporter Pdr5. By deleting nine ABC transporter-related genes, we generated a ABC9Δ yeast strain that is highly permeable to small molecules. In the ABC9Δ strain, p53-Mdm2 interaction inhibitors, like AMG232 and MI-773, completely inhibited the p53-Mdm2 interaction at nanomolar concentrations in the Y2H assay. In addition, we identified a conserved segment in the core DNA-binding domain of p53 that facilitates stable interaction with Mdm2 in yeast cells and in vitro. CONCLUSION: The Y2H assay can be utilized for high-throughput screening of small-molecule inhibitors of PPIs and to identify domains that stabilize PPIs.

The Rim15-endosulfine-PP2ACdc55 signalling module regulates entry into gametogenesis and quiescence via distinct mechanisms in budding yeast.

Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2ACdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2ACdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2ACdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2ACdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2ACdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2ACdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates.

Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I.

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore.

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions.

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4(Eme1). Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastrophe.

The cohesin ring concatenates sister DNA molecules.

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.

Mono-2-ethylhexylphthalate (MEHP) is a potent agonist of human TRPA1 channel.

Phthalates are extensively used as plasticizers in diverse consumer care products but have been reported to cause adverse health effects in humans. A commonly used phthalate, di-2-ethylhexylphthalate (DEHP) causes developmental and reproductive toxicities in humans, but the associated molecular mechanisms are not fully understood. Mono-2-ethylhexylphthalate (MEHP), a hydrolytic product of DEHP generated by cellular esterases, is proposed to be the active toxicant. We conducted a screen for sensory irritants among compounds used in consumer care using an assay for human Transient Receptor Potential A1 (hTRPA1). We have identified MEHP as a potent agonist of hTRPA1. MEHP-induced hTRPA1 activation was blocked by the TRPA1 inhibitor A-967079. Patch clamp assays revealed that MEHP induced inward currents in cells expressing hTRPA1. In addition, the N855S mutation in hTRPA1 associated with familial episodic pain syndrome decreased MEHP-induced hTRPA1 activation. In summary, we report that MEHP is a potent agonist of hTRPA1 which generates new possible mechanisms for toxic effects of phthalates in humans.

Sustainable production of lipids from cocoa fatty acid distillate fermentation driven by adaptive evolution in Yarrowia lipolytica.

Single cell oil production using oleaginous yeasts is a promising alternative to animal and plant-derived lipids. But substrate costs for microbial fermentation are a major bottleneck. Using side streams as alternative to substrates like glucose, for growing yeast, is a potential cost-effective solution. By combining a previously reported process of growing yeasts on a solid cocoa fatty acid distillate side stream with adaptive evolution techniques, the growth of oleaginous yeast Yarrowia lipolytica was improved by 2-fold. The lipid titre was also boosted by more than 3-fold. Using transcriptomics, key genes were identified that are possibly involved in tailoring of lipid composition, side stream utilisation and enhancement of lipid titres. Candidate genes were also identified that might enable efficient growth and utilization of fatty acids and triacylglycerides found in cocoa fatty acid distillate. In summary, this research has improved the understanding of side stream utilisation for lipid production in oleaginous yeast.

How to halve ploidy: lessons from budding yeast meiosis.

Maintenance of ploidy in sexually reproducing organisms requires a specialized form of cell division called meiosis that generates genetically diverse haploid gametes from diploid germ cells. Meiotic cells halve their ploidy by undergoing two rounds of nuclear division (meiosis I and II) after a single round of DNA replication. Research in Saccharomyces cerevisiae (budding yeast) has shown that four major deviations from the mitotic cell cycle during meiosis are essential for halving ploidy. The deviations are (1) formation of a link between homologous chromosomes by crossover, (2) monopolar attachment of sister kinetochores during meiosis I, (3) protection of centromeric cohesion during meiosis I, and (4) suppression of DNA replication following exit from meiosis I. In this review we present the current understanding of the above four processes in budding yeast and examine the possible conservation of molecular mechanisms from yeast to humans.

Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae.

Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, the mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing an in vivo TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first "canonical Rag guanosine triphosphatase (GTPase)-dependent pathway" requires conversion of glucose to fructose 1,6-bisphosphate, which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP-Gtr2GDP. The second "non-canonical Rag GTPase-dependent pathway" requires conversion of glucose to glucose 6-phosphate, which activates TORC1 via a process that involves Gtr1GTP-Gtr2GTP and mitochondrial function. The third "Rag GTPase-independent pathway" requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.

Floating sphere assay: A rapid qualitative method for microvolume analysis of gelation.

A huge, unprecedented demand for gelatin coupled with its implications on global sustainability has resulted in the need to discover novel proteins with gelling attributes for applications in the food industry. Currently used gelation assays require large sample volumes and thus the screening for novel gelling proteins is a formidable technical challenge. In this paper, we report the 'Floating Sphere Assay' which is a simple, economical, and miniaturized assay to detect minimum gelling concentration with volumes as low as 50 µl. Results from the Floating Sphere Assay are consistent with currently used methods for gelation tests and accurately estimate the Minimum Gelling Concentrations (MGCs) of gelatin, κ-carrageenan and gellan gum. The assay was also able to differentiate the strengths of strong and weak gellan gum gels prepared at pH 3.5 and pH 7.0 respectively. The Floating Sphere Assay can be utilized in high-throughput screens for gelling proteins and can accelerate the discovery of gelatin substitutes.

Diethyl phthalate (DEP) perturbs nitrogen metabolism in Saccharomyces cerevisiae.

Phthalates are ubiquitously used as plasticizers in various consumer care products. Diethyl phthalate (DEP), one of the main phthalates, elicits developmental and reproductive toxicities but the underlying mechanisms are not fully understood. Chemogenomic profiling of DEP in S. cerevisiae revealed that two transcription factors Stp1 and Dal81 involved in the Ssy1-Ptr5-Ssy5 (SPS) amino acid-sensing pathway provide resistance to DEP. Growth inhibition of yeast cells by DEP was stronger in poor nitrogen medium in comparison to nitrogen-rich medium. Addition of amino acids to nitrogen-poor medium suppressed DEP toxicity. Catabolism of amino acids via the Ehrlich pathway is required for suppressing DEP toxicity. Targeted metabolite analyses showed that DEP treatment alters the amino acid profile of yeast cells. We propose that DEP inhibits the growth of yeast cells by affecting nitrogen metabolism and discuss the implications of our findings on DEP-mediated toxic effects in humans.

Identification of pathways modulating vemurafenib resistance in melanoma cells via a genome-wide CRISPR/Cas9 screen.

Vemurafenib is a BRAF kinase inhibitor (BRAFi) that is used to treat melanoma patients harboring the constitutively active BRAF-V600E mutation. However, after a few months of treatment patients often develop resistance to vemurafenib leading to disease progression. Sequence analysis of drug-resistant tumor cells and functional genomic screens has identified several genes that regulate vemurafenib resistance. Reactivation of mitogen-activated protein kinase (MAPK) pathway is a recurrent feature of cells that develop resistance to vemurafenib. We performed a genome-scale CRISPR-based knockout screen to identify modulators of vemurafenib resistance in melanoma cells with a highly improved CRISPR sgRNA library called Brunello. We identified 33 genes that regulate resistance to vemurafenib out of which 14 genes have not been reported before. Gene ontology enrichment analysis showed that the hit genes regulate histone modification, transcription and cell cycle. We discuss how inactivation of hit genes might confer resistance to vemurafenib and provide a framework for follow-up investigations.

Macrocyclization of an all-d linear α-helical peptide imparts cellular permeability.

Peptide-based molecules hold great potential as targeted inhibitors of intracellular protein-protein interactions (PPIs). Indeed, the vast diversity of chemical space conferred through their primary, secondary and tertiary structures allows these molecules to be applied to targets that are typically deemed intractable via small molecules. However, the development of peptide therapeutics has been hindered by their limited conformational stability, proteolytic sensitivity and cell permeability. Several contemporary peptide design strategies are aimed at addressing these issues. Strategic macrocyclization through optimally placed chemical braces such as olefinic hydrocarbon crosslinks, commonly referred to as staples, may improve peptide properties by (i) restricting conformational freedom to improve target affinities, (ii) improving proteolytic resistance, and (iii) enhancing cell permeability. As a second strategy, molecules constructed entirely from d-amino acids are hyper-resistant to proteolytic cleavage, but generally lack conformational stability and membrane permeability. Since neither approach is a complete solution, we have combined these strategies to identify the first examples of all-d α-helical stapled and stitched peptides. As a template, we used a recently reported all d-linear peptide that is a potent inhibitor of the p53-Mdm2 interaction, but is devoid of cellular activity. To design both stapled and stitched all-d-peptide analogues, we used computational modelling to predict optimal staple placement. The resultant novel macrocyclic all d-peptide was determined to exhibit increased α-helicity, improved target binding, complete proteolytic stability and, most notably, cellular activity.

Cohesin's ATPase activity is stimulated by the C-terminal Winged-Helix domain of its kleisin subunit.

BACKGROUND: Cohesin, a multisubunit protein complex conserved from yeast to humans, holds sister chromatids together from the onset of replication to their separation during anaphase. Cohesin consists of four core subunits, namely Smc1, Smc3, Scc1, and Scc3. Smc1 and Smc3 proteins are characterized by 50-nm-long anti-parallel coiled coils flanked by a globular hinge domain and an ABC-like ATPase head domain. Whereas Smc1 and Smc3 heterodimerize via their hinge domains, the kleisin subunit Scc1 connects their ATPase heads, and this results in the formation of a large ring. Biochemical studies suggest that cohesin might trap sister chromatids within its ring, and genetic evidence suggests that ATP hydrolysis is required for the stable association of cohesin with chromosomes. However, the precise role of the ATPase domains remains enigmatic. RESULTS: Characterization of cohesin's ATPase activity suggests that hydrolysis depends on the binding of ATP to both Smc1 and Smc3 heads. However, ATP hydrolysis at the two active sites is not per se cooperative. We show that the C-terminal winged-helix domain of Scc1 stimulates the ATPase activity of the Smc1/Smc3 heterodimer by promoting ATP binding to Smc1's head. In contrast, we do not detect any effect of Scc1's N-terminal domain on Smc1/Smc3 ATPase activity. CONCLUSIONS: Our studies reveal that Scc1 not only connects the Smc1 and Smc3 ATPase heads but also regulates their ATPase activity.

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry.

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2' deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Chemical-genetic interaction landscape of mono-(2-ethylhexyl)-phthalate using chemogenomic profiling in yeast.

Integration of chemical-genetic interaction data with biological functions provides a mechanistic understanding of how toxic compounds affect cells. Mono-(2-ethylhexyl)-phthalate (MEHP) is an active metabolite of di-(2-ethylhexyl)-phthalate (DEHP), a commonly used plasticizer. MEHP adversely affects human health causing hepatotoxicity and reproductive toxicity. How MEHP affects cellular physiology is not fully understood. We utilized a genome-wide competitive fitness-based assay called 'chemogenomic profiling' to determine the genetic interaction map of MEHP in Saccharomyces cerevisiae. Gene Ontology enrichment analysis of 218 genes that provide resistance to MEHP indicated that MEHP affects seven cellular processes namely: (1) cellular amino acid biosynthetic process, (2) sterol biosynthetic process, (3) cellular transport, (4) transcriptional and translational regulation, (5) protein glycosylation, (6) cytokinesis and cell morphogenesis and (7) ionic homeostasis. We show that MEHP protects yeast cells from membrane perturbing agents such as amphotericin B, dihydrosphingosine and phytosphingosine. Moreover, we also demonstrate that MEHP compromises the integrity of the yeast plasma membrane and cell wall. Our work provides a basis for further investigation of MEHP toxicity in humans.