GFP Fusion to the N-Terminus of MotB Affects the Proton Channel Activity of the Bacterial Flagellar Motor in Salmonella

The bacterial flagellar motor converts the energy of proton flow through the MotA/MotB complex into mechanical works required for motor rotation. The rotational force is generated by electrostatic interactions between the stator protein MotA and the rotor protein FliG. The Arg-90 and Glu-98 from MotA interact with Asp-289 and Arg-281 of FliG, respectively. An increase in the expression level of the wild-type MotA/MotB complex inhibits motility of the gfp-motBfliG(R281V) mutant but not the fliG(R281V) mutant, suggesting that the MotA/GFP-MotB complex cannot work together with wild-type MotA/MotB in the presence of the fliG(R281V) mutation. However, it remains unknown why. Here, we investigated the effect of the GFP fusion to MotB at its N-terminus on the MotA/MotB function. Over-expression of wild-type MotA/MotB significantly reduced the growth rate of the gfp-motBfliG(R281V) mutant. The over-expression of the MotA/GFP-MotB complex caused an excessive proton leakage through its proton channel, thereby inhibiting cell growth. These results suggest that the GFP tag on the MotB N-terminus affects well-regulated proton translocation through the MotA/MotB proton channel. Therefore, we propose that the N-terminal cytoplasmic tail of MotB couples the gating of the proton channel with the MotA–FliG interaction responsible for torque generation.

A Review on the Possible Leakage of Electrons through the Electron Transport Chain within Mitochondria

The finding of electron leakage during the electron transport within the mitochondrial membrane (in eukaryotes) or in the cell membrane of the prokaryotes is an important issue for the accumulation of the Reactive Oxygen Species (ROS) in the cytosol which in turn induce the probable aging of cells. In eukaryotes, mitochondrion is known to be the major site of the ROS generation in different pathological processes which may further cause cell damages as evident through the ischemia-reperfusion (I/R) injury, respiratory diseases, cell apoptosis, and even the onset of cancer. Thus, the mitochondrial leakage and the physiological effect of leaked protons and electrons grow up with future interest in energy metabolism. Current review focused on the physiological impact of electron/ proton leakage particularly in the eukaryotic cells based on the previous reports; emphasized on the prospects of the eukaryotic mitochondrion as a modulator of proton and electron leakage; and finally attempted to assess the regulatory mechanisms of such electron/ proton leakage.

Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

Selective anion transport mediated by strap-extended calixpyrroles

Synthetic anion receptors that facilitate transmembrane chloride transport are of interest as potential therapeutic agents for cancer and cystic fibrosis. Transporters selective for chloride over protons are desired for therapeutic applications to avoid autophagy inhibition and cytotoxicity. Examples of such compounds are rare because the majority of anion transporters can interact with the carboxylate head groups of fatty acids leading to proton leakage. In this paper, we report the synthesis, anion binding and transmembrane anion transport properties of two novel bis-triazole-functionalized calixpyrroles with extended straps, and compare them to previously reported shorter-strap analogues known to exhibit high Cl [Formula: see text] H[Formula: see text] selectivity. We demonstrate improved chloride transport activities of the strap-extended compounds that likely benefit from increased lipophilicity, and reduced Cl [Formula: see text] H[Formula: see text] selectivity due to the larger anion binding cavities facilitating interaction with fatty acids. The results are instructive for future design of ideal anion transporters with potent activity and high selectivity against proton leakage.

Concentration of Sulfuric Acid from Spent Acidic Liquor by Cascaded Electrodialysis Using an Interpolymer Anion Exchange Membrane

Cascaded electrodialysis system has been developed for increasing the concentration of sulfuric acid from aqueous solution using an interpolymer anion exchange (IPA) membrane. Enrichment process was carried out in a cascade of six electrodialyzers at 20.2 mA/cm2 current density. The performance of the process was evaluated rigorously in terms of current efficiency, voltage requirements and processability to enrich the acid concentration. The bench-scale experimental results exhibited the ability of membrane to perform as a low proton leakage membrane and to enrich the sulfuric acid concentration up to 27.93 wt. %. The higher values of current efficiencies were reported to be in the range of 50 to 60 %. Proton leakage through the membrane, acid back diffusion, concentration polarization and solution conductivity were considered to be the limiting factors for acid enrichment and their effects were found significant on current efficiency and voltage requirement.

Candida albicansHyphal Expansion Causes Phagosomal Membrane Damage and Luminal Alkalinization

ABSTRACTMacrophages rely on phagosomal acidity to destroy engulfed microorganisms. To survive this hostile response, opportunistic fungi such asCandida albicansdeveloped strategies to evade the acidic environment.C. albicansis polymorphic and able to convert from yeast to hyphae, and this transition is required to subvert the microbicidal activity of the phagosome. However, the phagosomal lumen, which is acidic and nutrient deprived, is believed to inhibit the yeast-to-hypha transition. To account for this apparent paradox, it was recently proposed thatC. albicansproduces ammonia that alkalinizes the phagosome, thus facilitating yeast-to-hypha transition. We reexamined the mechanism underlying phagosomal alkalinization by applying dual-wavelength ratiometric pH measurements. The phagosomal membrane was found to be highly permeable to ammonia, which is therefore unlikely to account for the pH elevation. Instead, we find that yeast-to-hypha transition begins within acidic phagosomes and that alkalinization is a consequence of proton leakage induced by excessive membrane distension caused by the expanding hypha.IMPORTANCEC. albicansis the most common cause of nosocomial fungal infection, and over 3 million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that yeast-to-hypha transition occurs within the nutrient-deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause, of hyphal growth.

Candida albicans hyphal expansion causes phagosomal membrane damage and luminal alkalinization

ABSTRACTMacrophages rely on phagosomal acidity to destroy engulfed microorganisms. To survive this hostile response, opportunistic fungi such as Candida albicans developed strategies to evade the acidic environment. C. albicans is polymorphic, able to convert from yeast to hyphae, and this transition is required to subvert the microbicidal activity of the phagosome. However, the phagosomal lumen, which is acidic and nutrient-deprived, inhibits yeast-to-hypha transition. To account for this apparent paradox, it was recently proposed that C. albicans produces ammonia that alkalinizes the phagosome, thus facilitating yeast-to-hypha transformation. We re-examined the mechanism underlying phagosomal alkalinization by applying dual-wavelength ratiometric pH measurements. The phagosomal membrane was found to be highly permeable to ammonia, which is therefore unlikely to account for the pH elevation. Instead, we find that yeast-to-hypha transition begins within acidic phagosomes, and that alkalinization is a consequence of proton leakage induced by excessive membrane distension caused by the expanding hypha.IMPORTANCEC. albicans is the most common nosocomial fungal infection, and over three million people acquire life-threatening invasive fungal infections every year. Even if antifungal drugs exist, almost half of these patients will die. Despite this, fungi remain underestimated as pathogens. Our study uses quantitative biophysical approaches to demonstrate that the yeast-to-hypha transition occurs within the nutrient deprived, acidic phagosome and that alkalinization is a consequence, as opposed to the cause of hyphal growth.

The potential mechanism of mitochondrial dysfunction in septic cardiomyopathy

Septic cardiomyopathy is one of the most serious complications of sepsis or septic shock. Basic and clinical research has studied the mechanism of cardiac dysfunction for more than five decades. It has become clear that myocardial depression is not related to hypoperfusion. As the heart is highly dependent on abundant adenosine triphosphate (ATP) levels to maintain its contraction and diastolic function, impaired mitochondrial function is lethally detrimental to the heart. Research has shown that mitochondria play an important role in organ damage during sepsis. The mitochondria-related mechanisms in septic cardiomyopathy have been discussed in terms of restoring mitochondrial function. Mitochondrial uncoupling proteins located in the mitochondrial inner membrane can promote proton leakage across the mitochondrial inner membrane. Recent studies have demonstrated that proton leakage is the essential regulator of mitochondrial membrane potential and the generation of reactive oxygen species (ROS) and ATP. Other mechanisms involved in septic cardiomyopathy include mitochondrial ROS production and oxidative stress, mitochondria Ca2+ handling, mitochondrial DNA in sepsis, mitochondrial fission and fusion, mitochondrial biogenesis, mitochondrial gene regulation and mitochondria autophagy. This review will provide an overview of recent insights into the factors contributing to septic cardiomyopathy.

Structural basis of proton translocation and force generation in mitochondrial ATP synthase

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.