Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion

Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.     

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.Polyphosphoinositides are phosphorylated derivatives of phosphatidylinositol (PtdIns) implicated in many aspects of cell function. They control a surprisingly large number of processes in animal, yeast, and plant cells, including exocytosis, endocytosis, cytoskeletal adhesion, and signal transduction not only as second-messenger precursors but also as signaling molecules on their own by interacting with protein partners, allowing spatially selective regulation at the cytoplasm-membrane interface (for review, see Di Paolo and De Camilli, 2006). Polyphosphoinositides also control the activity of ion transporters and channels during biosynthesis or vesicle trafficking (Liu et al., 2005; Monteiro et al., 2005b). In plants, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is present in very small quantities (for review, see Stevenson et al., 2000; Meijer and Munnik, 2003) and was visualized in vivo by expressing a fluorescent protein (GFP or yellow fluorescent protein) fused to the pleckstrin homology (PH) domain of the human phospholipase C δ1 (PLCδ1) that specifically binds PtdIns(4,5)P₂. The fused protein yellow fluorescent protein-PHPLCδ1 was present in the cytoplasm but concentrated at the plant plasma membrane (PM) in response to salt stress or upon treatment with the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (van Leeuwen et al., 2007). In pollen tubes and root hairs, where spatially focused cell expansion occurs, highly localized PtdIns(4,5)P₂ has been evidenced at the membrane tip (Braun et al., 1999; Kost et al., 1999). PtdIns(4,5)P₂ likely functions as an effector of small G proteins at the apex of cells influencing membrane fusion events (Monteiro et al., 2005a). In guard cells, the level of PtdIns(4,5)P₂ increases at the PM upon illumination (Lee et al., 2007). Combining imaging, patch clamp, and genetic evidence, Lee et al. (2007) further proposed that PtdIns(4,5)P₂ is important for stomatal opening. Stomatal guard cells have also been reported to contain phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 4-phosphate (PtdIns4P), the products of PtdIns 3-kinase and PtdIns 4-kinase activities, respectively. Jung et al. (2002) demonstrated that PtdIns3P and PtdIns4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PtdIns3P and PtdIns4P enhance stomatal opening. Recently, the hyperosmotic stress response was studied in Arabidopsis (Arabidopsis thaliana). Several groups (Pical et al., 1999; DeWald et al., 2001; Konig et al., 2007, 2008b) have shown that plants exhibit a transient increase in polyphosphoinositides after hyperosmotic stress, providing a model for comparing constitutive and stress-inducible polyphosphoinositide pools. Under nonstress conditions, structural phospholipids and PtdIns contained 50 to 70 mol % polyunsaturated fatty acids (PUFA), whereas polyphosphoinositides were more saturated (10–20 mol % PUFA; Konig et al., 2007). Upon hyperosmotic stress, polyphosphoinositides with up to 70 mol % PUFA were formed that differed from constitutive species and coincided with a transient loss in unsaturated PtdIns. These patterns indicate the inducible turnover of an unsaturated PtdIns pool and the presence of distinct polyphosphoinositide pools in plant membranes (Konig et al., 2007).Since these biological phenomena are likely to occur in distinct regions of the PM, it has been our working hypothesis that in plant cells polyphosphoinositides are localized in various microdomains to participate in different cellular functions. Two decades ago, Metcalf et al. (1986) already suggested that the plant PM contains stable immiscible domains of fluid and gel-like lipids using fluorescent lipid and phospholipid probes incorporated into soybean (Glycine max) protoplasts prepared from cultured soybean cells. To this day, it has been generally accepted that lipids and proteins of the PM are not homogeneously distributed within membranes but rather form various domains of localized enrichment. The best-characterized membrane domains are membrane rafts (MRs; Pike, 2006). MRs are liquid-ordered subdomains within eukaryotic membranes that are hypothesized to play important roles in a variety of biological functions by coordinating and compartmentalizing diverse sets of proteins to facilitate signal transduction mechanisms, focal regulation of cytoskeleton, and membrane trafficking (for review, see Rajendran and Simons, 2005; Brown, 2006). Both evidenced in plants and animals, MRs are enriched in sphingolipids and sterols and largely deprived in phospholipids (for review, see Brown and London, 2000; Bhat and Panstruga, 2005). Sterols interact preferentially, although not exclusively, with sphingolipids due to their structure and the saturation of their hydrocarbon chains. Because of the rigid nature of the sterol group, sterols have the ability to pack in between the lipids in rafts, serving as molecular spacers and filling voids between associated sphingolipids (Binder et al., 2003). Acyl chains of MR lipids tend to be more rigid and in a less fluid state (Roche et al., 2008). In agreement, the hydrophobic chains of the phospholipids within the raft are more saturated and tightly packed than those of lipids in the surrounding bilayer (Mongrand et al., 2004). MRs can be isolated from PM by extraction with nonionic detergents such as Triton X-100 (TX100) or Brij-98 at low temperatures. Fluid nonraft domains will solubilize while the MRs remain intact and can be enriched after centrifugation, floating in a Suc density gradient. Floating purified fractions, therefore, are called detergent-insoluble membranes (DIMs) or detergent-resistant membranes and are thought to be the biochemical counterpart of in vivo MRs.In plants, a few results suggest the role in vivo of dynamic clustering of PM proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the PM, a process reminiscent of MRs (Bhat et al., 2005). The proteomic analysis of tobacco (Nicotiana tabacum) DIMs led to the identification of 145 proteins, among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (Morel et al., 2006). Therefore, these domains are likely to constitute, as in animal cells, signaling membrane platforms concentrating lipids and proteins necessary for the generation of signaling molecules of physiological relevance. This hypothesis was confirmed by a quantitative proteomic study describing the dynamic association of proteins with DIMs upon challenge of tobacco cells with an elicitor of defense reaction (Stanislas et al., 2009). Recently, Raffaele et al. (2009) showed that a group of proteins specific to vascular plants, called remorins (REMs), share the biochemical properties of other MR proteins and are clustered into microdomains of approximately 70 nm in diameter in the PM and plasmodesmata in tobacco, providing a link between biochemistry (DIM purification) and imaging (membrane microdomain observation).Several investigators have previously suggested that PtdIns(4,5)P₂-rich raft assemblies exist in animal cell membranes to provide powerful organizational principles for tight spatial and temporal control of signaling in motility. Laux et al. (2000) demonstrated that PtdIns(4,5)P₂ formed microdomains in the PM of animal cells, and at least part of these microdomains was colocalized with the myristoylated Ala-rich type C kinase substrate, a protein enriched in MRs, and involved in the regulation of the actin cytoskeleton. The relationship between the spatial organization of PtdIns(4,5)P₂ microdomains and exocytotic machineries has been evidenced in rat. Both PtdIns(4,5)P₂ and syntaxin, a protein essential for exocytosis, exhibited punctate clusters in isolated PM. PtdIns(4,5)P₂ also accumulated at sites of cell surface motility together with a Rho-type GTPase. Therefore, PtdIns(4,5)P₂ may coordinate membrane dynamics and actin organization as well as integrate signaling (Aoyagi et al., 2005). These results provide evidence of compartmentalization of PtdIns(4,5)P₂-dependent signaling in cell membranes.Little is known in plants about whether and how separate pools of polyphosphoinositides come about and how they are regulated. In this article, we have analyzed the lipid composition of DIMs enriched from tobacco PM, with a particular focus on phospholipids involved in signaling events, such as polyphosphoinositides. We showed that polyphosphoinositides were enriched in DIMs, whereas structural phospholipids were largely excluded. We were able to calculate that almost half of the PtdInsP and PtdIns(4,5)P₂ were present in MR domains. Fatty acid composition analyses demonstrate that this enrichment is accompanied by the presence of more saturated fatty acids in polyphosphoinositides. Consistently, using an electron microscopy approach with immunogold labeling and a pattern-identifying statistical analysis, we showed that more than half of the PtdIns(4,5)P₂ labeling is clustered into microdomains of approximately 25 nm in diameter in the PM. Finally, we measured the activities of lipid-using enzymes present in DIMs/PM and showed that activities responsible for polyphosphoinositide metabolism are present in the DIM fraction.     

Lipid Breakdown in Smooth Microsomal Membranes from Bean Cotyledons Alters Membrane Proteins and Induces Proteolysis

The effects of lipid degradation on proteins of smooth microsomalmembranes isolated from young bean cotyledons have been examinedby three techniques, viz. fluorescence energy transfer fromtryptophan to cis-parinaric acid; protein spin-labelling with3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was inducedin isolated membranes by activating phospholipase D and phosphatidicacid phosphatase through the addition of Ca²⁺, by treatmentwith exogenous phospholipase C to simulate the concerted actionsof phospholipase D and phosphatidic acid phosphatase or by treatmentwith exogenous phospholipase A₂ to generate endogenous substratefor lipoxygenase. All of the treatments induced time-dependentchanges in lipid-protein interaction and in protein conformation,and the treatment with phospholipase A₂ also engendered proteolysis.The effects of the Ca²⁺ and phospholipase C treatments on lipid-proteininteraction and protein conformation can presumably be partlyattributed to an accumulation of diacylglycerol in the membrane,whereas the induction of proteolysis by phospholipase A₂ appearsto be due to activated oxygen derived from the lipoxygenasereaction and ensuing lipid peroxidation. Lipid degradation inducedby these treatments simulates that which occurs during naturalsenescence of the cotyledons and hence these observations suggestthat loss of protein function and proteolysis in senescing membranesis facilitated by lipolytic and peroxidative activity withinthe lipid bilayer. Key words: Activated oxygen, lipids, membranes, proteins, senescence     

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli.The cells of living organisms contain different types of membranes performing uniquely specific functions that are largely dictated by their protein compositions. Membrane proteome typically contains integral membrane proteins (IMPs)¹ with one or more transmembrane domains (TM) and peripheral membrane proteins (PMPs) without TM. PMPs usually interact with IMPs and function together as protein complexes, as typically demonstrated by the peripheral subunits of the membrane protein complexes such as photosystem (PS) I, PSII, the F₁F₀-ATP synthase, and ABC type transporters (1–6). Identification of the PMPs is important for the understanding of the underlying mechanism of various membrane related functions, and could help to discover novel functionally important membrane protein complexes.Large-scale identification of PMPs were typically performed by identification of the total proteins from the isolated whole membranes from which PMPs were predicted by the absence of TM using topology prediction software such as TMHMM (7), or by identification of the proteins extracted from the intact whole membranes with chaotropic reagents such as high concentration salts, urea, or high pH solution (8–13). These methods can identify some non-TM containing proteins uniquely from the membrane fraction. However, in most cases the majority of the non-TM containing proteins identified with such methods can also be identified from the soluble fraction that is expected to consist of mainly cytoplasmic proteins. Therefore, it is necessary to evaluate whether the non-TM containing proteins identified from the membranes are true PMPs or just some carry-over contaminant from the soluble fraction during sample fractionation. Unfortunately, the high throughput method to perform such an evaluation is still lacking, and such a method is a pressing need considering the ever-increasing number of identified proteins from a single proteomic study.The unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis) is an ideal organism for studies in membrane proteomics. Synechocystis is the first cyanobacterium with a completely sequenced genome and contains large numbers of membrane structures (12–14). The organism can naturally take up foreign DNA from environment and integrate it into its genome through homologous recombination, making it simple to perform target mutagenesis for the validation of functional significance of proteins screened from high throughput approaches. The autotrophic growth ability allows Synechocystis to emerge as a potential cost-effective cell factory for producing clean and renewable biofuels to deal with the world-wide crisis of energy shortage and environmental pollution (15–18). Functional proteomics have great potential in the identification of novel target proteins and for discovering and optimizing novel protein networks for the generation of biofuel-producing strains with higher efficiency and less cost.We separated Synechocystis whole cell lysates into membrane and soluble fractions, and identified the proteins in each fraction with unprecedented coverage using high-resolution MS. We present a novel method and its rationale for evaluating the tightness of membrane association for all non-TM containing proteins identified in both fractions. This built a foundation for the large-scale identification of bona fide peripheral membrane proteins, particularly for the hypothetical and unknown proteins that are not known to be physically or functionally associated with the membranes.     

Lipid-protein interactions in mitochondria. The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis.

Phospholipase A₂ (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.     

Interactions of Cations with Membrane Fractions of Smooth and Rough Strains of Salmonella typhimurium and Other Gram-Negative Bacteria

Addition of cations (20 to 50 mM for Mg²⁺ or Ca²⁺ or 100 to 500 mM for Na⁺) to N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer during preparation of membranes from smooth and rough strains of Salmonella typhimurium LT2, Salmonella minnesota, and Escherichia coli O8 had two effects on the composition of the membranes isolated. First, in rough strains of chemotypes Ra to Re the “total membranes” (pellets from high-speed centrifugation) were deficient in the proteins of the outer membrane. The missing proteins were found to have been sedimented in a prior low-speed centrifugation in a fraction we call “cation-aggregated membranes.” Since these membranes were enriched for lipopolysaccharide and for outer membrane proteins, deficient in succinic dehydrogenase, and contained primarily the dense peak after sucrose gradient centrifugation, it appears to be relatively pure outer membrane. About 10% of the membrane protein of smooth strains and up to 50% that of rough strains were cation-aggregated membranes, appearing to contain most of the outer membrane of rough strains. Thus, cation aggregation may be a useful means of preparation of outer membrane samples. The second effect was that with cation addition, several high-molecular-weight proteins not seen when membranes were prepared without cation addition were found in the total membranes of both smooth and rough strains after high-speed centrifugation. These proteins were bound by cations to the inner membranes, since they were soluble in Triton X-100 and separated into the less dense peak upon sucrose gradient centrifugation. They originated from the cytoplasm or the periplasm, since they corresponded to soluble proteins found in the supernatant after high-speed centrifugation and were depleted from this supernatant when preparation was done in the presence of cations.     

Aggregation Modulators Interfere with Membrane Interactions of?β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β₂-microglobulin (β₂m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β₂m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.     

Membrane lipid composition defines membrane protein spectrum

In addition to glycerophospholipids, bacterial membranes often include amino acid-containing acyloxyacyl lipids. The functional implications of these aminolipids are largely unknown. However, a recent study by Stirrup et al. expands our understanding and shows that they are major determinants for membrane properties and the relative abundance of distinct membrane proteins in bacterial membranes.     

Geometrical Membrane Curvature as an Allosteric Regulator of Membrane Protein Structure and Function

Transmembrane proteins are embedded in cellular membranes of varied lipid composition and geometrical curvature. Here, we studied for the first time the allosteric effect of geometrical membrane curvature on transmembrane protein structure and function. We used single-channel optical analysis of the prototypic transmembrane β-barrel α-hemolysin (α-HL) reconstituted on immobilized single small unilamellar liposomes of different diameter and therefore curvature. Our data demonstrate that physiologically abundant geometrical membrane curvatures can enforce a dramatic allosteric regulation (1000-fold inhibition) of α-HL permeability. High membrane curvatures (1/diameter ∼1/40 nm⁻¹) compressed the effective pore diameter of α-HL from 14.2 ± 0.8 Å to 11.4 ± 0.6 Å. This reduction in effective pore area (∼40%) when combined with the area compressibility of α-HL revealed an effective membrane tension of ∼50 mN/m and a curvature-imposed protein deformation energy of ∼7 k_BT. Such substantial energies have been shown to conformationally activate, or unfold, β-barrel and α-helical transmembrane proteins, suggesting that membrane curvature could likely regulate allosterically the structure and function of transmembrane proteins in general.     

Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane

RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.     

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Arabidopsis CURVATURE THYLAKOID1 Proteins Modify Thylakoid Architecture by Inducing Membrane Curvature

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.     

Phospholipase D1 Regulates Lymphocyte Adhesion via Upregulation of Rap1 at the Plasma Membrane

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated. We report here that phospholipase D1 (PLD1) is expressed on the same vesicular compartment in T cells as Rap1 and is translocated to the plasma membrane along with Rap1. Moreover, PLD activity is required for both translocation and activation of Rap1. Increased T-cell adhesion in response to stimulation of the antigen receptor depended on PLD1. C3G, a Rap1 guanine nucleotide exchange factor located in the cytosol of resting cells, translocated to the plasma membranes of stimulated T cells. Our data support a model whereby PLD1 regulates Rap1 activity by controlling exocytosis of a stored, vesicular pool of Rap1 that can be activated by C3G upon delivery to the plasma membrane.Regulated adhesion of lymphocytes is required for immune function. The β2 integrin lymphocyte function-associated antigen 1 (LFA-1) mediates lymphocyte adhesion to endothelium, antigen-presenting cells, and virally infected target cells (14). These cell-cell adhesions enable lymphocyte trafficking in and out of lymphoid organs, T-cell activation, and cytotoxicity, respectively (2, 34). Thus, the regulation of LFA-1 adhesiveness is central to adaptive immunity.LFA-1 is a bidirectional receptor in that it mediates both outside-in and inside-out signaling (30). Outside-in signaling is analogous to signaling by conventional receptors and is defined as stimulation of intracellular signaling pathways as a consequence of ligation of LFA-1 with any of its extracellular ligands, such as intracellular adhesion molecule 1 (ICAM-1). Inside-out signaling refers to intracellular signaling events that result in a higher-affinity state of the ectodomain of LFA-1 for its cognate ligands. Regulatory events that mediate inside-out signaling converge on the cytoplasmic tails of the LFA-1 α and β chains, which transduce signals to their ectodomains (14). Signaling molecules implicated in inside-out signaling through LFA-1 include talin, Vav1, PKD1, several adaptor proteins (SLP-76, ADAP, and SKAP-55), the Ras family GTPase Rap1, and two of its effectors, RAPL and RIAM (26). How these proteins interact to activate LFA-1 remains poorly understood.Rap1 is a member of the Ras family of GTPases and has been implicated in growth control, protein trafficking, polarity, and cell-cell adhesion (6). The ability of activated Rap1 to promote LFA-1-mediated lymphocyte adhesion is well established (33). The physiologic relevance of this pathway is highlighted by leukocyte adhesion deficiency type III (LAD III), where immunocompromised patients have a congenital defect in GTP loading of Rap1 in leukocytes (24). LFA-1 is a plasma membrane protein, consistent with its role in cell-cell adhesion, which by definition is a cell surface phenomenon. Paradoxically, the bulk of Rap1 is expressed on intracellular vesicles. We have characterized these vesicles as recycling endosomes and have shown that the intracellular pool of Rap1 can be mobilized by exocytosis to augment the expression of Rap1 at the plasma membranes of lymphocytes, leading to increased adhesion (5). We used a fluorescent probe of activated Rap1 in live cells to show that only the pool of Rap1 at the plasma membrane becomes GTP bound upon lymphocyte activation. Thus, it appears that delivery of Rap1 via vesicular transport to the plasma membrane and activation of the GTPase on that compartment are linked. Among the signaling enzymes known to regulate vesicular trafficking is phospholipase D (PLD). Whereas PLD type 2 (PLD2) is expressed at the plasma membranes of lymphocytes, PLD1 is expressed on intracellular vesicles (29). We now show that PLD1 resides on the same vesicles as Rap1, is delivered along with Rap1 to the plasma membranes of stimulated T cells, and is required for Rap1 activation and T-cell adhesion.     

In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.     

Lipid-Mediated Regulation of Extrinsic Membrane Protein Activities

Abstract

It is increasingly apparent that lipids function not only in the membranous compartmentalization of cell components, but also in the regulation of activities of soluble proteins involved in key cellular events. There are several mechanisms by which membranes and their component lipids affect protein function. Certain proteins are activated by a conformational change that occurs upon association with lipid bilayers. Others appear to be influenced by being recruited to membranes so that they can interact with regulatory factors, or by being sequestered at membranes and thus incapable of interacting with soluble proteins or factors necessary for their function. Finally, membranes regulate many proteins by mechanisms yet to be elucidated. In addition to the lipids in membrane bilayers, products of glycerophospholipid and sphingolipid metabolism, functioning as second messengers, influence certain cytosolic proteins involved in cellular signal signaling pathways. This form of regulation, while important, is not the focus of this review and will only briefly be discussed.     

The Endoplasmic Reticulum Provides the Membrane Platform for Biogenesis of the Flavivirus Replication Complex

The cytoplasmic replication of positive-sense RNA viruses is associated with a dramatic rearrangement of host cellular membranes. These virus-induced changes result in the induction of vesicular structures that envelop the virus replication complex (RC). In this study, we have extended our previous observations on the intracellular location of West Nile virus strain Kunjin virus (WNV_KUN) to show that the virus-induced recruitment of host proteins and membrane appears to occur at a pre-Golgi step. To visualize the WNV_KUN replication complex, we performed three-dimensional (3D) modeling on tomograms from WNV_KUN replicon-transfected cells. These analyses have provided a 3D representation of the replication complex, revealing the open access of the replication complex with the cytoplasm and the fluidity of the complex to the rough endoplasmic reticulum. In addition, we provide data that indicate that a majority of the viral RNA species housed within the RC is in a double-stranded RNA (dsRNA) form.West Nile virus (WNV) belongs to the Flaviviridae, which is a large family of enveloped, positive-strand RNA viral pathogens that are responsible for causing severe disease and mortality in humans and animals each year. The Australian WNV strain Kunjin virus (WNV_KUN) is a relatively low-pathogenic virus that is closely related to the pathogenic WNV strain New York 99 (WNV_NY99), the causative agent of the 1999 epidemic of encephalitis in New York City (11).It has become increasingly known that the replication of most, if not all, positive-sense RNA viruses, whether they infect plants, insects, or humans, is associated with dramatic membrane alterations resulting in the formation of membranous microenvironments that facilitate efficient virus replication. In most cases the induced membrane structures house the actively replicating viral RNA and comprise 70- to 100-nm membrane “vesicles” (sometimes referred to as spherules). Although this distinct morphology is shared across virus families, the cellular origins of these membranes is diverse: the endoplasmic reticulum (ER), mitochondria, peroxisomes, and trans-Golgi membranes have been implicated in different viral systems (1, 8, 13, 23, 31, 38, 41, 45). This diversity implies that the processes involved in inducing the membrane vesicles/spherules are shared, rather than the composition of the membrane itself, although the exact purpose for utilizing membranes derived from different cellular compartments is still not completely resolved or understood.The replication of the flavivirus WNV_KUN is associated with the induction of morphologically distinct membrane structures that have defined roles during the WNV_KUN replication cycle. Three well-defined structures can be seen as large convoluted membranes (CM), paracrystalline arrays (PC), or membrane sacs containing small vesicles, termed vesicle packets (VP) (18, 20, 48). Based on localization studies with viral proteins of specific functions, we observed that components of the virus protease complex (namely, nonstructural protein 3 [NS3] with cofactor NS2B) localize specifically to the CM/PC, whereas viral double-stranded RNA (dsRNA) and the viral RNA-dependent RNA polymerase (RdRp) NS5 localized primarily to VP (20-22, 47, 48). Additionally, we observed that the CM and PC originate from and are modified membranes of the intermediate compartment (IC) and rough endoplasmic reticulum (RER), whereas the VP appear to be derived from trans-Golgi network (TGN) membranes (19). Recently, we have found that the WNV_KUN NS4A protein by itself has the intrinsic capacity to induce the CM and PC structures (35), a property also subsequently shown for Dengue virus (DENV) NS4A (29). Additionally, we have shown that upon WNV infection cellular cholesterol and cholesterol-synthesizing proteins are redistributed to the virus-induced membranes and that this redistribution severely disrupted the formation of cholesterol-rich microdomains (23). Furthermore, we have shown that the membranous structures induced during WNV replication provide partial protection of the WNV replication components from the interferon (IFN)-induced antiviral MxA protein, suggesting that the distinct compartmentalization of viral replication and components of the cellular antiviral response may be an evolutionary mechanism by which flaviviruses can protect themselves from host surveillance (6).In this study we focused on three-dimensional (3D) modeling to give insight into the 3D structure of the VP and provide evidence of how these complexes are organized and formed within the RER membrane. These results add valuable information to our understanding of how the WNV replication complex (RC) functions.     

Co-opted Oxysterol-Binding ORP and VAP Proteins Channel Sterols to RNA Virus Replication Sites via Membrane Contact Sites

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.

Authors Summary

Cellular proteins and cellular membranes are usurped by positive-stranded RNA viruses to assemble viral replicase complexes required for their replication. Tombusviruses, which are small RNA viruses of plants, depend on sterol-rich membranes for replication. The authors show that the tombusviral replication protein binds to cellular oxysterol-binding ORP proteins. Moreover, the endoplasmic reticulum resident cellular VAP proteins also co-localize with viral replication proteins. These protein interactions likely facilitate the formation of membrane contact sites that are visible in cells replicating tombusvirus RNA. The authors also show that sterols are recruited and enriched to the sites of viral replication. In vitro replication assay was used to show that sterols indeed stimulate tombusvirus replication. In summary, tombusviruses use subverted cellular proteins to build sterol-rich membrane microdomain to promote the assembly of the viral replicase complex. The paper connects efficient virus replication with cellular lipid transport and membrane structures.     

Hydrophobic Compounds Reshape Membrane Domains

Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.