The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene.

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC. The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene. ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB. YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

On the lipid dependence of bacterial mechanosensitive channel gating in situ.

For bacterial mechanosensitive channels acting as turgor-adjusting osmolyte release valves, membrane tension is the primary stimulus driving opening transitions. Because tension is transmitted through the surrounding lipid bilayer, it is possible that the presence or absence of different lipid species may influence the function of these channels. In this work, we characterize the lipid dependence of chromosome-encoded MscS and MscL in E. coli strains with genetically altered lipid composition. We use two previously generated strains that lack one or two major lipid species (PE, PG, or CL) and engineer a third strain that is highly enriched in CL due to the presence of hyperactive cardiolipin synthase ClsA. We characterize the functional behavior of these channels using patch-clamp and quantify the relative tension midpoints, closing rates, inactivation depth, and the rate of recovery back to the closed state. We also measure the osmotic survival of lipid-deficient strains, which characterizes the functional consequences of lipid-mediated channel function at the cell level. We find that the opening and closing behavior of MscS and MscL tolerate the absence of specific lipid species remarkably well. The lack of cardiolipin (CL), however, reduces the active MscS population relative to MscL and decreases the closing rate, slightly increasing the propensity of MscS toward inactivation and slowing the recovery process. The data points to the robustness of the osmolyte release system and the importance of cardiolipin for the adaptive behavior of MscS.

Preparation of Uniformly Oriented Inverted Inner (Cytoplasmic) Membrane Vesicles from Gram-Negative Bacterial Cells.

The complex double-membrane organization of the envelope in Gram-negative bacteria places unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on cytoplasm facing leaflet of cytoplasmic (inner) membrane (IM), across IM and between IM and outer membrane (OM). Uniformly oriented inside-out (ISO) vesicles became functional requisite for many biochemical reconstitution functional assays, vectorial proteomics, and vectorial lipidomics. Due to these demands, it is necessary to develop simple and reliable approaches for preparation of uniformly oriented IM membrane vesicles and validation of their sidedness. The uniformly ISO oriented membrane vesicles which have the cytoplasmic face of the membrane on the outside and the periplasmic side facing the sealed lumen can be obtained following intact cell disruption by a single passage through a French pressure cell (French press) at desired total pressure. Although high-pressure lysis leads to the formation of mostly inverted membrane vesicles (designated and abbreviated usually as ISO vesicles, everted or inverted membrane vesicles (IMVs)), inconclusive results are quite common. This uncertainty is due mainly by applying a different pressures, using either intact cells or spheroplasts and presence or absence of sucrose during rupture procedure. Many E. coli envelope fractionation techniques result in heterogeneity among isolated IM membrane vesicles. In part, this is due to difficulties in simple validation of sidedness of oriented membrane preparations of unknown sidedness. The sidedness of various preparations of membrane vesicles can be inferred from the orientation of residing uniformly oriented transmembrane protein. We outline the method in which the orientation of membrane vesicles can be verified by mapping of uniform or mixed topologies of essential protein E. coli protein leader peptidase (LepB) by advanced SCAM™. Although the protocol discussed in this chapter has been developed using Escherichia coli and Yersinia pseudotuberculosis, it can be directly adapted to other Gram-negative bacteria including pathogens.

Exploring Uniform, Dual, and Dynamic Topologies of Membrane Proteins by Substituted Cysteine Accessibility Method (SCAM™).

A described simple and advanced protocol for Substituted Cysteine Accessibility Method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform or identify and quantify the degree of mixed topology or establish transmembrane assembly dynamics from relatively static experimental data such as endpoint topologies of membrane proteins. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at the time by cysteine residue, and the orientation with respect to the membrane is evaluated by using a pair of membrane-impermeable non-detectable and detectable thiol-reactive labeling reagents. For the most water-exposed cysteine residues in proteins, the thiol pKa lies in the range of 8-9, and formation of cysteinyl thiolate ions is optimum in aqueous rather in a nonpolar environment. These features and the ease of specific chemical modification with thiol reagents are central to SCAM™. Membrane side-specific sulfhydryl labeling allows to discriminate "exposed, protected or dynamic" cysteines strategically "implanted" at desired positions throughout cysteine less target protein template. The strategy described is widely used to map the topology of membrane protein and establish its transmembrane dynamics in intact cells of both diderm (two-membraned) Gram-negative and monoderm (one-membraned) Gram-positive bacteria, cell-derived oriented membrane vesicles, and proteoliposomes.

Differentiation of gram-negative intermembrane phospholipid transporter function by intrinsic substrate preference.

The outer membrane of diderm Gram-negative bacteria acts as a barrier from chemical and physical stress. Anterograde phospholipid transport to the outer membrane has long been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by three related proteins: YhdP, TamB, and YdbH, which appear to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct phospholipid transport functions. We investigated these functions using the synthetic cold sensitivity of a strain with ΔyhdP (but not ΔtamB or ΔydbH) and ΔfadR, a transcriptional regulator allowing switching between fatty acid degradation and synthesis and regulating unsaturated fatty acid production. Deletion of tamB, forcing phospholipid transport to YdbH, suppresses the ΔyhdP ΔfadR cold sensitivity suggesting this phenotype is due to TamB dysfunction. Increased levels of cardiolipin and fatty acid saturation are necessary for cold sensitivity and lowering levels of either suppresses this sensitivity. Our data support a model where YhdP primarily transports more saturated phospholipids, TamB primarily transports phospholipids with more than one carbon unsaturation, and cardiolipin obstructs TamB by selectively clogging its channel. Thus, the multiple phospholipid transporters may allow the saturation state of the outer membrane to be regulated independently of the inner membrane by altering the YhdP-TamB activity ratio. Maintaining membrane physical integrity and function under changing conditions may require envelope remodeling including altered phospholipid composition and intermembrane trafficking. Our data provide a potential mechanism for this regulation.

The power and challenge of lipid (a)symmetry across the membrane and cell.

Membrane asymmetry means that the two sides of membrane are structurally, physically and functionally different. Membrane asymmetry is largely related to the lipid sidedness and particularly to compositional (lipid head and acyl group) and physical (lipid packing order, charge, hydration and H-bonding interactions) differences in the inner and outer leaflets of lipid bilayer. Chemically, structurally and conformationally different non-covalent bound lipid molecules are physically fluid and deformable and enable to interact dynamically to form transient arrangements with asymmetry both perpendicular and parallel to the plane of the lipid bilayer. Although biological membranes are almost universally asymmetric however the asymmetry is not absolute since only drastic difference in the number of lipids per leaflet is found and symmetric arrangements are possible. Asymmetry is thought to direct and influence many core biological functions by altering the membrane's collective biochemical, biophysical and structural properties. Asymmetric transbilayer lipid distribution is found across all lipid classes, cells and near all endomembrane compartments. Why cell membranes are (a)symmetric and adopt almost exclusively highly entropically disfavored asymmetric state?

Renovating a double fence with or without notifying the next door and across the street neighbors: why the biogenic cytoplasmic membrane of Gram-negative bacteria display asymmetry?

The complex two-membrane organization of the envelope of Gram-negative bacteria imposes an unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on the cytoplasm facing leaflet of the cytoplasmic (inner) membrane (IM), across the IM and between the IM and outer membrane (OM). Balanced growth of two membranes and continuous loss of phospholipids in the periplasmic leaflet of the IM as metabolic precursors for envelope components and for translocation to the OM requires a constant supply of phospholipids in the IM cytosolic leaflet. At present we have no explanation as to why the biogenic E. coli IM displays asymmetry. Lipid asymmetry is largely related to highly entropically disfavored, unequal headgroup and acyl group asymmetries which are usually actively maintained by active mechanisms. However, these mechanisms are largely unknown for bacteria. Alternatively, lipid asymmetry in biogenic IM could be metabolically controlled in order to maintain uniform bilayer growth and asymmetric transmembrane arrangement by balancing temporally the net rates of synthesis and flip-flop, inter IM and OM bidirectional flows and bilayer chemical and physical properties as spontaneous response. Does such flippase-less or 'lipid only", 'passive' mechanism of generation and maintenance of lipid asymmetry exists in the IM? The driving force for IM asymmetry can arise from the packing requirements imposed upon the bilayer system during cell division through disproportional distribution of two negatively curved phospholipids, phosphatidylethanolamine and cardiolipin, with consistent reciprocal tendency to increase and decrease lipid order in each membrane leaflet respectively.

Efficacy of a Relaxation Scenario in Virtual Reality for the Comorbid Symptoms of Anxiety and Asthenia in a General Hospital Setting: A Pilot Comparative Randomized Open-Label Study.

BACKGROUND: Patients in general hospitals often display concomitant signs of mental maladjustment: low mood, anxiety, apathy, asthenia, all of which can have a negative impact on the course of the underlying disease and the recovery process. One of the non-pharmacological approaches that has gained wider acceptance in medical practice in recent years is the use of procedures based on virtual reality. AIM: Assess the efficacy of the new domestic, virtual reality application Flow as relates to symptoms of anxiety and asthenia in patients undergoing inpatient treatment. METHODS: The study was open-label and had a comparison group; the patients were assigned to the experimental or control group using a randomization table. The patients were assessed using the Spielberger State Anxiety Inventory; the Fatigue Symptom Rating Scale; the Well-being, Activity, Mood questionnaire; the Depression Anxiety Stress Scale; and the Clinical Global Impression Scale. Physical parameters were measured before and after each virtual reality session. The obtained data were statistically processed. RESULTS: The study involved 60 patients. In 40 patients, the treatment program included a course of five daily relaxation sessions in virtual reality; the control group consisted of 20 patients, who were treated in accordance with the usual practice of the institution. The addition of virtual reality sessions to the standard treatment course yielded significant advantage in terms of affective symptoms reduction in patients both after a single session and as a result of undergoing the full course, and several days after its completion. The patients in the experimental group also showed a significant decrease in blood pressure after the sessions, and this was most pronounced in individuals who initially had elevated and high blood pressure. CONCLUSION: The use of relaxation program courses in the virtual reality application Flow is an effective and promising means of non-pharmacological care for non-psychiatric inpatients showing symptoms of anxiety, apathy, depressive mood, as well as hypertension.

Toward a Topology-Based Therapeutic Design of Membrane Proteins: Validation of NaPi2b Topology in Live Ovarian Cancer Cells.

NaPi2b is a sodium-dependent phosphate transporter that belongs to the SLC34 family of transporters which is mainly responsible for phosphate homeostasis in humans. Although NaPi2b is widely expressed in normal tissues, its overexpression has been demonstrated in ovarian, lung, and other cancers. A valuable set of antibodies, including L2 (20/3) and MX35, and its humanized versions react strongly with an antigen on the surface of ovarian and other carcinoma cells. Although the topology of NaPi2b was predicted in silico, no direct experimental data are available for the orientation of NaPi2b extracellular domains in cancer cells. The presented results of antibody mapping of untagged NaPi2b in live ovarian carcinoma cells OVCAR-4 provide a platform for current and future epitope-based cancer therapies and serological diagnostics.

Characterization of SLC34A2 as a Potential Prognostic Marker of Oncological Diseases.

The main goal of this study is to consider SLC34A2 as a potential prognostic marker of oncological diseases using the mutational, expression, and survival data of cancer studies which are publicly available online. We collected data from four databases (cBioPortal, The Cancer Genome Atlas; cBioPortal, Genie; International Cancer Genome Consortium; ArrayExpress). In total, 111,283 samples were categorized according to 27 tumor locations. Ninety-nine functionally significant missense mutations and twelve functionally significant indel mutations in SLC34A2 were found. The most frequent mutations were SLC34A2-ROS1, p.T154A, p.P506S/R/L, p.G257A/E/R, p.S318W, p.A396T, p.P410L/S/H, p.S461C, p.A473T/V, and p.Y503H/C/F. The upregulation of SLC34A2 was found in samples of myeloid, bowel, ovarian, and uterine tumors; downregulation was found in tumor samples of breast, liver, lung, and skin cancer tumors. It was found that the life expectancy of breast and thymus cancer patients with an SLC34A2 mutation is lower, and it was revealed that SLC34A2 overexpression reduced the life span of patients with brain, ovarian, and pancreatic tumors. Thereby, for these types of oncological diseases, the mutational profile of SLC34A2 can be a potential prognostic marker for breast and thymus cancers, and the upregulation of SLC34A2 can be a potential prognostic marker for brain, ovarian, and pancreatic cancers.

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.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Eugene P. Kennedy's Legacy: Defining Bacterial Phospholipid Pathways and Function.

In the 1950's and 1960's Eugene P. Kennedy laid out the blueprint for phospholipid biosynthesis in somatic cells and Escherichia coli, which have been coined the Kennedy Pathways for phospholipid biosynthesis. His research group continued to make seminal contributions in the area of phospholipids until his retirement in the early 1990's. During these years he mentored many young scientists that continued to build on his early discoveries and who also mentored additional scientists that continue to make important contributions in areas related to phospholipids and membrane biogenesis. This review will focus on the initial E. coli Kennedy Pathways and how his early contributions have laid the foundation for our current understanding of bacterial phospholipid genetics, biochemistry and function as carried on by his scientific progeny and others who have been inspired to study microbial phospholipids.

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.

Phospholipid distribution in the cytoplasmic membrane of Gram-negative bacteria is highly asymmetric, dynamic, and cell shape-dependent.

The distribution of phospholipids across the inner membrane (IM) of Gram-negative bacteria is unknown. We demonstrate that the IMs of Escherichia coli and Yersinia pseudotuberculosis are asymmetric, with a 75%/25% (cytoplasmic/periplasmic leaflet) distribution of phosphatidylethanolamine (PE) in rod-shaped cells and an opposite distribution in E. coli filamentous cells. In initially filamentous PE-lacking E. coli cells, nascent PE appears first in the periplasmic leaflet. As the total PE content increases from nearly zero to 75%, cells progressively adopt a rod shape and PE appears in the cytoplasmic leaflet of the IM. The redistribution of PE influences the distribution of the other lipids between the leaflets. This correlates with the tendency of PE and cardiolipin to regulate antagonistically lipid order of the bilayer. The results suggest that PE asymmetry is metabolically controlled to balance temporally the net rates of synthesis and translocation, satisfy envelope growth capacity, and adjust bilayer chemical and physical properties.

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.

Cardiolipin is required in vivo for the stability of bacterial translocon and optimal membrane protein translocation and insertion.

Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane ß-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomerization properties of SecA were strongly compromised while the levels of individual SecYEG translocon components, SecA and insertase YidC were largely unaffected. These results demonstrate that CL is required in vivo for the stability of the bacterial translocon and its efficient function in co-translational insertion into and translocation across the inner membrane of E. coli.

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 lipid-dependent structure and function of LacY can be recapitulated and analyzed in phospholipid-containing detergent micelles.

Membrane proteins play key roles in cellular functions, their activity mainly depending on their topological arrangement in membranes. Structural studies of membrane proteins have long adopted a protein-centric view regarding the determinants of membrane protein topology and function. Several studies have shown that the orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer can be largely determined by membrane lipid composition. However, the mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is still unknown. Here we show that lipid-dependent structural and functional assessment of a membrane protein can be conducted in detergent micelles, opening the possibility for the determination of lipid-dependent high-resolution crystal structures. We found that the lactose permease purified from Escherichia coli cells exhibiting varied phospholipid compositions exhibits the same topology and similar function as in its membrane of origin. Furthermore, we found several conditions, including protein mutations and micelle lipid composition, that lead to increased protein stability, correlating with a higher yield of two-dimensional crystal formation. Altogether, our results demonstrate how the membrane lipid environment influences membrane protein topology and arrangement, both in native membranes and in mixed detergent micelles.

Lipid-Assisted Membrane Protein Folding and Topogenesis.

Due to the heterogenous lipid environment in which integral membrane proteins are embedded, they should follow a set of assembly rules, which govern transmembrane protein folding and topogenesis accordingly to a given lipid profile. Recombinant strains of bacteria have been engineered to have different membrane phospholipid compositions by molecular genetic manipulation of endogenous and foreign genes encoding lipid biosynthetic enzymes. Such strains provide a means to investigate the in vivo role of lipids in many different aspects of membrane function, folding and biogenesis. In vitro and in vivo studies established a function of lipids as molecular chaperones and topological determinants specifically assisting folding and topogenesis of membrane proteins. These results led to the extension of the Positive Inside Rule to Charge Balance Rule, which incorporates a role for lipid-protein interactions in determining membrane protein topological organization at the time of initial membrane insertion and dynamically after initial assembly. Membrane protein topogenesis appears to be a thermodynamically driven process in which lipid-protein interactions affect the potency of charged amino acid residues as topological signals. Dual topology for a membrane protein can be established during initial assembly where folding intermediates in multiple topological conformations are in rapid equilibrium (thus separated by a low activation energy), which is determined by the lipid environment. Post-assembly changes in lipid composition or post-translational modifications can trigger a reorganization of protein topology by inducing destabilization and refolding of a membrane protein. The lipid-dependent dynamic nature of membrane protein organization provides a novel means of regulating protein function.