Identification of a regulatory element for yeast tryptophan permease Tat2 ubiquitination using high hydrostatic pressure

ABSTRACT

Tryptophan uptake in the yeast Saccharomyces cerevisiae is extremely sensitive to high pressure; therefore, the growth of tryptophan auxotrophic strains is impaired. Degradation of tryptophan permease Tat2 is enhanced at 25?MPa, depending on Rsp5 ubiquitin ligase. Any defect in Tat2 ubiquitination confers high pressure growth capacity, which is a luminous phenotype of the yeast used to explore the mechanism by which Rsp5 mediates Tat2 ubiquitination. Here we show that the N-terminal four (K17, K20, K29, and K31) among five lysines are required for efficient Tat2 degradation under high pressure. We found that a domain spanning D70 to S76 is also critical for Tat2 degradation at 25?MPa probably because of the recognition by Bul1, an adaptor protein of Rsp5. Defects in Tat2 ubiquitination do not produce any remarkable mutant phenotype at 0.1?MPa. Therefore, we suggest that high pressure is a unique and advanced tool to explore ubiquitination-dependent Tat2 regulation.     

The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25?MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10–25?MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25?MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25?MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10–25?MPa.

ABBREVIATIONS: Trp⁺: the phenotype of tryptophan prototrophic strains that synthesize tryptophan de novo; Trp^?: the phenotype of tryptophan auxotrophic strains that require tryptophan from the medium; YPR153W: an yeast poorly characterized gene; Ypr153w: a protein encoded by the YPR153W gene; ypr153wΔ: a deletion mutant for the YPR153W gene; Tat2: the high-affinity tryptophan permease; MCT10: a human aromatic amino acid transporter     

Dissociation of the light-harvesting membrane protein complex I from Rhodobacter sphaeroides under high hydrostatic pressure

The light-harvesting complex 1 (LH1) from Rhodobacter sphaeroides is an excellent model system for investigating the stability of oligomeric membrane proteins under high hydrostatic pressure. The currently investigated LH1 forms a 16-meric ring structure of B825 subunits. B825 is a heterodimer of transmembrane α- and β-polypeptide chains, which non-covalently binds two bacteriochlorophyll a molecules. These pigment molecules were used as intrinsic spectroscopic sensors to follow the dissociation reaction. Our results demonstrate that the LH1 dissociates into B825 subunits through an intermediary tetrameric unit B845. The dissociation mechanism depends on pressure. At ～200–500?MPa the dissociation corresponds to a pseudo-first-order reaction, characterised by the apparent reaction rate at atmospheric pressure k₀?=?3·10^?5?s^?1, activation volume ΔV^??=??4?mL/mol, and free energy of activation ΔG^??=?26?kJ/mol. Below 200?MPa and above 500?MPa, the reaction is more complex, including further dissociation of B825 into monomers B777.     

The permittivities of PVDF/PMMA blends at hydrostatic pressure

The complex permittivities of poly(vinylidene fluoride)/poly(methyl methacrylate) (PVDF/PMMA) blends have been measured under variation of temperature T(20°-60°C), frequency v(5 Hz-300 kHz), and hydrostatic pressure p (0-260 MPa). The values can be represented by a master curve with the shift factors △p/△ log (v/Hz) = ?140 MPa at room temperature and △ (1 /T) /△ log (v/Hz) = ?2. 10^?4 K^?1 at atmospheric pressure. The dependence of the activation energy △E _A on pressure p is then given by △E _A=(100 + 0.02 MPa^?1 p) kJ/mol. Furthermore, the results indicate that the β-relaxation of PVDF is due to motions in the crystal-amorphous interphase. The interactions between the two polymers, which are miscible at all compositions, disturb the correlations between the PVDF monomer units at that location as well as the mobility of the PMMA side group.     

Possibility of Campylobacter jejuni inactivation in smoked salmon by high-pressure treatment

The sterile samples of cold-smoked salmon were placed in polyamide–polyethylene pouches and inoculated with three-strain composite of Campylobacter jejuni (inoculum ca 10⁷ CFU g^?1). The inoculated samples were sealed under vacuum and subjected to 200, 300 and 400 MPa of hydrostatic pressure for 0, 5, 10 and 15 min. The number of surviving C. jejuni per gram was determined by the 10-fold dilution method followed by plating on Karmali agar. D ₁₀ values were calculated. This work has shown that for reducing C. jejuni in cold-smoked salmon by 6 log units, the application of 200 MPa for 64.26 min or 300 MPa for 17.10 min or 400 MPa for less than 5 min is needed. Applying such parameters of high-pressure processing should not change significantly the organoleptic properties of the product.     

1H NMR studies on reduced high potential iron protein (HIPIP) from Chromatium Vinosum

¹H NMR studies were performed on reduced high potential iron protein from Chromatium Vinosum, focusing on the assignment of the resonances of the aromatic residues. Despite the residual paramagnetism of the iron sulfur cluster, the complete ring proton spin system and sequence specific assignment of all tryptophan residues (Trp⁸⁰, Trp⁷⁶, Trp⁶⁰) was achieved. Tentative assignments are proposed for Tyr¹⁹ and one Phe residue. Implications of the above assignments are discussed. 2D EXSY experiments permitted correlation of the resonances in the reduced and the oxidized form. Similar NOESY pattern for the indole NH’s were observed, suggesting substantial retention of the gross structural features upon oxidation.     

Effect of temperature and high pressure on Mn2+ EPR spectra in K3H(SO4)2 fast-proton conductor

The linewidth ΔH _pp and spin-Hamiltonian parameters under temperature and high hydrostatic pressure by X-band continuous wave electron paramagnetic resonance in the K₃H(SO₄)₂ crystal were studied. Spin-Hamiltonian parameters, direction cosines and coordination of Mn²⁺ ion were determined at room temperature. The pressure at 300?MPa leads to the change of hydrogen bond potential and the transition from double well to single well potential moves about 10?K towards a higher temperature.     

Hydrostatic pressure effects on the fluorescence of tyrosine solution with different concentrations of copper ion

The effects of hydrostatic pressure on the fluorescence of tyrosine (Tyr) solution with different concentrations of copper ion (Cu²⁺) were investigated. The fluorescent intensity of pure Tyr aqueous solution enhanced with the increase of hydrostatic pressure, the fluorescent intensity increased by 8.8% when the pressure was up to 60 MPa. The fluorescence of Tyr aqueous solution was quenched obviously due to complex formation with Cu²⁺ and the quenching became stronger when the concentration of Cu²⁺ was higher. When the concentration ratio of Cu²⁺ and Tyr ([Cu²⁺]/[Tyr]) increased from 0 to 40, the fluorescent intensity decreased to 19.0% at 0.1 MPa and 24.2% at 60 MPa. It was also found that the effects of pressure on the fluorescent intensity of Tyr aqueous solution were different at various [Cu²⁺]/[Tyr]. The fluorescent intensity increased by 14.4% and 38.4% for 1 and 40 ratio respectively when the pressure was changed from 0.1 MPa to 60 MPa.     

The effect of high hydrostatic pressure on the survival of Saccharomyces cerevisiae in model suspensions and beetroot juice

The inactivation of Saccharomyces cerevisiae NCFB 3191 using high hydrostatic pressure of 300 MPa at 20°C with a holding time of 0, 1, 5 and 10 min was investigated with model suspensions in phosphate-buffered saline and in beetroot juice. The reduction in S. cerevisiae NCFB 3191 in model suspensions was about 5 log after 10 min of pressurization, irrespective of the initial level of cell concentration in the samples (5.4–8.7 log cfu/mL). The baroprotective effect of beetroot juice on yeast cells during pressurization was observed; the reduction was lower and was only 3.5 log (the inoculum was 5.4 log cfu/mL). No sublethal injury among the surviving cells of the studied yeast strain was found.     

Critical events leading to cellular mortality of yeast cells following hydrostatic pressure treatment

ABSTRACT

Hydrostatic pressures of 40–150?MPa are sub-lethal conditions for yeast cells. On exposure to this pressure range, damaged yeast cells will attempt to recover, but a critical cellular event may ultimately lead to cell mortality. We employed yeast strains whose cellular organelles were labeled with green fluorescent protein to investigate this critical event. We were able to visualize the nuclear membrane, nucleus, endoplasmic reticulum, Golgi body, and plasma membranes. Of the cellular organelles tested, the nuclear membrane was the weakest, displaying damage following only 50?MPa of pressure. This nuclear membrane rupture correlated with cellular viability. Thus, we hypothesize that nuclear membrane damage is the critical event leading to cellular mortality of yeast cells following hydrostatic pressure treatment.     

石油醚在流体静高压下的超声声速、衰减、密度和绝热压缩系数

 专门设计了用脉冲-回波-重合法在不同静水压下测量液体声速的装置，并对石油醚在0.1~650 MPa流体静压力范围内测量了其超声声速和衰减系数。还通过测量体积压缩量和质量得到石油醚密度ρ随流体静压力的变化。最后，给出了石油醚的绝热压缩系数β与流体静压力的关系。     

Moderate hydrostatic pressure–temperature combinations for inactivation of Bacillus subtilis spores

We report the effect of using moderate hydrostatic pressure, 40–140?MPa, at moderate temperature (38–58°C) to inactivate Bacillus subtilis spores in McIlvaine's citric phosphate buffer at pH 6. We have investigated several parameters: pressure applied, holding time, pressure cycling, and temperature. The kinetics of spore inactivation is reported. The results show that spore inactivation is exponentially proportional to the time the sample is exposed to pressure. Spore germination and inactivation occur at the hydrostatic pressures/temperature combinations we explored. Cycling the pressure while keeping the total time at high pressure constant does not significantly increase spore inactivation. We show that temperature increases spore inactivation at two different rates; a slow rate below 33°C, and at a more rapid rate at higher temperatures. Increasing pressure leads to an increase in spore inactivation below 95?MPa; however, further increases in pressure give a similar rate kill. The time dependence of the effect of pressure is consistent with the first-order model (R²?>?0.9). The thermal resistance values (Z_T) of B. subtilis spores are 30°C, 37°C, and 40°C at 60, 80, 100?MPa. The increase in Z_T value at higher pressures indicates lower temperature sensitivity. The pressure resistance values (Z_P) are 125, 125 and 143?MPa at 38°C, 48°C, and 58°C. These Z_P values are lower than those reported for B. subtilis spores in the literature, which indicates higher sensitivity at pressures less than about 140?MPa. We show that at temperatures <60°C, B. subtilis spores are inactivated at pressures below 100?MPa. This finding could have implications for the design of the sterilization equipment.     

X‐ray reflectivity measurements of liquid/solid interfaces under high hydrostatic pressure conditions

A high‐pressure cell for in situ X‐ray reflectivity measurements of liquid/solid interfaces at hydrostatic pressures up to 500 MPa (5 kbar), a pressure regime that is particularly important for the study of protein unfolding, is presented. The original set‐up of this hydrostatic high‐pressure cell is discussed and its unique properties are demonstrated by the investigation of pressure‐induced adsorption of the protein lysozyme onto hydrophobic silicon wafers. The presented results emphasize the enormous potential of X‐ray reflectivity studies under high hydrostatic pressure conditions for the in situ investigation of adsorption phenomena in biological systems.     

Study of the high pressure effect on nanoparticles GdVO4: Eu3+ optical properties

This study considers the effects of hydrostatic pressure on the line position and fluorescence lifetime τ for ⁵D₀ → ⁷F₂ transitions in GdVO₄: Eu³⁺ nanocrystals. The results indicate that the pressure induced the red shift toward longer wavelengths for all the considered lines with different rate. The fluorescence lifetime τ nonlinearly decreases with pressure in the considered pressure range. High pressure induced the fluorescence lifetime τ that can be explained with a simple theoretical model. The measured line position and τ are in a satisfactory agreement with the theoretical calculations.     

Effect of Hydrostatic Pressure on the Dielectric Response of Pb(Mg1/3Nb2/3)O3 Relaxor

The effect of hydrostatic pressure on the dielectric response of Pb(Mg_1/3Nb_2/3)O₃ relaxor single crystal was studied. An increase in the dispersion of the dielectric anomaly, characteristic of the relaxors, was observed: pressure-induced downward shift of the temperature T _m of permittivity maximum at 1 kHz amounts to dT _m /dp = ? 3.8 K/100 MPa, whereas that measured at 1 MHz equals ? 3.3 K/100 MPa. Analysis of the dielectric response of the highly polarizable Pb(Mg_1/3Nb_2/3)O₃ shows that hydrostatic pressure results in a decrease of the correlation radius r _c of polar nanodomains, as well as their activation energy.     

Dc electrical conductivity of Ge2S3Agx glasses at high hydrostatic pressure

The paper deals with the influence of hydrostatic pressure on d.c. electrical conductivity in Ge₂S₃Ag _x glasses forx10%. The initial material exhibits high resistivity and the presence of Ag impurity yields strong increase in electrical conductivity. The experimental results suggest that there is a non-linear decrease of electrical resistivity at pressure ranging from 0·1 to 10³ MPa. The pressure coefficient of resistivity is a function ofx. All measurements were performed on bulk samples using graphite contacts. The experimental results are interpreted by means of ionic conductivity.     

Decoupling of the order-disorder and displacive contributions to the phase transition in NH4H(ClH2CCOO)2

The effects of hydrostatic pressure and substitution of Rb⁺for the ammonium cations on the ferroelectric phase transition temperature in NH₄H(ClH₂CCOO)₂ have been studied by electric permittivity measurements. The transition temperature (T_c) decreases with increasing pressure up to 800 MPa and the pressure coefficient dT_c/dp=−1.4×10⁻² [K/MPa] has been experimentally determined. The substitution of Rb⁺ for the ammonium cations has been shown to considerably lower the ferroelectric phase transition temperature T_c. In mixed crystals, additional electric permittivity anomaly has been clearly evidenced. The results are discussed assuming a model, which combines polarizability effects, related to the heavy ion units, with the pseudo-spin tunnelling.     

水稻种子萌发期高压诱导的SSH-cDNA文库构建与分析

 为了研究高压处理对高等植物基因表达的影响,以萌发过程中经高压处理后的水稻种子的mRNA作为检测子（Tester）,以对照样品的mRNA作为驱动子（Driver）,采用抑制差减杂交（SSH）结合镜像选择技术（MOS）构建了高压处理后水稻种子萌发过程中诱导表达的差减cDNA文库,并通过蓝白斑筛选获得了580个重组克隆。菌液PCR检测的结果表明这些克隆中的插入片段的大小主要分布在200～1000 bp之间,平均大小约为575 bp,其中单一扩增条带的有效重组克隆数为542个,有效的重组克隆率为93.3%。对部分重组克隆进行测序发现,受高压诱导表达的基因主要涉及到代谢、胁迫响应、转录调控等方面。为高压处理后水稻种子基因表达模式变化的研究打下了基础。     

Hydrostatic pressure driven spin,volume and band gap collapses in SmFeO3: a GGA + U study

The crystal structure, magnetic and electronic properties of SmFeO₃ under hydrostatic pressure have been studied by first-principles calculations within the generalized gradient approximation plus Hubbard U (GGA + U). The iso-structural phase transition with spin, volume and band gap collapses can be induced by a large enough hydrostatic pressure. The high-spin (HS) state of Fe³⁺, with the magnetic moment of ~4 μ_B, is retained at low pressure. The spin crossover occurs at a transition pressure (~68 GPa) with the magnetic moment of Fe³⁺ decreasing to ~1 μ_B in low-spin (LS) state. Meanwhile, the reductions of cell volume (by ~?5.43%) and band gap (from >2 eV to ~1.6 eV) of SmFeO₃ are obtained when the HS–LS transition happens. Finally, the critical pressure of HS–LS transition, magnetic and electronic properties are found to be Hubbard U dependent.     

Electromechanical-induced antiferroelectricben ferroelectric phase transition in PbLa(Zr,Sn,Ti)O3 ceramic

Antiferroelectric—ferroelectric (AFE—FE) phase transition in ceramic Pb_0.97La_0.02(Zr_0.75Sn_0.136Ti_0.114)O₃ (PLZST) was studied by dielectric spectroscopy as functions of frequency (10²—10⁵ Hz) and pressure (0—500 MPa) under a DC electric field. The hydrostatic pressure-dependent remnant polarization and dielectric constant were measured. The results show that remnant polarization of the metastable rhombohedral ferroelectric PLZST poled ceramic decreases sharply and depoles completely at phase transition under hydrostatic pressure. The dielectric constant undergoes an abrupt jump twice during a load and unload cycle under an electric field. The two abrupt jumps correspond to two phase transitions, FE—AFE and AFE—FE.