Membrane lipid composition must be dynamically adjusted to preserve bilayer physical properties, yet the cellular mechanisms that support bulk lipid remodeling under physical stress remain incompletely understood. Here, we identify Csf1 as a regulator of membrane lipid remodeling functionally associated with endoplasmic reticulum-plasma membrane (ER-PM) contact sites in Saccharomyces cerevisiae, with features consistent with bridge-like lipid transfer proteins. Using high hydrostatic pressure as a defined physical perturbation that constrains membrane packing, we reveal a requirement for Csf1-dependent lipid remodeling linked to ER-PM contact sites that is masked under standard growth conditions. Quantitative lipidomic and membrane biophysical analyses show that, under hydrostatic compression, loss of Csf1 disrupts coordinated lipid remodeling, leading to reduced phospholipid unsaturation, increased membrane rigidity, and destabilization of PM permeases. We further show that Csf1 cooperates with Osh6/7 to sustain lipid flux and bilayer re-equilibration linked to ER-PM contact sites under conditions permissive for Osh6/7 activity. These findings identify Csf1 as a stress-dependent lipid-remodeling factor that enables adaptive membrane remodeling and preserves membrane protein stability under conditions of constrained membrane flexibility.

ABSTRACT High-temperature stress is a major constraint on yeast growth and fermentation, and has traditionally been interpreted primarily in terms of intracellular molecular damage such as protein denaturation and aggregation. Despite this extensive focus on intracellular mechanisms, how physical factors within the extracellular environment influence yeast thermotolerance remains poorly understood. Here we demonstrate that increases in extracellular osmolarity markedly attenuate growth inhibition under high-temperature conditions in yeast. This protective effect was consistently observed in multiple laboratory and industrial strains of Saccharomyces cerevisiae , as well as in ascomycetous and basidiomycetous yeasts, indicating that osmotic pressure-dependent thermotolerance is a broadly conserved phenomenon. We also found that extracellular osmolarity dynamically increases during growth and then decreases in a diauxic shift-like pattern after growth arrest. At high temperature, the secretion of glucose-derived metabolites decreased, but that of other solutes increased, suggesting that heat stress alters the composition of extracellular solutes contributing to osmolarity. In addition, intracellular glycerol levels increased at high temperature, and this increase was further enhanced under high-osmolarity conditions. Notably, expression of a constitutively active Hog1 mutant exhibited raised intracellular glycerol levels, enhanced nuclear localization of Hog1, and improved growth under high-temperature conditions. Collectively, these findings support a model in which extracellular osmolarity is modulated to avoid excessive intracellular osmolarity under high-temperature conditions, while the intracellular accumulation of glycerol contributes to yeast adaptation at high-temperature. Our results highlight extracellular–intracellular osmotic coordination as an additional physiological layer of high-temperature stress adaptation in yeast.

Biological membranes continuously remodel their lipid composition to preserve functionality under environmental stress, yet the molecular basis of this process in eukaryotes remains incompletely understood. Here, we identify the tunnel-like lipid transfer protein Csf1 as a central factor mediating adaptive lipid remodeling that enables Saccharomyces cerevisiae to tolerate high hydrostatic pressure and low temperature. Quantitative lipidomic and membrane biophysical analyses revealed that loss of Csf1 markedly reduces the unsaturation of phosphatidylserine (PS) and phosphatidylethanolamine (PE), leading to rigidification of the endoplasmic reticulum (ER) membrane. Whereas OLE1 overexpression partially mitigated this defect at low temperature, no compensatory response occurred under pressure. Overexpression of the PS/phosphatidylinositol 4-phosphate exchanger Osh6/7 restored PS and PE unsaturation and rescued growth of the Csf1-deficient mutant, indicating a cooperative role in sustaining PS flux at ER–plasma membrane (PM) contact sites. Pressure-induced degradation of the amino-acid permease Bap2 further linked lipid imbalance to membrane-protein instability. By supplying unsaturated PS and PE, Csf1 preserves ER and PM flexibility, defining a conserved mechanism of eukaryotic membrane adaptation to extreme physical stress. ### Competing Interest Statement The authors have declared no competing interest. * MPa : megapascals BLTP : Bridge-like Lipid Transfer Protein PLCI : phospholipid curvature index DBI : double bond index S : order parameter < τ > : mean fluorescence lifetime of TMA-DPH Japan Society for the Promotion of Science, 18K05397, 22H02247

Pressure is a thermodynamic parameter that influences chemical equilibrium and reaction kinetics; however, its effects on complex cellular mechanisms remain largely unexplored. In this study, we investigated the role of Ehg1 (also known as May24), a novel endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae, in the stabilization of tryptophan permease Tat2, which ensures cell growth under high hydrostatic pressure (∼25 MPa, megapascals). We show that Ehg1 in the cortical ER (cER) physically interacts with the plasma membrane Tat2 in trans and plays a vital role in preserving its localization in the plasma membrane by facilitating its partitioning into the plasma membrane microdomains, lipid rafts. This stabilization depends on the contact between the cER and plasma membrane, which is critical for effective nutrient transport under pressure, as evidenced from the fact that Tat2 was destabilized in Δtether and Δ-super-tether strains lacking such contact. These insights into the regulation of nutrient permease under high pressure contribute to our understanding of microbial adaptation to extreme environments.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrates that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates the proper folding of nutrient permeases in the ER under stress conditions. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity. Graphical Abstract

Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25 MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species. Graphical Abstract Working hypothesis for the role of Mtc6/Ehg2 on the stability and transport function of Tat2 under high pressure.

The fungal cell wall is the initial barrier for the fungi against diverse external stresses, such as osmolarity changes, harmful drugs, and mechanical injuries. This study explores the roles of osmoregulation and the cell-wall integrity (CWI) pathway in response to high hydrostatic pressure in the yeast Saccharomyces cerevisiae. We demonstrate the roles of the transmembrane mechanosensor Wsc1 and aquaglyceroporin Fps1 in a general mechanism to maintain cell growth under high-pressure regimes. The promotion of water influx into cells at 25 MPa, as evident by an increase in cell volume and a loss of the plasma membrane eisosome structure, activates the CWI pathway through the function of Wsc1. Phosphorylation of Slt2, the downstream mitogen-activated protein kinase, was increased at 25 MPa. Glycerol efflux increases via Fps1 phosphorylation, which is initiated by downstream components of the CWI pathway, and contributes to the reduction in intracellular osmolarity under high pressure. The elucidation of the mechanisms underlying adaptation to high pressure through the well-established CWI pathway could potentially translate to mammalian cells and provide novel insights into cellular mechanosensation.

Tryptophan is a relatively rare amino acid whose influx is strictly controlled to meet cellular demands. The yeast Saccharomyces cerevisiae has two tryptophan permeases, namely Tat1 (low-affinity type) and Tat2 (high-affinity type). These permeases are differentially regulated through ubiquitination based on inducible conditions and dependence on arrestin-related trafficking adaptors, although the physiological significance of their degradation remain unclear. Here, we demonstrated that Tat2 was rapidly degraded in an Rsp5-Bul1-dependent manner upon the addition of tryptophan, phenylalanine, or tyrosine, whereas Tat1 was unaffected. The expression of the ubiquitination-deficient variant Tat25K>R led to a reduction in cell yield at 4 μg/mL tryptophan, suggesting the occurrence of an uncontrolled, excessive consumption of tryptophan at low tryptophan concentrations. Eisosomes are membrane furrows that are thought to be storage compartments for some nutrient permeases. Tryptophan addition caused rapid Tat2 dissociation from eisosomes, whereas Tat1 distribution was unaffected. The 5 K > R mutation had no marked effect on Tat2 dissociation, suggesting that dissociation is independent of ubiquitination. Interestingly, the D74R mutation, which was created within the N-terminal acidic patch, stabilized Tat2 while reducing the degree of partitioning into eisosomes. Moreover, the hyperactive I285V mutation in Tat2, which increases Vmax/Km for tryptophan import by 2-fold, reduced the degree of segregation into eisosomes. Our findings illustrate the coordinated activity of Tat1 and Tat2 in the regulation of tryptophan transport at various tryptophan concentrations and suggest the positive role of substrates in inducing a conformational transition in Tat2, resulting in its dissociation from eisosomes and subsequent ubiquitination-dependent degradation.

Pressures found in the biosphere (up to 110 MPa) can affect cellular processes including motility, cell division, nutrient uptake, fermentation and respiration, translation and transcription, protein synthesis, and viability. The pressure sensitivity of cellular respiration, for example, has significance in bioscience and biotechnology as indicated by the existence of pressure-regulated respiratory oxidases and cytochromes, the discovery of piezotolerant obligate aerobes, and the use of pressure in bioethanol production and brewed products. Here, we consider the use of reduced pyridine nucleotide conformation as a metabolic indicator under pressurized conditions. Spectral and time-resolved analysis of autofluorescence signals can identify multiple intracellular NAD(P)H conformations, significant because the distribution of these forms depends on metabolic conditions. This suggests detailed information regarding mitochondrial status and function resides in autofluorescence signals, leading to a renewed interest in developing NAD(P)H conformation as a metabolic indicator and endogenous biomarker. For this study, we use a high-pressure micro-perfusion system and nanosecond-gated spectroscopy setup to monitor the UV-excited autofluorescence spectrum from pressurized samples of Saccharomyces cerevisiae (baker's yeast) during chemically induced metabolic response. Specifically, we compare the pressure-dependent response (up to 30 MPa) induced by two respiratory inhibitors – cyanide and ethanol – each having well-known but different mechanisms of inhibition. We assess the two-component nature of the spectral response using phasor analysis, finding distinct pressure effects on the cyanide- and ethanol-induced responses. Overall, the development of cellular NAD(P)H conformation as a high-pressure fluorescence-based metabolic indicator provides a new tool for investigating the dynamics of cellular metabolism under extreme conditions.

Cellular conformation of reduced pyridine nucleotides NADH and NADPH sensed using autofluorescence spectroscopy is presented as a real-time metabolic indicator under pressurized conditions. The approach provides information on the role of pressure in energy metabolism and antioxidant defense with applications in agriculture and food technologies. Here, we use spectral phasor analysis on UV-excited autofluorescence from Saccharomyces cerevisiae (baker's yeast) to assess the involvement of one or multiple NADH- or NADPH-linked pathways based on the presence of two-component spectral behavior during a metabolic response. To demonstrate metabolic monitoring under pressure, we first present the autofluorescence response to cyanide (a respiratory inhibitor) at 32 MPa. Although ambient and high-pressure responses remain similar, pressure itself also induces a response that is consistent with a change in cellular redox state and ROS production. Next, as an example of an autofluorescence response altered by pressurization, we investigate the response to ethanol at ambient, 12 MPa, and 30 MPa pressure. Ethanol (another respiratory inhibitor) and cyanide induce similar responses at ambient pressure. The onset of non-two-component spectral behavior upon pressurization suggests a change in the mechanism of ethanol action. Overall, results point to new avenues of investigation in piezophysiology by providing a way of visualizing metabolism and mitochondrial function under pressurized conditions.

High hydrostatic pressure is common mechanical stress in nature and is also experienced by the human body. Organisms in the Challenger Deep of the Mariana Trench are habitually exposed to pressures up to 110 MPa. Human joints are intermittently exposed to hydrostatic pressures of 3–10 MPa. Pressures less than 50 MPa do not deform or kill the cells. However, high pressure can have various effects on the cell’s biological processes. Although Saccharomyces cerevisiae is not a deep-sea piezophile, it can be used to elucidate the molecular mechanism underlying the cell’s responses to high pressures by applying basic knowledge of the effects of pressure on industrial processes involving microorganisms. We have explored the genes associated with the growth of S. cerevisiae under high pressure by employing functional genomic strategies and transcriptomics analysis and indicated a strong association between high-pressure signaling and the cell’s response to nutrient availability. This review summarizes the occurrence and significance of high-pressure effects on complex metabolic and genetic networks in eukaryotic cells and how the cell responds to increasing pressure by particularly focusing on the physiology of S. cerevisiae at the molecular level. Mechanosensation in humans has also been discussed.

High hydrostatic pressure in the range of several dozen MPa, are generally assumed to be nonlethal but exert adverse impacts on growth of organisms that are adapted to atmospheric pressure. Deep-sea organisms have established intracellular mechanisms to cope with such extreme environments. A living cell is composed of myriad molecules including nucleic acids, proteins, lipids, ions, and various low molecular compounds. These molecules interact with each other transiently or statically, eventually eliciting innumerable intermolecular interactions even in a small microbial cell. The complexity hampers the understandings of adaptive mechanisms to high pressure employed by deep-sea organisms. Studies with model organisms such as a bacterium Escherichia coli and a yeast Saccharomyces cerevisiae offer breakthroughs to unravel the effects of high pressure on the complex intermolecular networks in living cells. This review summarizes recent advances in high-pressure biology as well as classic issues in this field, especially focusing on remodeling of intracellular systems to adapted to high-pressure environments.

Living organisms are subject to various mechanical stressors, such as high hydrostatic pressure. Empirical evidence shows that under high pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. Here, we demonstrate that superoxide dismutase 1 (Sod1) plays a role in resisting high pressure for cell growth. Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. When these mutant cells were cultured under 25 MPa, their intracellular O2•- levels increased while sod1∆ mutant genome stability was unaffected. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O2•- generator paraquat. The sod1∆ mutant is known to exhibit methionine and lysine auxotrophy. However, excess methionine addition or overexpression of the lysine permease gene LYP1 did not counteract high-pressure sensitivity in the sod1 mutants, suggesting that their amino acid availability might be intact under 25 MPa. Interestingly, an exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria appeared to partially restore the high-pressure growth ability in the sod1 mutants. Taken these results together, we suggest that high pressure enhances O2•- production and Sod1 within the IMS plays a role in scavenging O2•- allowing the cells to grow under high pressure. BACKGROUND:Empirical evidence shows that under high hydrostatic pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. In the current study, we aimed to explore the role of superoxide dismutase 1 (Sod1) in yeast able to grow under high pressure. METHODS:Wild type and sod1 mutant cells were cultured in high-pressure chambers under 25 MPa (~250 kg/cm2). The SOD activity in whole cell extracts and 6His-tagged Sod1 recombinant proteins was analyzed using an SOD assay kit. The O2•- generation in cells was estimated by fluorescence staining. RESULTS:Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O2•- generator paraquat. Exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria partially restored the high-pressure growth ability in the sod1 mutants. CONCLUSIONS:High pressure enhances O2•- production and Sod1 within the IMS plays a role in scavenging O2•- allowing the cells to grow under high pressure. GENERAL SIGNIFICANCE:Unlike external free radical-generating compounds, high-pressure treatment appeared to increase endogenous O2•- levels in yeast cells. Our experimental system offers a unique approach to investigating the physiological responses to mechanical and oxidative stresses in human body.

The draft genome sequence of the deep-sea yeast Naganishia liquefaciens strain N6, isolated from the Japan Trench, is reported here. This strain was previously classified into a Cryptococcus clade. Phylogenetic analysis using the presented sequence suggests that strain N6 is in the clade of the genus Naganishia .

Hsp70: J-domain protein (JDP) machines, along with the cellular protein degradation systems play a central role in regulating cellular proteostasis. An equally robust surveillance system operates at the plasma membrane too that affects proper sorting, stability as well as the turnover of membrane proteins. Although plausible, a definitive role of the Hsp70: JDP machine in regulating the stability of plasma membrane proteins is not well understood in Saccharomyces cerevisiae. Here we show that a moderate over-expression of Caj1, one of the thirteen JDPs residing in the nucleo-cytosolic compartment of S. cerevisiae reduced the cold sensitivity of tryptophan auxotrophic yeast cells by stabilizing tryptophan permeases, Tat1 and Tat2 in a J-domain dependent manner. Concomitantly, higher Caj1 levels also caused slow growth and increased plasma membrane damage at elevated temperatures possibly due to the stabilization of thermolabile plasma membrane proteins. Finally, we show that although majorly cytosolic, Caj1 also co-localizes with the membrane dye FM4-64 at the cellular periphery suggesting that Caj1 might interact with the plasma membrane. Based on the results presented in this study, we implicate the Hsp70: Caj1 chaperone machine in regulating the stability or turnover of plasma membrane proteins in budding yeast.

Mechanical stresses including high hydrostatic pressure elicit diverse physiological effects on organisms. Gtr1/Gtr2 and Ego1/Ego3, central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased the glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ, and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1, and PIB2 with GLN3. These results suggested that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrated the regulatory circuit that maintained the intracellular amino acid homeostasis and suggested the critical roles the EGOC-TORC1 and Pib2-TORC1 complexes played in the growth of yeast under high hydrostatic pressure.

Location: Plaza Verde, Numazu, Shizuoka, JapanDate: September 18th to 22nd, 2018After its inception in the year 1998, the international association of High Pressure Bioscience and Biotechnology (HP...