Face Of Membrane Research Articles

Analysis of human T-cell leukemia virus type 1 particles by using cryo-electron tomography.

The particle structure of human T-cell leukemia virus type 1 (HTLV-1) is poorly characterized. Here, we have used cryo-electron tomography to analyze HTLV-1 particle morphology. Particles produced from MT-2 cells were polymorphic, roughly spherical, and varied in size. Capsid cores, when present, were typically poorly defined polyhedral structures with at least one curved region contacting the inner face of the viral membrane. Most of the particles observed lacked a defined capsid core, which likely impacts HTLV-1 particle infectivity.

Biosynthesis and Translocation of Unsulfated Acyltrehaloses in Mycobacterium tuberculosis

A number of species-specific polymethyl-branched fatty acid-containing trehalose esters populate the outer membrane of Mycobacterium tuberculosis. Among them, 2,3-diacyltrehaloses (DAT) and penta-acyltrehaloses (PAT) not only play a structural role in the cell envelope but also contribute to the ability of M. tuberculosis to multiply and persist in the infected host, promoting the intracellular survival of the bacterium and modulating host immune responses. The nature of the machinery, topology, and sequential order of the reactions leading to the biosynthesis, assembly, and export of these complex glycolipids to the cell surface are the object of the present study. Our genetic and biochemical evidence corroborates a model wherein the biosynthesis and translocation of DAT and PAT to the periplasmic space are coupled and topologically split across the plasma membrane. The formation of DAT occurs on the cytosolic face of the plasma membrane through the action of PapA3, FadD21, and Pks3/4; that of PAT occurs on the periplasmic face via transesterification reactions between DAT substrates catalyzed by the acyltransferase Chp2 (Rv1184c). The integral membrane transporter MmpL10 is essential for DAT to reach the cell surface, and its presence in the membrane is required for Chp2 to be active. Disruption of mmpL10 or chp2 leads to an important build-up of DAT inside the cells and to the formation of a novel form of unsulfated acyltrehalose esterified with polymethyl-branched fatty acids normally found in sulfolipids that is translocated to the cell surface.

Surface hierarchical porosity in poly (ɛ-caprolactone) membranes with potential applications in tissue engineering prepared by foaming in supercritical carbon dioxide

This article describes the preparation of porous poly (ɛ-caprolactone), PCL, membranes by supercritical CO2 (SCCO2) foaming, displaying surface hierarchical macroporosity which could be tailored by careful control of the pressure, in the range of 150–250bar, and depressurization processes in several steps, showing also pore interconnectivity between both membrane faces. The membranes exhibited two distinct types of surface macroporosity, the larger with diameter sizes of 300–500μm were surrounded by and also composed of smaller pores of 15–50μm (same size as inner pores). Membranes were prepared by solvent casting and submitted to different SCCO2 foaming. Parameters such as membrane thickness, CO2 flow, foaming time, pressure, temperature and the depressurization processes (rate and profiles), were varied to determine their influence on final porosity and to decipher which parameters were the most critical ones in terms of surface hierarchical pore organization. No remarkable changes in PCL crystallinity were found when membranes were processed under SCCO2. Finally, biological evaluation of the porous membranes was achieved by seeding human skin fibroblasts on the prepared membranes. The results, in terms of cell adhesion, spreading, proliferation and metabolic activity indicate that these membranes could hold promise for the fabrication of meshes with controlled porosity for tissue engineering applications.

A novel member of lymphocyte-specific protein tyrosine kinase protein identified in lamprey, Lampetra japonica.

Lymphocyte-specific protein tyrosine kinase p56 (Lck) is a member of the Src family of non-receptor protein tyrosine kinase. Lck plays an important role in mediating T-cell receptor (TCR) signal transduction and the development, differentiation, proliferation, and activation of T-cells [1]. Lck contains N-terminus of myristylation sequence, a unique aminoterminus region, followed by Src homology 3 (SH3) and SH2 domains and a C-terminus of tyrosine kinase catalytic domain [2]. Lck can associate with the inner face of the plasma membrane through its myristoyl glycine and palmitoyl cysteines in the amino-terminus [3].Following the myristylation sequence, there is the unique region, a short region of 80 amino acids. This unique region is involved in the interaction of Lck with specific cellular proteins [4,5]. Downstream of this unique region are SH3 and SH2 domains which are involved in protein–protein interactions [6]. The tyrosine kinase domain is the catalytic domain of Lck catalyzing the transfer of the gamma-phosphate from ATP to tyrosine residues in proteins. The catalytic domain of human Lck contains a site of autophosphorylation (Tyr-394), which plays an important role in regulating the protein kinase activity [7]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently identified possessing the adaptive immune defenses [8]. A recent study of jawless vertebrate has provided a clue for the origin of adaptive immune defense. Though TCR and B-cell receptor system do not exist in jawless vertebrates, lamprey has been confirmed to possess an alternative immune system that could specifically recognize and respond to external pathogens [9]. The handling of lamprey (Lampetra japonica) and all experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University. Adult lampreys were purchased from Tongjiang section of the Heilongjiang River (Tongjiang, China) in December. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group); one group of animals was immunized with 0.1 mg of lipopolysaccharide (LPS) (Sigma-Aldrich, St Louis, USA) in 0.1 ml phosphate-buffered saline (PBS), and the control animals were injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections. Based on the expressed sequence tag analysis of the cDNA library which was constructed with lamprey lymphocyte-like cells in our laboratory, a Lck homolog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (NCBI; http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [10] using RNAiso (TaKaRa Biotechnology, Dalian, China) reagent following the manufacturer instructions, and dissolved in Diethyl pyrocarbonate-treated water and stored at 2808C. First strand 30and 50-RACEcDNAs were synthesized from 5 mg of total RNA by Reverse Transcriptase M-MLV at 308C for 10 min, 428C for 30 min, 708C for 15 min, 958C for 5 min, 48C for 60 min with the 30-coding sequence primer and 50-coding sequence primer and Random 9-mers primer following the manufacturer instructions (TaKaRa Biotechnology). The 30and 50-end sequences of Lj-Lck were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa Biotechnology), and specific primers (Supplementary Table S1). LA Taq DNA polymerase (TaKaRa Biotechnology) was used for amplification with the following cycling conditions: 948C for 3 min, followed by 40 amplification cycles at 948C for 30 s, 558C for 30 s, 728C for 2 min, and a final extension step at 728C for 10 min. Products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. The target band of PCR product was isolated and purified, subcloned into Acta Biochim Biophys Sin 2014, 46: 820–825 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu066. Advance Access Publication 25 July 2014

Nutrient generation and retrieval from the host cell cytosol by intra-vacuolar Legionella pneumophila.

Nutrient generation and retrieval from the host cell cytosol by intra-vacuolar Legionella pneumophila.

Structure and function of the CagA oncoprotein from Helicobacter pylori

CagA is known as a major bacterial virulence determinant from Helicobacter pylori and is critical for gastric cancer. Upon delivery into the gastric epithelial cells, CagA localizes to the inner leaflet of the plasma membrane and promiscuously interacts with host proteins such as PAR1b and SHP2. The CagA-PAR1-SHP2 complex potentiates oncogenic signaling. Biochemical and physicochemical analyses revealed that CagA is comprises a structured N-terminal region (residues 1-876) and an intrinsically disordered C-terminal region (residues 877-1186). To understand the structure and function relationship of CagA, we determined the crystal structure of the N-terminal region (residues 1-876) of CagA [1]. The N-terminal CagA is rich in α-helices and composed of three domains. Domain I (residues 24-221) is linked to domain II (residues 303-644) by a disordered loop with about 80 amino acid residues. Domain II has a basic patch composed of 14 lysine and 2 arginine residues. Biological experiments revealed that the basic patch mediates the CagA-phosphatidylserine interaction to localize the inner face of the plasma membrane. In addition, we found that C-terminal disordered region forms a lariat-like loop by the interaction between NBS (residues 645 - 824) and CBS (residues 998 - 1038) in the disordered C-terminal region. The formation of the lariat-like loop facilitates promiscuous interaction of CagA with target protein such as SHP2.

Bacterial cell wall. MurJ is the flippase of lipid-linked precursors for peptidoglycan biogenesis.

Peptidoglycan (PG) is a polysaccharide matrix that protects bacteria from osmotic lysis. Inhibition of its biogenesis is a proven strategy for killing bacteria with antibiotics. The assembly of PG requires disaccharide-pentapeptide building blocks attached to a polyisoprene lipid carrier called lipid II. Although the stages of lipid II synthesis are known, the identity of the essential flippase that translocates it across the cytoplasmic membrane for PG polymerization is unclear. We developed an assay for lipid II flippase activity and used a chemical genetic strategy to rapidly and specifically block flippase function. We combined these approaches to demonstrate that MurJ is the lipid II flippase in Escherichia coli.

Building the cell wall is flipping difficult

Bacteria Cell Wall The cell wall of bacteria is constructed from a polysaccharide called peptidoglycan (PG). It forms a matrix that surrounds cells and is essential for the integrity of the cytoplasmic membrane. Many of our most successful antibiotics target PG synthesis. The synthetic pathway involves the assembly of sugar building blocks on a lipid carrier at the inner face of the cytoplasmic membrane. The reactions that produce this so-called lipid II precursor and the enzymes that catalyze them have been known for decades. However, the identity of the flippase enzyme that “flips” lipid II in the membrane to expose the sugar building blocks on the cell surface for polymerization has remained highly controversial. Sham et al. now show that the essential protein MurJ is the long sought-after flippase responsible for the translocation of lipid-linked cell wall precursors across the bacterial cytoplasmic membrane (see the Perspective by Young). The work completes the cell wall biogenesis pathway and defines the function of an attractive target for new antibiotics. Science , this issue p. [220][1]; see also p. [139][2] [1]: /lookup/volpage/345/220?iss=6193 [2]: /lookup/doi/10.1126/science.1256585

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ϕX174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg-Trp-x-x-Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC50 200-600 μM), and demonstrated antimicrobial activity against E. coli (MIC 31-125 μg mL(-1)). Cationic antimicrobial peptides at a concentration of 100 μg mL(-1) containing Arg-Trp sequences also showed 30-60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg-Trp-octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.

Ca2 + regulation of mitochondrial function in neurons

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca2+ signal or to Ca2+ itself. [Na+]i, [Ca2+]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca2+ clearly showed that Ca2+-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca2+ may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca2+ entry in mitochondria through the calcium uniporter (MCU). Two Ca2+-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate–glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate–aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca2+ of 300nM and 3.4μM, respectively. The lack of SCaMC-3 results in a smaller Ca2+-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca2+-dependent responses to all workloads: a reduction of about 65–70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K+-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K+-depolarization, this occurs in spite of the presence of large [Ca2+]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca2+-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca2+ is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.

Sec62 as a novel gating modulator for protein translocation and ion permeability of the mammalian Sec61 complex (LB196)

In mammalian cells the endoplasmic reticulum (ER) plays a key role during the biogenesis of secretory and membrane proteins and acts as a major regulator for intracellular Ca2+ signaling. Sec62, a component of the ER‐protein translocase, acts during post‐translational translocation of small precursor proteins and in ER‐Ca2+ leakage‐control. Our project aims to elucidate the molecular mechanisms how it regulates the translocation machinery concerning these two fundamental and seemingly opposing functions. We used siRNA mediated depletion of target proteins from human cells followed by functional assays for protein transport and live cell calcium imaging.Depletion of Sec62 in combination with proteasomal inhibition led to accumulation of the precursor of the ER resident protein ERj3 at the cytosolic face of the ER membrane. Control experiments using different siRNA as well as a plasmid rescue approach confirmed the specificity for Sec62. Follow‐up experiments revealed a pattern of dependencies upon translocon components which is characteristic for a post‐translational translocation pathway.Decrease of Sec62 level led to an increased ER calcium leakage that could be attributed to Sec61α. We observed that Sec62 binds the N‐terminus of Sec61α in a Ca2+ sensitive manner and identified a crucial putative EF‐hand in the C‐terminal part of Sec62.These experiments demonstrated for the first time that human Sec62 is important for the early steps of translocation of a large precursor protein indicating that Sec62 participation is not restricted to small proteins as suggested in previous studies.Additionally, Sec62 acts as a modulator, shifting the translocon from the open to the closed state after translocation is completed in order to maintain the ion impermeability of the ER membrane.Further experiments will allow us to determine the recently proposed role of Sec62 for various cancer entities.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability

The cell wall peptidoglycan (PG) of Burkholderia cenocepacia, an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia. Our results show that BCAL2764, herein referred to as murJBc, is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJBc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJBc underwent cell lysis, while partial MurJBc depletion sensitized the mutant to the action of β-lactam antibiotics. Depletion of MurJBc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJBc and MurJEc are functional homologs. Together, our results are consistent with the notion that MurJBc is a PG lipid II flippase in B. cenocepacia.

Cation Binding Site of cytochrome c oxidase: Progress report

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed earlier the determination of the kinetic and equilibrium characteristics of the binding, and, as shown recently, the binding of calcium to the site inhibits cytochrome oxidase activity at low turnover rates of the enzyme [Vygodina, Т., Kirichenko, A., Konstantinov, A.A (2013). Direct Regulation of Cytochrome c Oxidase by Calcium Ions. PloS ONE 8, e74436]. This paper summarizes further progress in the studies of the Cation Binding Site in this group presenting the results to be reported at 18th EBEC Meeting in Lisbon, 2014. The paper revises specificity of the bovine oxidase Cation Binding Site for different cations, describes dependence of the Ca2+-induced inhibition on turnover rate of the enzyme and reports very high affinity binding of calcium with the “slow” form of cytochrome oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

A Structural Framework for a Near-Minimal Form of Life: Mass and Compositional Analysis of the Helical Mollicute Spiroplasma melliferum BC3

Spiroplasma melliferum is a wall-less bacterium with dynamic helical geometry. This organism is geometrically well defined and internally well ordered, and has an exceedingly small genome. Individual cells are chemotactic, polar, and swim actively. Their dynamic helicity can be traced at the molecular level to a highly ordered linear motor (composed essentially of the proteins fib and MreB) that is positioned on a defined helical line along the internal face of the cell’s membrane. Using an array of complementary, informationally overlapping approaches, we have taken advantage of this uniquely simple, near-minimal life-form and its helical geometry to analyze the copy numbers of Spiroplasma’s essential parts, as well as to elucidate how these components are spatially organized to subserve the whole living cell. Scanning transmission electron microscopy (STEM) was used to measure the mass-per-length and mass-per-area of whole cells, membrane fractions, intact cytoskeletons and cytoskeletal components. These local data were fit into whole-cell geometric parameters determined by a variety of light microscopy modalities. Hydrodynamic data obtained by analytical ultracentrifugation allowed computation of the hydration state of whole living cells, for which the relative amounts of protein, lipid, carbohydrate, DNA, and RNA were also estimated analytically. Finally, ribosome and RNA content, genome size and gene expression were also estimated (using stereology, spectroscopy and 2D-gel analysis, respectively). Taken together, the results provide a general framework for a minimal inventory and arrangement of the major cellular components needed to support life.

Redesigning the procaspase‐8 dimer interface for improved dimerization

Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

Diversity of O-Antigen Repeat Unit Structures Can Account for the Substantial Sequence Variation of Wzx Translocases

The most common system for synthesis of cell surface polysaccharides is the Wzx/Wzy-dependent pathway, which involves synthesis, on the cytoplasmic face of the cell membrane, of repeat units, which are then translocated to the periplasmic face by a Wzx translocase and then polymerized by Wzy to generate the polysaccharide. One such polysaccharide is O antigen, which is incorporated into lipopolysaccharide (LPS). The O antigen is extremely variable, with over 186 forms in Escherichia coli. Wzx proteins are also very diverse, but they have been thought to be specific only for the first sugar of the repeat units. However, recent studies demonstrated examples in which Wzx translocases have considerable preference for their native repeat unit, showing that specificity can extend well beyond the first sugar. These results appear to be in conflict with the early conclusions, but they involved specificity for side branch residues and could be a special case. Here we take six Wzx translocases that were critical in the earlier studies on the importance of the first sugar and assess their ability to translocate the Escherichia coli O16 and O111 repeat units. We use gene replacements to optimize maintenance of expression level and show that under these conditions the native translocases are the most effective for their native repeat unit, being, respectively, 64-fold and 4-fold more effective than the next best. We conclude that Wzx translocases are commonly adapted to their native repeat unit, which provides an explanation for the great diversity of wzx genes.

Roles for Protons in Living Membranes: Cardiolipin

Cardiolipin (CL) in mitochondria has 2 unique headgroup conformations. In lipid bilayers (pK >7.0) it traps 2 protons forming patches. It also binds supercomplexes together where its phosphates bind cations. Patches trap protons with short, strong H-bonds (SSHBs) as described by Al Mildvan (Meth Enz 2007). CL's SSHBs trap protons its phosphates. They're tightly held due to insertion of CL's 4 chains into the bilayer. The H+, not available to water, has no affect the pH. Yet patches may act as a H+ antenna, pK >7.0, on both sides of the IM. X. Periole (JACS & Nature 2013) has identified CL-patch-binding-sites on the Xray structures of cyt bc1 & cyt oxidase, near the entrance to their putative H+ channels. Thus H+ delivery is on a microsecond (μs) timescale, like the chloroplast's chlorophyll antenna. The complexes pump on a msec timescale. CL patches are on the matrix side are near the F0 of the F0, F1 ATPase, the protons would charge F0 of the F0, F1, ATPase. SSHB formation minimally changes the pH on both membrane faces. Pumped protons have little ΔpH but a maximum ΔΨ. Junge & Oster have each shown that the F0 protons are driven by ΔΨ, not ΔpH.Living membrane surfaces are polyanionic, mostly on the lipid headgroups, tightly held together by their chains. Guoy-Chapman tells us that this decreases the surface pH. SHHB trapped protons have no effect on the pH. Finally, the ΔΨ of most living membranes is created by proton pumps on a msec timescale, but is adjusted by K-channels on a μs timescale. ΔΨ provokes wasteful leakage of H+s across the membrane. ATP energy pumps them back! Lipids, polyisoprenes, sterols, iso- and anteiso- chains, hopanoids and squalene inhibit this leakage.

Regulation of the Ion Channel TRPM3 by Phosphoinositides

TRPM3 belongs to the Melastatin family of Transient Receptor Potential (TRP) ion channels. It is a Ca2+ permeable outwardly rectifying nonselective cation channel. TRPM3 is expressed in sensory neurons, brain, pancreas, kidney and vascular smooth muscle. In sensory dorsal root ganglion (DRG) neurons it was shown to function as a sensor for noxious heat. TRPM3-/- mice have decreased ability to detect noxious heat. In DRG neurons as well as pancreatic beta cells, the neurosteroid pregnenolone sulfate has been shown to activate TRPM3.Many members of the TRP family are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Thus we decided to test if TRPM3 is also regulated by PtdIns(4,5)P2. PtdIns(4,5)P2 is predominantly present at the cytoplasmic face of the plasma membrane and it is an important signaling molecule. In the current study we modulated the levels of PtdIns(4,5)P2 in Xenopus laevis oocytes and HEK cells heterologously expressing TRPM3. In inside-out patches, TRPM3 currents ran down rapidly after excision. This current could be restored by the exogenous application of water soluble diC8 PtdIns(4,5)P2 and the naturally occurring arachidonyl-stearyl (AASt) PtdIns(4,5)P2. Also, application of MgATP to excised inside-out patches reactivated the TRPM3 current. This effect of MgATP was inhibited by LY294002 at a concentration where it inhibits phosphatidylinositol 4 kinase (300 µM) but not at a concentration where it inhibits phosphatidylinositol 3 kinase (10 µM). This suggests that MgATP acted via replenishing PtdIns(4,5)P2 and PtdIns(4)P. In addition, inducing the activity of PtdIns(4,5)P2-5-phosphatases in whole-cell patch clamp experiments also decreased TRPM3 currents. Overall our data collected through excised inside-out and whole-cell patch clamp measurements suggest that TRPM3 requires PtdIns(4,5)P2 as a cofactor.

Live cell imaging of phosphoinositide dynamics during Legionella infection.

The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.