Biology subjects

Ernst, R.

Publications and source records attributed to Ernst, R..

14 recordsLinked to original sources

bioRxiv2026

Lipid remodelling enables adaptation to chronic hyperosmotic stress

Lipid droplets (LDs) accumulate in response to diverse cellular stresses. However, their regulation and physiological roles remain poorly understood in most contexts. Here, we show that, in budding yeast, chronic hyperosmotic stress induces sustained LD accumulation. Unlike the transient LD response observed during acute osmotic shock, chronic stress triggers prolonged, Dga1-dependent triacylglycerol synthesis. In the absence of triacylglycerol synthesis cellular fitness is severely affected. Lipidomic profiling reveals extensive membrane remodelling during chronic hyperosmotic stress, most notably a shift from phosphatidylethanolamine to phosphatidylcholine. In LD-deficient cells, the stress-induced PC increase is blunted and manipulation of PC synthesis modulates the fitness of triacylglycerol-deficient cells under hyperosmotic stress. Thus, LD accumulation and phospholipid remodelling underlie an adaptive response to chronic hyperosmotic stress. SummaryThis work demonstrates that membrane remodelling occurs in cells experiencing chronic hyperosmotic stress. Both triacylglycerol and phosphatidylcholine levels are increased. Cell fitness depends upon increased triacylglycerol synthesis and is further modulated by manipulating phosphatidylcholine levels.

cell biology↗

bioRxiv2026

Mammalian MemPrep establishes the lipid composition of ER membranes in HEK293T cells

The endoplasmic reticulum (ER) forms a dynamic network of sheets and tubules, whose molecular lipid composition remains incompletely defined. Using an optimized MemPrep workflow, we establish a high-confidence lipidome of the mammalian ER and selectively enrich membrane vesicles originating from ER tubules as a major ER subdomain. Quantitative lipidomics show that ER membranes are dominated by phosphatidylcholine and mono-unsaturated glycerophospholipids, consistent with a highly compressible bilayer. Although proteomics suggests a functional specialization of ER tubules and an enrichment of tubule-associated proteins therein, the lipidome of an ER tubule-enriched isolate is indistinguishable from the general ER, indicating that principal ER architectures share a common lipid composition. Integration of lipidomic data with bioinformatic analyses of transmembrane helices further demonstrates that the physicochemical features of ER lipids mirror those of ER-resident membrane proteins, including reduced hydrophobicity and increased polarity compared to plasma membrane proteins. These findings support a coordinated evolution of ER proteins and lipids based on shared biophysical constraints. Together, this work provides a definitive characterization of the mammalian ER lipidome and suggest that membrane properties are maintained across the entire ER.

cell biology↗

bioRxiv2025

Ubiquitin-dependent signal amplification in lipid saturation sensing

Cellular membranes are dynamic platforms whose composition and biophysical properties are surveyed by sensor proteins to maintain homeostasis. Failure to preserve membrane homeostasis, however, results in cellular stress and organelle dysfunction. Using the prototypical lipid saturation sensor Mga2, we explored how weak physical cues that modulate rotational movements in the transmembrane region are converted into decisive biochemical outputs that ultimately control the production of unsaturated fatty acids. Quantitative in vitro ubiquitylation assays and kinetic modeling reveal vastly distinct rates of Mga2 ubiquitylation controlled by the membrane environment. Mga2 ubiquitylation dominates in tightly packed, saturated membranes, while loosely packed environments favor an inhibitory autoubiquitylation of the cognate E3 ligase Rsp5. This mechanism provides a means of signal amplification, which can function even in the absence of deubiquitylating enzymes. Our findings provide a mechanistic framework for how membrane property sensors convert weak, fluctuating physical cues into robust biochemical outcomes, and put a spotlight on the regulatory potential of E3 ligase autoubiquitylation in cellular surveillance.

biochemistry↗

bioRxiv2025

Pex30-dependent membrane contacts sites maintain ER lipid homeostasis

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the Dysferlin domain (DysF) on Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo . The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that ER-Vacuole MCS requires Pex30 C-terminal Domain of Unknown Function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

cell biology↗

bioRxiv2025

Laboratory yeast strains are highly diverse in lipid metabolism

The impact of the genetic background on the lipidome of yeast strains remains underexplored. This study systematically compares the lipidomes of five commonly used laboratory yeast strains: BY4741, W303, D273-10B, RM11-1a, and CEN.PK2-1c. Shotgun lipidomics reveals significant variations in lipid class and acyl chain composition down to the level of molecular species. Notably, the most abundant lipid class differed between the strains: phosphatidylinositol (PI) lipids are predominant in BY4741, while phosphatidylethanolamine (PE) lipids are in D273. Ergosterol esters, which are the storage form of the major yeast sterol ergosterol, are at higher levels in all strains other than BY4741, correlating with a low gene expression of lipid metabolic enzymes Hmg1 and Are2 in BY4741. Despite these lipidomic differences, transcriptomic analysis did not show significant changes in most genes related to lipid metabolism, suggesting post-transcriptional modifications, protein abundance, and metabolic flux as potential regulatory mechanisms. This study underscores the complexity of lipidome regulation and the need for further investigation into the underlying mechanisms.

biochemistry↗

bioRxiv2024

Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting

Large unilamellar vesicles (LUVs) are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of individual membrane proteins. The functional reconstitution of transmembrane in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the surrounding lipid bilayer. During the reconstitution procedure Such hydrophobic mismatch causes low yields and protein aggregation, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune membrane sterol contents proteoliposomes after their formation. Both cholesterol delivery and extraction are mediated by methyl-{beta}-cyclodextrin in a dialysis setting, which maintains (proteo)liposomes in a confined compartment. This makes it possible to reversibly tune the cholesterol level without losing membrane material simply by placing the dialysis cassette in a new bath containing either empty or cholesterol-loaded methyl-{beta}-cyclodextrin. Cholesterol delivery and removal is monitored with the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Forster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while the subsequent removal of cholesterol demonstrates the full reversibility. We propose that tuning membrane compressibility by methyl-{beta}-cyclodextrin-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study its impact on membrane protein structure, function, and dynamics. Statement of significanceGenerating complex, sterol-rich, biomimetic membranes for studying the structure and function of reconstituted membrane proteins is challenging. As an important step towards asymmetric, sterol-rich, complex model membrane systems, we have established a procedure to control the membrane sterol level of liposomes and proteoliposomes using methyl-{beta}-cyclodextrin in a dialysis setup. We demonstrate the feasibility of this approach by C-Laurdan and dehydroergosterol fluorescence spectroscopy and gain control over the membrane sterol content. We explore several parameters that affect the rate of cholesterol delivery and show that the oligomerization of a membrane property sensor, which is on the unfolded protein response sensor protein Ire1, is controlled by the sterol content of the surrounding lipid bilayer.

biophysics↗

bioRxiv2022

Remodeling of yeast vacuole membrane lipidomes from the log (1-phase) to stationary stage (2-phases)

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micron-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids ([~]50%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These results fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols. SIGNIFICANCE STATEMENTWhen budding yeast shift from growth to quiescence, the membrane of one of their organelles (the vacuole) undergoes liquid-liquid phase separation. What changes in the membranes lipids cause this phase transition? Here, we conduct lipidomics of immuno-isolated vacuole membranes. We analyze our data in the context of lipid melting temperatures, inspired by observations that liquid-liquid phase separation in model membranes requires a mixture of lipids with high melting temperatures, lipids with low melting temperatures, and sterols. We find that phase-separated vacuole membranes have higher concentrations of PC lipids, and that those lipids have higher melting temperatures. To conduct our experiments, we developed a tagged version of a protein (Mam3) for immuno-isolation of vacuole membranes.

biophysics↗

bioRxiv2022

A new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work provided insights into the subcellular distribution of lipids, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remained insufficiently characterized. Here we describe a method for purifying organellar membranes from yeast, MemPrep. We demonstrate the purity of our ER preparations by quantitative proteomics and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings have important implications for understanding the role of lipids in membrane protein insertion, folding, and their sorting along the secretory pathway. Application of the combined preparative and analytical platform to acutely stressed cells reveals dynamic ER membrane remodeling and establishes molecular fingerprints of lipid bilayer stress.

cell biology↗

bioRxiv2021

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

cell biology↗

bioRxiv2019

Systematic cysteine-crosslinking in native membranes establishes the transmembrane architecture in Ire1 clusters

The endoplasmic reticulum (ER) is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine crosslinking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1. SummaryThe endoplasmic reticulum (ER) is a hotspot of lipid biosynthesis and crucial for the folding of membrane and secretory proteins. The unfolded protein response (UPR) controls the size and folding capacity of the ER. The conserved UPR transducer Ire1 senses both unfolded proteins and aberrant lipid compositions to mount adaptive responses. Using a biochemical assay to study Ire1 in signaling-active clusters, Vath et al. provide evidence that the neighboring transmembrane helices of clustered Ire1 form an X irrespectively of the primary cause of ER stress. Hence, different forms of ER stress converge in a common, signaling-active transmembrane architecture of Ire1.

biochemistry↗

bioRxiv2019

Regulation of lipid saturation without sensing membrane fluidity

Abstract/SummaryCells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here, we have reconstituted the core machinery for sensing and regulating lipid saturation in bakers yeast to directly characterize its response to defined membrane environments. Using spectroscopic techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains and provide unprecedented insight into the molecular rules of membrane adaptivity. Our data challenge the prevailing hypothesis that membrane viscosity serves as the measured variable for regulating lipid saturation. Rather, we show that the signaling output of Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which senses the lateral pressure and/or compressibility profile in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such membrane properties in the future.

biophysics↗

bioRxiv2018

Sexual Dichromatism Drives Diversification Within a Major Radiation of African Amphibians

Theory predicts that sexually dimorphic traits under strong sexual selection, particularly those involved with intersexual signaling, can accelerate speciation and produce bursts of diversification. Sexual dichromatism (sexual dimorphism in color) is widely used as a proxy for sexual selection and is associated with rapid diversification in several animal groups, yet studies using phylogenetic comparative methods to explicitly test for an association between sexual dichromatism and diversification have produced conflicting results. Sexual dichromatism is rare in frogs, but it is both striking and prevalent in African reed frogs, a major component of the diverse frog radiation termed Afrobatrachia. In contrast to most other vertebrates, reed frogs display female-biased dichromatism in which females undergo color transformation, often resulting in more ornate coloration in females than in males. We produce a robust phylogeny of Afrobrachia to investigate the evolutionary origins of sexual dichromatism in this radiation and examine whether the presence of dichromatism is associated with increased rates of net diversification. We find that sexual dichromatism evolved once within hyperoliids and was followed by numerous independent reversals to monochromatism. We detect significant diversification rate heterogeneity in Afrobatrachia and find that sexually dichromatic lineages have double the average net diversification rate of monochromatic lineages. By conducting trait simulations on our empirical phylogeny, we demonstrate our inference of trait-dependent diversification is robust. Although sexual dichromatism in hyperoliid frogs is linked to their rapid diversification and supports macroevolutionary predictions of speciation by sexual selection, the function of dichromatism in reed frogs remains unclear. We propose that reed frogs are a compelling system for studying the roles of natural and sexual selection on the evolution of sexual dichromatism across both micro- and macroevolutionary timescales.

evolutionary biology↗

bioRxiv2018

Homeostatic remodeling of mammalian membranes in response to dietary lipids is essential for cellular fitness

Biological membranes form the functional, dynamic interface that hosts a major fraction of all cellular bioactivity. Proper membrane physiology requires maintenance of a narrow range of physicochemical properties, which must be buffered from external perturbations. While homeostatic adaptation of membrane fluidity to temperature variation is a ubiquitous design feature of ectothermic organisms, such responsive membrane adaptation to external inputs has not been directly observed in mammals. Here, we report that challenging mammalian membrane homeostasis by dietary lipids leads to robust lipidomic remodeling to preserve membrane physical properties. Specifically, exogenous polyunsaturated fatty acids (PUFAs) are rapidly and extensively incorporated into membrane lipids, inducing a reduction in membrane packing. These effects are rapidly compensated both in culture and in vivo by lipidome-wide remodeling, most notably upregulation of saturated lipids and cholesterol. These lipidomic changes result in recovery of membrane packing and permeability. This lipidomic and biophysical compensation is mediated in part by lipid regulatory machinery, whose pharmacological or genetic abrogation results in cytotoxicity when membrane homeostasis is challenged by dietary lipids. These results reveal an essential mammalian mechanism for membrane homeostasis wherein lipidome remodeling in response to dietary lipid inputs preserves functional membrane phenotypes.

cell biology↗

bioRxiv2018

The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important lipid intermediate and signaling lipid at the branch point of these pathways and constantly monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. Here, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for the selective binding to membranes containing PA over phosphatidylserine (PS). The insertion of the AH into the hydrophobic core of the membrane renders Opi1 sensitive to the lipid acyl chain composition as an important factor contributing to the regulation of membrane biogenesis. Based on these findings, we rationally designed the membrane binding properties of Opi1 to control its responsiveness in the physiological context. Using extensive molecular dynamics (MD) simulations, we identified two PA-selective three-finger grips that tightly bind the phosphate headgroup, while interacting less intimately and more transiently with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

biochemistry↗