The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of DeltapH and Deltapsi. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-beta-D-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,DeltapH and Deltapsi, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.

When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.

The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpHenv), whether the concentration of ionophores used can effectively abolish the ΔpHenv is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpHenv can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r-square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpHenv can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin required to collapse the ΔpHenv was then studied. The establishment of a non-destructive method of monitoring the stromal pH will be valuable for studying the roles of the ΔpHenv in chloroplast physiology.

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

Accumulation of PINK1 on the outer mitochondrial membrane (OMM) is necessary for PINK-mediated mitophagy. The proton ionophores, like carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), inhibit PINK1 import into mitochondrial matrix and induce PINK1 OMM accumulation. Here, we show that the CHCHD4/GFER disulfide relay system in the mitochondrial intermembrane space (IMS) is required for PINK1 stabilization when mitochondrial membrane potential is lost. Activation of CHCHD4/GFER system by mitochondrial oxidative stress or inhibition of CHCHD4/GFER system with antioxidants can promote or suppress PINK1 accumulation, respectively. Thus data suggest a pivotal role of CHCHD4/GFER system in PINK1 accumulation. The amyotrophic lateral sclerosis-related superoxide dismutase 1 mutants dysregulated redox state and CHCHD4/GFER system in the IMS, leading to inhibitions of PINK1 accumulation and mitophagy. Thus, the redox system in the IMS is involved in PINK1 accumulation and damaged mitochondrial clearance, which may play roles in mitochondrial dysfunction-related neurodegenerative diseases.

Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Deltapsim, in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of DeltaPsim which utilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (<1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of DeltaPsim, as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, DeltaPsip, also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Deltapsim by flow cytometry.

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.

The functional characteristics of a nonacidic, inositol 1,4,5-trisphosphate- and thapsigargin-insensitive Ca2+ pool have been characterized in mammalian cells derived from the rat pituitary gland (GH3, GC, and GH3B6), the adrenal tissue (PC12), and mast cells (RBL-1). This Ca2+ pool is released into the cytoplasm by the Ca2+ ionophores ionomycin or A23187 after the discharge of the inositol 1,4,5-trisphosphate-sensitive store with an agonist coupled to phospholipase C activation and/or thapsigargin. The amount of Ca2+ trapped within this pool increased significantly after a prolonged elevation of intracellular Ca2+ concentration elicited by activation of Ca2+ influx. This pool was affected neither by caffeine-ryanodine nor by mitochondrial uncouplers. Probing mitochondrial Ca2+ with recombinant aequorin confirmed that this pool did not coincide with mitochondria, whereas its homogeneous distribution across the cytosol, as revealed by confocal microscopy, and its insensitivity to brefeldin A make localization within the Golgi complex unlikely. A proton gradient as the driving mechanism for Ca2+ uptake was excluded since ionomycin is inefficient in releasing Ca2+ from acidic pools and Ca2+ accumulation/release in/from this store was unaffected by monensin or NH4Cl, drugs known to collapse organelle acidic pH gradients. Ca2+ sequestration inside this pool, thus, may occur through a low-affinity, high-capacity Ca2+-ATPase system, which is, however, distinct from classical endosarcoplasmic reticulum Ca2+-ATPases. The cytological nature and functional role of this Ca2+ storage compartment are discussed.

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury.

Nervous necrosis virus (NNV) is the etiological agent of viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER), which results in heavy economic losses to the aquaculture industry worldwide. Dramatic cytoplasmic vacuoles were observed during NNV infection both in vitro and in vivo; however, the origin and mechanism of cytoplasmic vacuolization remains unknown. In this report, we found that the cytoplasmic vacuole morphology became fused and enlarged during infection with red spotted grouper nervous necrosis virus (RGNNV), which was accompanied by increased cell death. Notably, Lyso-Tracker, but not Mito-Tracker or ER-Tracker, was accumulated in the vacuoles, and abnormal lysosome swelling was observed in RGNNV-infected cells, suggesting that the cytoplasmic vacuoles originated from lysosomal organelles. Cytoplasmic vacuolization and cell death in RGNNV-infected cells was completely blocked by the vacuolar H+-ATPase inhibitor (bafilomycin A1), and was significantly weakened by chloroquine (CQ), a lysosomotropic agent that induces the acidification of the lysosomes. This suggests that lysosome acidification was essential for vacuole formation. Significant inhibitory effects on vacuolization and cell death were also observed in the RGNNV-infected cells following treatment with nigericin and monensin (ionophores that uncouple the proton gradient present in lysosomes). This indicated that lysosome function was tightly associated with RGNNV infection-induced cell death. In addition, vacuoles were found to be partially co-localized with GFP-LC3II punctate dots during RGNNV infection. Moreover, the severity of vacuolization and cell death were both significantly decreased after treatment with the autophagy inhibitor, 3-MA, suggesting that autophagy was involved in lysosomal vacuolization and cell death evoked by RGNNV infection. Thus, our results demonstrate that autophagy participates in lysosomal vacuolation-mediated cell death during RGNNV infection, and provides new insight into our understanding of the potential mechanisms underlying nodavirus pathogenesis in vitro.