Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Lipid-dependent membrane protein topogenesis.

The topology of polytopic membrane proteins is determined by topogenic sequences in the protein, protein-translocon interactions, and interactions during folding within the protein and between the protein and the lipid environment. Orientation of transmembrane domains is dependent on membrane phospholipid composition during initial assembly as well as on changes in lipid composition postassembly. The membrane translocation potential of negative amino acids working in opposition to the positive-inside rule is largely dampened by the normal presence of phosphatidylethanolamine, thus explaining the dominance of positive residues as retention signals. Phosphatidylethanolamine provides the appropriate charge density that permits the membrane surface to maintain a charge balance between membrane translocation and retention signals and also allows the presence of negative residues in the cytoplasmic face of proteins for other purposes.

Erythrocyte Metabolic Reprogramming by Sphingosine 1-Phosphate in Chronic Kidney Disease and Therapies.

RATIONALE: Hypoxia promotes renal damage and progression of chronic kidney disease (CKD). The erythrocyte is the only cell type for oxygen (O2) delivery. Sphingosine 1-phosphate (S1P)-a highly enriched biolipid in erythrocytes-is recently reported to be induced under high altitude in normal humans to enhance O2 delivery. However, nothing is known about erythrocyte S1P in CKD. OBJECTIVE: To investigate the function and metabolic basis of erythrocyte S1P in CKD with a goal to explore potential therapeutics. METHODS AND RESULTS: Using erythrocyte-specific SphK1 (sphingosine kinase 1; the only enzyme to produce S1P in erythrocytes) knockout mice (eSphK1-/-) in an experimental model of hypertensive CKD with Ang II (angiotensin II) infusion, we found severe renal hypoxia, hypertension, proteinuria, and fibrosis in Ang II-infused eSphk1-/- mice compared with controls. Untargeted metabolomics profiling and in vivo U-13C6 isotopically labeled glucose flux analysis revealed that SphK1 is required for channeling glucose metabolism toward glycolysis versus pentose phosphate pathway, resulting in enhanced erythroid-specific Rapoport-Luebering shunt in Ang II-infused mice. Mechanistically, increased erythrocyte S1P functioning intracellularly activates AMPK (AMP-activated protein kinase) 1α and BPGM (bisphosphoglycerate mutase) by reducing ceramide/S1P ratio and inhibiting PP2A (protein phosphatase 2A), leading to increased 2,3-bisphosphoglycerate (an erythrocyte-specific metabolite negatively regulating Hb [hemoglobin]-O2-binding affinity) production and thus more O2 delivery to counteract kidney hypoxia and progression to CKD. Preclinical studies revealed that an AMPK agonist or a PP2A inhibitor rescued the severe CKD phenotype in Ang II-infused eSphK1-/- mice and prevented development of CKD in the control mice by inducing 2,3-bisphosphoglycerate production and thus enhancing renal oxygenation. Translational research validated mouse findings in erythrocytes of hypertensive CKD patients and cultured human erythrocytes. CONCLUSIONS: Our study elucidates the beneficial role of eSphk1-S1P in hypertensive CKD by channeling glucose metabolism toward Rapoport-Luebering shunt and inducing 2,3-bisphosphoglycerate production and O2 delivery via a PP2A-AMPK1α signaling pathway. These findings reveal the metabolic and molecular basis of erythrocyte S1P in CKD and new therapeutic avenues.

TTAPE-Me dye is not selective to cardiolipin and binds to common anionic phospholipids nonspecifically.

Identification, visualization, and quantitation of cardiolipin (CL) in biological membranes is of great interest because of the important structural and physiological roles of this lipid. Selective fluorescent detection of CL using noncovalently bound fluorophore 1,1,2,2-tetrakis[4-(2-trimethylammonioethoxy)-phenylethene (TTAPE-Me) has been recently proposed. However, this dye was only tested on wild-type mitochondria or liposomes containing negligible amounts of other anionic lipids, such as phosphatidylglycerol (PG) and phosphatidylserine (PS). No clear preference of TTAPE-Me for binding to CL compared to PG and PS was found in our experiments on artificial liposomes, Escherichia coli inside-out vesicles, or Saccharomyces cerevisiae mitochondria in vitro or in situ, respectively. The shapes of the emission spectra for these anionic phospholipids were also found to be indistinguishable. Thus, TTAPE-Me is not suitable for detection, visualization, and localization of CL in the presence of other anionic lipids present in substantial physiological amounts. Our experiments and complementary molecular dynamics simulations suggest that fluorescence intensity of TTAPE-Me is regulated by dynamic equilibrium between emitting dye aggregates, stabilized by unspecific but thermodynamically favorable electrostatic interactions with anionic lipids, and nonemitting dye monomers. These results should be taken into consideration when interpreting past and future results of CL detection and localization studies with this probe in vitro and in vivo. Provided methodology emphasizes minimal experimental requirements, which should be considered as a guideline during the development of novel lipid-specific probes.

Single Amino Acid Replacements in RocA Disrupt Protein-Protein Interactions To Alter the Molecular Pathogenesis of Group A Streptococcus.

Group A Streptococcus (GAS) is a human-specific pathogen and major cause of disease worldwide. The molecular pathogenesis of GAS, like many pathogens, is dependent on the coordinated expression of genes encoding different virulence factors. The control of virulence regulator/sensor (CovRS) two-component system is a major virulence regulator of GAS that has been extensively studied. More recent investigations have also involved regulator of Cov (RocA), a regulatory accessory protein to CovRS. RocA interacts, in some manner, with CovRS; however, the precise molecular mechanism is unknown. Here, we demonstrate that RocA is a membrane protein containing seven transmembrane helices with an extracytoplasmically located N terminus and cytoplasmically located C terminus. For the first time, we demonstrate that RocA directly interacts with itself (RocA) and CovS, but not CovR, in intact cells. Single amino acid replacements along the entire length of RocA disrupt RocA-RocA and RocA-CovS interactions to significantly alter the GAS virulence phenotype as defined by secreted virulence factor activity in vitro and tissue destruction and mortality in vivo In summary, we show that single amino acid replacements in a regulatory accessory protein can affect protein-protein interactions to significantly alter the virulence of a major human pathogen.

Metabolic dysfunctions in the kynurenine pathway, noradrenergic and purine metabolism in schizophrenia and bipolar disorders.

BACKGROUND: We aimed at exploring potential pathophysiological processes across psychotic disorders, applying metabolomics in a large and well-characterized sample of patients and healthy controls. METHODS: Patients with schizophrenia and bipolar disorders (N = 212) and healthy controls (N = 68) had blood sampling with subsequent metabolomics analyses using electrochemical coulometric array detection. Concentrations of 52 metabolites including tyrosine, tryptophan and purine pathways were compared between patients and controls while controlling for demographic and clinical characteristics. Significant findings were further tested in medication-free subsamples. RESULTS: Significantly decreased plasma concentrations in patients compared to healthy controls were found for 3-hydroxykynurenine (3OHKY, p = 0.0008), xanthurenic acid (XANU, p = 1.5×10-5), vanillylmandelic acid (VMA, p = 4.5×10-5) and metanephrine (MN, p = 0.0001). Plasma concentration of xanthine (XAN) was increased in the patient group (p = 3.5×10-5). Differences of 3OHKY, XANU, VMA and XAN were replicated across schizophrenia spectrum disorders and bipolar disorders subsamples of medication-free individuals. CONCLUSIONS: Although prone to residual confounding, the present results suggest the kynurenine pathway of tryptophan metabolism, noradrenergic and purinergic system dysfunction as trait factors in schizophrenia spectrum and bipolar disorders. Of special interest is XANU, a metabolite previously not found to be associated with bipolar disorders.

The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack.

Secretory phospholipases A2 (sPLA2s) are potent components of mammalian innate-immunity antibacterial mechanisms. sPLA2 enzymes attack bacteria by hydrolyzing bacterial membrane phospholipids, causing membrane disorganization and cell lysis. However, most Gram-negative bacteria are naturally resistant to sPLA2 Here we report a novel resistance mechanism to mammalian sPLA2 in Escherichia coli, mediated by a phospholipid repair system consisting of the lysophospholipid transporter LplT and the acyltransferase Aas in the cytoplasmic membrane. Mutation of the lplT or aas gene abolished bacterial lysophospholipid acylation activity and drastically increased bacterial susceptibility to the combined actions of inflammatory fluid components and sPLA2, resulting in bulk phospholipid degradation and loss of colony-forming ability. sPLA2-mediated hydrolysis of the three major bacterial phospholipids exhibited distinctive kinetics and deacylation of cardiolipin to its monoacyl-derivative closely paralleled bacterial death. Characterization of the membrane envelope in lplT- or aas-knockout mutant bacteria revealed reduced membrane packing and disruption of lipid asymmetry with more phosphatidylethanolamine present in the outer leaflet of the outer membrane. Moreover, modest accumulation of lysophospholipids in these mutant bacteria destabilized the inner membrane and rendered outer membrane-depleted spheroplasts much more sensitive to sPLA2 These findings indicated that LplT/Aas inactivation perturbs both the outer and inner membranes by bypassing bacterial membrane maintenance mechanisms to trigger specific interfacial activation of sPLA2 We conclude that the LplT/Aas system is important for maintaining the integrity of the membrane envelope in Gram-negative bacteria. Our insights may help inform new therapeutic strategies to enhance host sPLA2 antimicrobial activity.

Structural and functional characterization of protein-lipid interactions of the Salmonella typhimurium melibiose transporter MelB.

BACKGROUND: Membrane lipids play critical roles in the structure and function of membrane-embedded transporters. Salmonella typhimurium MelB (MelBSt) is a symporter coupling melibiose translocation with a cation (Na+, Li+, or H+). We present an extensive study on the effects of specific phospholipids on the structure of MelBSt and the melibiose transport catalyzed by this protein. RESULTS: Lipidomic analysis and thin-layer chromatography (TLC) experiments reveal that at least one phosphatidylethanolamine (PE) and one phosphatidylglycerol (PG) molecule associate with MelBSt at high affinities. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy experiments confirmed the presence of lipid tails and glycerol backbones that co-purified with MelBSt; headgroups of PG were also observed. Studies with lipid-engineered strains, including PE-deficient, cardiolipin (CL)- and PG-deficient, or CL-deficient strains, show that lack of PE or PG, however not CL, largely inhibits both H+- and Na+-coupled melibiose active transport to different extents. Interestingly, neither the co-substrate binding (melibiose or Na+) nor MelBSt folding and stability are affected by changing lipid compositions. Remarkably, the delipidated MelBSt with only 2-3 bound lipids, regardless of the headgroup species, also exhibits unchanged melting temperature values as shown by circular dichroism spectroscopy. CONCLUSIONS: (1) Lipid tails and glycerol backbones of interacting PE and PG may contribute to the stability of the structure of MelBSt. (2) The headgroups of PE and PG, but not of CL, play important roles in melibiose transport; however, lipid headgroups do not modulate the folding and stability of MelBSt.

Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation.

Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.

Dynamic membrane protein topological switching upon changes in phospholipid environment.

A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.

Substrate Selectivity of Lysophospholipid Transporter LplT Involved in Membrane Phospholipid Remodeling in Escherichia coli.

Lysophospholipid transporter (LplT) was previously found to be primarily involved in 2-acyl lysophosphatidylethanolamine (lyso-PE) recycling in Gram-negative bacteria. This work identifies the potent role of LplT in maintaining membrane stability and integrity in the Escherichia coli envelope. Here we demonstrate the involvement of LplT in the recycling of three major bacterial phospholipids using a combination of an in vitro lysophospholipid binding assay using purified protein and transport assays with E. coli spheroplasts. Our results show that lyso-PE and lysophosphatidylglycerol, but not lysophosphatidylcholine, are taken up by LplT for reacylation by acyltransferase/acyl-acyl carrier protein synthetase on the inner leaflet of the membrane. We also found a novel cardiolipin hydrolysis reaction by phospholipase A2 to form diacylated cardiolipin progressing to the completely deacylated headgroup. These two distinct cardiolipin derivatives were both translocated with comparable efficiency to generate triacylated cardiolipin by acyltransferase/acyl-acyl carrier protein synthetase, demonstrating the first evidence of cardiolipin remodeling in bacteria. These findings support that a fatty acid chain is not required for LplT transport. We found that LplT cannot transport lysophosphatidic acid, and its substrate binding was not inhibited by either orthophosphate or glycerol 3-phosphate, indicating that either a glycerol or ethanolamine headgroup is the chemical determinant for substrate recognition. Diacyl forms of PE, phosphatidylglycerol, or the tetra-acylated form of cardiolipin could not serve as a competitive inhibitor in vitro. Based on an evolutionary structural model, we propose a "sideways sliding" mechanism to explain how a conserved membrane-embedded α-helical interface excludes diacylphospholipids from the LplT binding site to facilitate efficient flipping of lysophospholipid across the cell membrane.

Structural Insight into Substrate Selection and Catalysis of Lipid Phosphate Phosphatase PgpB in the Cell Membrane.

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro Here we report the crystal structure of the lipid-bound form of PgpB. The structure shows that a PE molecule is stabilized in a membrane-embedded tunnel formed by TM3 and the "PSGH" fingerprint peptide near the catalytic site, providing structural insight into PgpB substrate binding mechanism. Noteworthy, in silico docking of varied lipid phosphates exhibited similar substrate binding modes to that of PE, and the residues in the lipid tunnel appear to be important for PgpB catalysis. The catalytic triad in the active site is essential for dephosphorylating substrates lysophosphatidic acid, phosphatidic acid, or sphingosine-1-phosphate but surprisingly not for the native substrate PGP. Remarkably, residue His-207 alone is sufficient to hydrolyze PGP, indicating a specific catalytic mechanism for PgpB in PG biosynthesis. We also identified two novel sensor residues, Lys-93 and Lys-97, on TM3. Our data show that Lys-97 is essential for the recognition of lyso-form substrates. Modification at the Lys-93 position may alter substrate specificity of lipid phosphate phosphatase proteins in prokaryotes versus eukaryotes. These studies reveal new mechanisms of lipid substrate selection and catalysis by PgpB and suggest that the enzyme rests in a PE-stabilized state in the bilayer.

Effects of mixed proximal and distal topogenic signals on the topological sensitivity of a membrane protein to the lipid environment.

The final topology of membrane proteins is thought to be dictated primarily by the encoding sequence. However, according to the Charge Balance Rule the topogenic signals within nascent membrane proteins are interpreted in agreement with the Positive Inside Rule as influenced by the protein phospholipid environment. The role of long-range protein-lipid interactions in establishing a final uniform or dual topology is unknown. In order to address this role, we determined the positional dependence of the potency of charged residues as topological signals within Escherichia coli sucrose permease (CscB) in cells in which the zwitterionic phospholipid phosphatidylethanolamine (PE), acting as topological determinant, was either eliminated or tightly titrated. Although the position of a single or paired oppositely charged amino acid residues within an extramembrane domain (EMD), either proximal, central or distal to a transmembrane domain (TMD) end, does not appear to be important, the oppositely charged residues exert their topogenic effects separately only in the absence of PE. Thus, the Charge Balance Rule can be executed in a retrograde manner from any cytoplasmic EMD or any residue within an EMD most likely outside of the translocon. Moreover, CscB is inserted into the membrane in two opposite orientations at different ratios with the native orientation proportional to the mol % of PE. The results demonstrate how the cooperative contribution of lipid-protein interactions affects the potency of charged residues as topological signals, providing a molecular mechanism for the realization of single, equal or different amounts of oppositely oriented protein within the same membrane.

Biogenesis, transport and remodeling of lysophospholipids in Gram-negative bacteria.

Lysophospholipids (LPLs) are metabolic intermediates in bacterial phospholipid turnover. Distinct from their diacyl counterparts, these inverted cone-shaped molecules share physical characteristics of detergents, enabling modification of local membrane properties such as curvature. The functions of LPLs as cellular growth factors or potent lipid mediators have been extensively demonstrated in eukaryotic cells but are still undefined in bacteria. In the envelope of Gram-negative bacteria, LPLs are derived from multiple endogenous and exogenous sources. Although several flippases that move non-glycerophospholipids across the bacterial inner membrane were characterized, lysophospholipid transporter LplT appears to be the first example of a bacterial protein capable of facilitating rapid retrograde translocation of lyso forms of glycerophospholipids across the cytoplasmic membrane in Gram-negative bacteria. LplT transports lyso forms of the three bacterial membrane phospholipids with comparable efficiency, but excludes other lysolipid species. Once a LPL is flipped by LplT to the cytoplasmic side of the inner membrane, its diacyl form is effectively regenerated by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase (Aas). LplT-Aas also mediates a novel cardiolipin remodeling by converting its two lyso derivatives, diacyl or deacylated cardiolipin, to a triacyl form. This coupled remodeling system provides a unique bacterial membrane phospholipid repair mechanism. Strict selectivity of LplT for lyso lipids allows this system to fulfill efficient lipid repair in an environment containing mostly diacyl phospholipids. A rocker-switch model engaged by a pair of symmetric ion-locks may facilitate alternating substrate access to drive LPL flipping into bacterial cells. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

In vitro reconstitution of lipid-dependent dual topology and postassembly topological switching of a membrane protein.

Phospholipids could exert their effect on membrane protein topology either directly by interacting with topogenic signals of newly inserted proteins or indirectly by influencing the protein assembly machinery. In vivo lactose permease (LacY) of Escherichia coli displays a mixture of topological conformations ranging from complete inversion of the N-terminal helical bundle to mixed topology and then to completely native topology as phosphatidylethanolamine (PE) is increased from 0% to 70% of membrane phospholipids. These topological conformers are interconvertible by postassembly synthesis or dilution of PE in vivo. To investigate whether coexistence of multiple topological conformers is dependent solely on the membrane lipid composition, we determined the topological organization of LacY in an in vitro proteoliposome system in which lipid composition can be systematically controlled before (liposomes) and after (fliposomes) reconstitution using a lipid exchange technique. Purified LacY reconstituted into preformed liposomes of increasing PE content displayed inverted topology at low PE and then a mixture of inverted and proper topologies with the latter increasing with increasing PE until all LacY adopted its native topology. Interconversion between topological conformers of LacY was observed in a PE dose-dependent manner by either increasing or decreasing PE levels in proteoliposomes postreconstitution of LacY, clearly demonstrating that membrane protein topology can be changed simply by changing membrane lipid composition independent of other cellular factors. The results provide a thermodynamic-based lipid-dependent model for shifting the equilibrium between different conformational states of a membrane protein.

Cardiolipin is dispensable for oxidative phosphorylation and non-fermentative growth of alkaliphilic Bacillus pseudofirmus OF4.

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from (32)P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28-33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.

Lipids and topological rules governing membrane protein assembly.

Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid-protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Biosynthetic preparation of selectively deuterated phosphatidylcholine in genetically modified Escherichia coli.

Phosphatidylcholine (PC) is a major component of eukaryotic cell membranes and one of the most commonly used phospholipids for reconstitution of membrane proteins into carrier systems such as lipid vesicles, micelles and nanodiscs. Selectively deuterated versions of this lipid have many applications, especially in structural studies using techniques such as NMR, neutron reflectivity and small-angle neutron scattering. Here we present a comprehensive study of selective deuteration of phosphatidylcholine through biosynthesis in a genetically modified strain of Escherichia coli. By carefully tuning the deuteration level in E. coli growth media and varying the deuteration of supplemented carbon sources, we show that it is possible to achieve a controlled deuteration for three distinct parts of the PC lipid molecule, namely the (a) lipid head group, (b) glycerol backbone and (c) fatty acyl tail. This biosynthetic approach paves the way for the synthesis of specifically deuterated, physiologically relevant phospholipid species which remain difficult to obtain through standard chemical synthesis.

Discovery of a cardiolipin synthase utilizing phosphatidylethanolamine and phosphatidylglycerol as substrates.

Depending on growth phase and culture conditions, cardiolipin (CL) makes up 5-15% of the phospholipids in Escherichia coli with the remainder being primarily phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). In E. coli, the cls and ybhO genes (renamed clsA and clsB, respectively) each encode a CL synthase (Cls) that catalyzes the condensation of two PG molecules to form CL and glycerol. However, a ΔclsAB mutant still makes CL in the stationary phase, indicating the existence of additional Cls. We identified a third Cls encoded by ymdC (renamed clsC). ClsC has sequence homology with ClsA and ClsB, which all belong to the phospholipase D superfamily. The ΔclsABC mutant lacks detectible CL regardless of growth phase or growth conditions. CL can be restored to near wild-type levels in stationary phase in the triple mutant by expressing either clsA or clsB. Expression of clsC alone resulted in a low level of CL in the stationary phase, which increased to near wild-type levels by coexpression of its neighboring gene, ymdB. CL synthesis by all Cls is increased with increasing medium osmolarity during logarithmic growth and in stationary phase. However, only ClsA contributes detectible levels of CL at low osmolarity during logarithmic growth. Mutation of the putative catalytic motif of ClsC prevents CL formation. Unlike eukaryotic Cls (that use PG and CDP-diacylglycerol as substrates) or ClsA, the combined YmdB-ClsC used PE as the phosphatidyl donor to PG to form CL, which demonstrates a third and unique mode for CL synthesis.