Fumiyoshi Abe

Functional Interactions between Sphingolipids and Sterols in Biological Membranes Regulating Cell Physiology

Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.

Tryptophan permease gene TAT2 confers high-pressure growth in Saccharomyces cerevisiae.

ABSTRACT Hydrostatic pressure in the range of 15 to 25 MPa was found to cause arrest of the cell cycle in G1 phase in an exponentially growing culture of Saccharomyces cerevisiae, whereas a pressure of 50 MPa did not. We found that a plasmid carrying the TAT2 gene, which encodes a high-affinity tryptophan permease, enabled the cells to grow under conditions of pressure in the range of 15 to 25 MPa. Additionally, cells expressing the Tat2 protein at high levels became endowed with the ability to grow under low-temperature conditions at 10 or 15°C as well as at high pressure. Hydrostatic pressure significantly inhibited tryptophan uptake into the cells, and the Tat2 protein level was down-regulated by high pressure. The activation volume associated with tryptophan uptake was found to be a large positive value, 46.2 ± 3.85 ml/mol, indicating that there was a net volume increase in a rate-limiting step in tryptophan import. The results showing cell cycle arrest in G1 phase and down-regulation of the Tat2 protein seem to be similar to those observed upon treatment of cells with the immunosuppressive drug rapamycin. Although rapamycin treatment elicited the rapid dephosphorylation of Npr1 and induction of Gap1 expression, hydrostatic pressure did not affect the phosphorylation state of Npr1 and it decreased the level of Gap1 protein, suggesting that the pressure-sensing pathway may be independent of Npr1 function. Here we describe high-pressure sensing in yeast in comparison with the TOR-signaling pathway and discuss an important factor involved in adaptation of organisms to high-pressure environments.

The biotechnological potential of piezophiles

Microorganisms that prefer high-pressure conditions are termed piezophiles (previously termed barophiles). The molecular basis of piezophily is now being investigated extensively focusing on aspects of gene regulation and the function of certain proteins in deep-sea isolates. Little attention has been paid, however, to the potential biotechnological applications of piezophiles compared with other extremophiles. Based on the fundamental knowledge available, we will try to answer the following questions: How can we exploit the biotechnological potential of piezophiles? What can be understood by the application of high-pressure in biological systems?

Exploration of the Effects of High Hydrostatic Pressure on Microbial Growth, Physiology and Survival: Perspectives from Piezophysiology

The discovery of piezophiles (previously referred to as barophiles) prompted researchers to investigate the survival strategies they employ in high-pressure environments. There have been innovative high-pressure studies on biological processes applying modern techniques of genetics and molecular biology in bacteria and yeasts as model organisms. Recent advanced studies in this field have shown unexpected outcomes in microbial growth, physiology and survival when living cells are subjected to high hydrostatic pressure. The effects are conceptually dependent on the sign and magnitude of volume changes associated with any chemical reaction in the cells. Nevertheless, it is difficult to explain the pressure effects on complex metabolic networks based on a simple volume law. The challenges in piezophysiology are to discover whether the physiological responses of living cells to high pressure are relevant to their growth and to identify the critical factors in cell viability and lethality under high pressure from the general and organism-specific viewpoints.

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergDelta mutants, the erg2Delta mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Delta mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D(w)) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC(50) of cycloheximide was closely correlated with S/D(w) in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Delta strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu2+ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO4. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO4. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU μg−1 total protein) when the strain was grown in the absence of CuSO4. However, the activity was stimulated (25.8 mU μg−1 total protein) when cells were grown with 1 mM CuSO4 and further enhanced to 110 mU μg−1 total protein with 10 mM CuSO4. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO4 in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO4 in strain N6.

Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

Microorganisms display an optimal temperature and hydrostatic pressure for growth. To establish the molecular basis of piezo- and psychroadaptation, we elucidated global genetic defects that give rise to susceptibility to high pressure and low temperature in Saccharomyces cerevisiae. Here we present 80 genes including 71 genes responsible for high-pressure growth and 56 responsible for low-temperature growth with a significant overlap of 47 genes. Numerous previously known cold-sensitive mutants exhibit marked high-pressure sensitivity. We identified critically important cellular functions: (i) amino acid biosynthesis, (ii) microautophagy and sorting of amino acid permease established by the exit from rapamycin-induced growth arrest/Gap1 sorting in the endosome (EGO/GSE) complex, (iii) mitochondrial functions, (iv) membrane trafficking, (v) actin organization mediated by Drs2-Cdc50, and (vi) transcription regulated by the Ccr4-Not complex. The loss of EGO/GSE complex resulted in a marked defect in amino acid uptake following high-pressure and low-temperature incubation, suggesting its role in surface delivery of amino acid permeases. Microautophagy and mitochondrial functions converge on glutamine homeostasis in the target of rapamycin (TOR) signaling pathway. The localization of actin requires numerous associated proteins to be properly delivered by membrane trafficking. In this study, we offer a novel route to gaining insights into cellular functions and the genetic network from growth properties of deletion mutants under high pressure and low temperature.

Fluconazole Modulates Membrane Rigidity, Heterogeneity, and Water Penetration into the Plasma Membrane in Saccharomyces cerevisiae

Azole anitifungal drugs such as fluconazole inhibit 14alpha-demethylase. The mechanism of fluconazole action on the plasma membrane is assumed to be ergosterol depletion and accumulation of a toxic sterol, 14alpha-methyl-3,6-diol, that differs in C-6 hydroxylation, B-ring saturation, C-14 methylation, and side-chain modification. Nevertheless, little is known about how these sterol modifications mechanically influence membrane properties and hence fungal viability. Employing time-resolved measurement with a fluorescence anisotropy probe, 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), we demonstrated that fluconazole administration decreased the rigidity of the plasma membrane of Saccharomyces cerevisiae, leading to a dramatic reduction in the order parameter (S) from 0.965 to 0.907 and a 5-fold acceleration of the rotational lipid motion. This suggests that the altered sterol has a deleterious impact on membrane packing, resulting in increased fluidity. Deletion of ERG3 confers hyperresistance to fluconazole by circumventing the accumulation of 14alpha-methyl-3,6-diol and instead produces 14alpha-methylfecosterol lacking the 6-OH group. We found that ERG3 deletion mitigated the fluconazole-induced loss of membrane rigidity with S remaining at a higher value (=0.922), which could contribute to the fluconazole resistance in the erg3Delta mutant. The reduced ability of the 6-OH sterol to stiffen lipid bilayers was supported by the finding that 30 mol % of 6alpha-hydroxy-5alpha-cholestanol marginally increased the S value of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes, while cholesterol and dihydrocholesterol markedly increased it. The decay of the TMA-DPH fluorescence was bimodal in the wild-type strain. This heterogeneity could have arisen from varying degrees of water penetration into the plasma membrane. Fluconazole eliminated the heterogeneity of the dielectric characteristic of the membrane interfacial region, and concomitantly the TMA-DPH lifetime was shortened. Therefore, we conclude that 14alpha-methyl-3,6-diol is insufficient to pack the plasma membrane, allowing water penetration, which is consistent with membrane disorder after fluconazole administration. Our findings illustrate the role of ergosterol in maintaining membrane heterogeneity and preventing water penetration as well as maintaining the rigidity of the plasma membrane interfacial region.

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Abstract Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H+-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO2 production is involved in the pressure-induced acidification. Hydration and ionization of CO2 is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H+-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology.

Precise expression of the cAMP receptor gene, CAR1, during transition from growth to differentiation in Dictyostelium discoideum.

The gene expressions associated with the switch‐over from cell proliferation of Dictyostelium discoideum to differentiation have been analyzed using temperature shift and differential plaque hybridization methods. Quit 1 was cloned as a specifically expressed gene when cells were starved just before the PS point (putative shift point from growth to differentiation). The coding region of the Quit 1 gene is identical to that of the cAMP receptor 1 CAR1 gene, which is essential for development. Quit1 mRNA was specifically expressed just after starvation of cells at around the PS point, thus indicating the importance of CAR1 for cellular differentiation as well as the actual existence of the PS point.