Processing and maturation of carboxypeptidase Y and alkaline phosphatase in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.     

Genetic characterization of <Emphasis Type="Italic">psp</Emphasis> encoding the DING protein in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> SBW25

Background  

DING proteins constitute a conserved and broadly distributed set of proteins found in bacteria, fungi, plants and animals (including humans). Characterization of DING proteins from animal and plant tissues indicated ligand-binding ability suggesting a role for DING proteins in cell signaling and biomineralization. Surprisingly, the genes encoding DING proteins in eukaryotes have not been identified in the eukaryotic genome or EST databases. Recent discovery of a DING homologue (named Psp here) in the genome of Pseudomonas fluorescens SBW25 provided a unique opportunity to investigate the physiological roles of DING proteins. P. fluorescens SBW25 is a model bacterium that can efficiently colonize plant surfaces and enhance plant health. In this report we genetically characterize Psp with a focus on conditions under which psp is expressed and the protein exported.     

The mechanism of coupling between oxido‐reduction and proton translocation in respiratory chain enzymes

The respiratory chain of mitochondria and bacteria is made up of a set of membrane‐associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane‐associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co‐operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X‐ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone‐cytochrome c oxidoreductase and NADH‐ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.     

Kinase Activity of ArcB from Escherichia coli Is Subject to Regulation by Both Ubiquinone and Demethylmenaquinone

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in ‘respiratory’ electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.     

Decarbonylated cyclophilin A Cpr1 protein protects <Emphasis Type="BoldItalic">Saccharomyces cerevisiae</Emphasis> KNU5377Y when exposed to stress induced by menadione

Cyclophilins are conserved cis–trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.     

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G₁ DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.     

In vivo redox state of the ubiquinone pool in the spadices of the thermogenic skunk cabbage, <Emphasis Type="Italic">Symplocarpus renifolius</Emphasis>

In vivo ubiquinone (UQ) reduction levels were determined in thermogenic stigma and post-thermogenic male stages of spadices of the skunk cabbage, Symplocarpus renifolius. In contrast to Arum maculatum, in which the UQ pool is almost fully reduced during thermogenesis, the reduction levels of UQ9 and UQ10 were not affected by the thermogenic status or developmental stage of individual S. renifolius spadices. Moreover, these levels were controlled within the ranges 40–75% and 35–60%, respectively. These results suggest that the reduction state of the UQ pool per se is not primarily involved in thermoregulation in S. renifolius.     

Induction changes in photosystems I and II in plant leaves upon modulation of membrane ion transport

The steady-state regime of linear photosynthetic electron transport implies concerted operation of photosystems I and II (PSI and PSII) in plant leaves. Acidification of the thylakoid lumen is known to cause down-regulation of PSII photochemical activity but it is not yet clear how the proton accumulation in the lumen affects the PSI activity and coordinated operation of the two photosystems in intact leaves. Chlorophyll fluorescence and absorbance of oxidized chlorophyll P700 in the near-infrared region ΔA _810–870 (ΔA ₈₁₀) are convenient noninvasive indicators of the redox state of PSII and PSI components, respectively. Simultaneous measurements of chlorophyll fluorescence and ΔA ₈₁₀ in pea leaves revealed that some kinetic stages in the induction curves occur synchronously both in dark-adapted and preilluminated leaves. After the treatment of leaves with ionophores promoting or inhibiting the light-induced thylakoid pH gradient (valinomycin, nigericin, monensin), the induction curves of ΔA ₈₁₀ and chlorophyll fluorescence were consistently modified. The results suggest that characteristic stages of ΔA ₈₁₀ induction curve, representing the second and the third waves of P700 photooxidation, are closely related to ΔpH generation, although the bases of ΔpH dependence differ for these two stages. The second wave of ΔA ₈₁₀ depends presumably on stroma alkalinization as a precondition for photoactivation of electron flow from PSI to terminal acceptors. The third wave of ΔA ₈₁₀ is apparently due to retardation of electron flow between PSII and PSI upon acidification of the lumen.     

Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from <Emphasis Type="Italic">Thraustochytrium aureum</Emphasis> ATCC 34304

In Thraustochytrids, Thraustochytrium aureum ATCC 34304 was able to produce high levels of several polyunsaturated fatty acids. In the present study, a novel gene encoding protein was cloned from the DHA rich microbe, T. aureum ATCC 34304. The functional analysis of a novel gene was demonstrated by its heterologous expression in Pichia pastoris. The gene was able to synthesize C20 and C22 PUFAs, as well as, to mediate different elongations (Δ9, Δ6, and Δ5) and one Δ5 desaturation activities. The conversion rates of the Δ9 elongation (n-3) and Δ5 desaturation products were found to be higher in response to the novel enzyme than the controls (TaElo and Tad5, respectively). The other Δ9 elongation (n-6) and Δ5 elongation products were slightly lower than those of the control (TaElo). The full length of the 1,374 bp gene contained 458 amino acids that showed very limited homology with desaturases and elongases from various organisms. In addition, the rate of synthesis of PUFAs was evaluated at temperatures ranging from 10 to 30°C. The elongation products were found to decrease dramatically and the desaturation products were found to increase dramatically at 10°C. TaNE was confirmed to be a multifunctional enzyme with higher activity towards Δ6 elongations than Δ9, Δ5 elongations, and Δ5 desaturation.     

Dependence of chlorophyll P700 redox transients during the induction period on the transmembrane distribution of protons in chloroplasts of pea leaves

Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H⁺ conductance in thylakoid membranes) and nigericin (a K⁺/H⁺ antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curves of chlorophyll fluorescence in pea (Pisum sativum L., cv. Premium) leaves. The treatment of leaves with DCCD strongly modified the kinetics of P700⁺ absorbance changes (ΔA ₈₁₀) by promoting rapid photooxidation of P700. These characteristic changes in ΔA ₈₁₀ induction kinetics and P700⁺ accumulation did not appear when the leaves were treated with DCCD in the presence of nigericin. In addition to opposite modifications of ΔA ₈₁₀ kinetics evoked by permeability-modifying agents, the fluorescence induction curves differed conspicuously depending on leaf incubation in DCCD solutions with or without nigericin. The observed modifications of fluorescence induction curves and ΔA ₈₁₀ indicate that DCCD suppresses electron transport from photosystem II (PSII) to P700, whereas this inhibition is removed by nigericin. The results suggest that slowing down of the electron transport rate in the presence of DCCD was caused by elevation of ΔpH in thylakoids. The prevention of pH gradient formation in the presence of protonophore lowered also the steady-state P700⁺ level in far-red irradiated leaves and accelerated the subsequent dark reduction of P700. These findings indicate that PSI-driven cyclic electron flow is accelerated after the removal of the pH gradient.     

Modification of quinone electrochemistry by the proteins in the biological electron transfer chains: examples from photosynthetic reaction centers

Quinones such as ubiquinone are the lipid soluble electron and proton carriers in the membranes of mitochondria, chloroplasts and oxygenic bacteria. Quinones undergo controlled redox reactions bound to specific sites in integral membrane proteins such as the cytochrome bc₁ oxidoreductase. The quinone reactions in bacterial photosynthesis are amongst the best characterized, presenting a model to understand how proteins modulate cofactor chemistry. The free energy of ubiquinone redox reactions in aqueous solution and in the Q_A and Q_B sites of the bacterial photosynthetic reaction centers (RCs) are compared. In the primary Q_A site ubiquinone is reduced only to the anionic semiquinone (Q^•−) while in the secondary Q_B site the product is the doubly reduced, doubly protonated quinol (QH₂). The ways in which the protein modifies the relative energy of each reduced and protonated intermediate are described. For example, the protein stabilizes Q^•− while destabilizing Q⁼ relative to aqueous solution through electrostatic interactions. In addition, kinetic and thermodynamic mechanisms for stabilizing the intermediate semiquinones are compared. Evidence for the protein sequestering anionic compounds by slowing both on and off rates as well as by binding the anion more tightly is reviewed.