Evidence of the involvement of membrane-bound steroids in the photoperiodic induction of flowering in Xanthium

Summary Filipin, which interferes with sterol stabilization of phospholipid membrane layers, inhibits the photoperiodic induction of flowering in Xantium; the inhibition is reversed to a large extent by cholesterol.We are thankful to Upjohn Co., Kalamazoo, Mich., USA, for the gift of a sample of filipin used in this investigation.     

Are shape and morphogenesis independent phenomena?

Summary Amphibian embryos can develop inspite of dramatic deformations produced by constraints in the surrounding medium, and can even ultimately recover a normal morphology. Consequently, no sequential change of shape is necessary for normal morphogenesis and the form of the embryo appears to be determined by cell activities intrinsic to each stage.I wish to thank Prof. A. Pisano for encouragement and Dr. J. Faber (Hubreacht Laboratory, Utrecht, Netherlands), for advice and editorial help. I am also grateful to A. M. Andreetta for skilful technical assistance. The electron microscopy work was done in the SEM unit of CONICET (Argentina) with the skilful technical assistance of N. De Vicenzo.     

Are shape and morphogenesis independent phenomena?

Amphibian embryos can develop inspite of dramatic deformations produced by constraints in the surrounding medium, and can even ultimately recover a normal morphology. Consequently, no sequential change of shape is necessary for normal morphogenesis and the form of the embryo appears to be determined by cell activities intrinsic to each stage.     

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.     

Use of 3H-QNB in the isolation of plasma membrane from smooth muscle of the urinary bladder: effect of oxalate on calcium uptake by the membrane fractions

Specific binding of tritiated quinuclidinyl benzilate (3H-QNB) to surface membrane muscarinic receptors was utilized to identify plasma membrane (PM) fractions from smooth muscle of the rabbit urinary bladder. Accumulation of 3H-QNB in the PM fraction was 4-5-fold higher than that in fractions of endoplasmic reticulum (EM) or mitochondria (M). A similar pattern of distribution was found for 5'-nucleotidase. 3H-QNB binding therefore appears to be a suitable marker for plasma membrane of the urinary bladder. Data on ATP-dependent calcium uptake by PM and ER fractions showed that oxalate highly potentiated calcium uptake by both fractions and consequently this feature cannot be used to identify ER fractions specifically.     

Kinetics of chemo-attraction of polymorphonuclear leukocytes towards N-formyl peptide studied with a novel polycarbonate (Nucleopore) membrane in the Boyden chamber

The motile responses of human polymorphonuclear leukocytes (PMN) to N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the Boyden chamber using a new sparse-pore polycarbonate membrane (pores 3 micron in diameter and occupying 0.1% of surface area) were compared with those demonstrated by using a standard polycarbonate (Nuclepore) filtration membrane (pores 3 micron in diameter and occupying 5% of surface area). Motility of PMN in gradients of FMLP using the new membrane was not influenced by chemokinetic effects of the factor, and the 'background' migration of the cells was minimal. However, motility of PMN in gradients of FMLP using the standard membrane was found to be influenced by chemokinetic effects of the chemotactic factor, and the 'background' or 'control' migration (in the absence of chemotactic factor) of the cells was substantial. Greater directional migration of PMN according to steepness of the gradient of chemotactic factor was demonstrated with the use of the new membrane. The new membrane may be of considerable value in the further study of the chemotactic responses of PMN.     

Changes in erythrocyte membrane lipid composition affect the transient decrease in membrane order which accompanies insulin receptor down-regulation.

We have recently demonstrated, using electron paramagnetic resonance (EPR) spectroscopy, that insulin receptor internalization in response to insulin incubation (down-regulation) in human erythrocytes is accompanied by a transient decrease in membrane order, as measured by the 2T' parallel order parameter. Since membrane lipids play such an important role in receptor internalization, we investigated the possible effects that an alteration of the normally-occurring lipid profile might have on down-regulation and the concomitant transient decrease in membrane order. Consequently, human erythrocytes enriched with cholesterol and erythrocytes from cirrhotic patients were examined, because both of these groups of cells have a higher cholesterol/phospholipid molar ratio (CH/PL) than controls. The 5-nitroxystearate spin label, which inserts into the lipid bilayer of cell membranes, was used to monitor changes in 2T' parallel for a 3-h period at 37 degrees C. We report here that both cholesterol-enriched and cirrhotic erythrocytes do not down-regulate, as demonstrated by binding assays, and that they do not show the typical transient decrease in membrane order observed in controls. The results seem to indicate that a more ordered membrane inhibits internalization of the insulin receptor in erythrocytes, and that an increase in membrane disorder is necessary for insulin receptor down-regulation.     

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P₂ relies on mechanisms ensuring transient appearance of PI(4,5)P₂ clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P₂ by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P₂ participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P₂ in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P₂ can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.     

The plasma membrane proton-translocating ATPase

Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.     

P-glycoprotein and ‘lipid rafts’: some ambiguous mutual relationships (floating on them, building them or meeting them by chance?)

P-glycoprotein (P-gp) is an active membrane transporter responsible for cell detoxification against numerous amphiphilic compounds, leading to multidrug resistance in tumor cells. It displays entangled connections with its membrane environment since it recognizes its substrates within the cytosolic leaflet and it also translocates some endogenous lipids to the exoplasmic leaflet. Regarding its relationships with membrane microdomains, ‘lipid rafts’, a literature analysis concludes that (i) P-gp also exists in rafts and non-raft membrane domains, depending on the cell considered, the experimental conditions and the method used to test it; (ii) cholesterol has a positive influence on P-gp function, and this may be a direct effect of the free cholesterol present in membrane or an indirect effect mediated by the cholesterol-enriched microdomains; (iii) when present in rafts, P-gp interacts with protein partners regulating its activity; (iv) P-gp is a lipid translocase that handles the raft-constituting lipids with particular efficiency, and it also influences membrane trafficking in the cell. Received 18 November 2005; received after revision 23 December 2005; accepted 12 January 2006     

Functions of intrinsic disorder in transmembrane proteins

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.     

Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C₅₅-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C₅₅-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.     

The biogenesis and function of eukaryotic porins.

Like most other mitochondrial proteins porin is synthesized in the cytosol and imported posttranslationally into the outer mitochondrial membrane. This transport follows the general rules for mitochondrial protein import with a few aberrations: a) porin contains an uncleaved NH2-terminal signal sequence, b) also its carboxyterminus might be involved in the import process, and c) this transport does not seem to require a membrane potential delta psi, although it is ATP-dependent. Most likely the actual import step occurs at contact sites between the outer and the inner mitochondrial membrane and involves at least one receptor protein. Although porin is known to be the major gate through the outer mitochondrial membrane, its absence only causes transient respiratory problems in yeast cells. This could mean a) that there is a bypass for some mitochondrial functions in the cytosol and/or b) that there are alternative channel proteins in the outer membrane. The first idea is supported by the overexpression of cytosolic virus-like particles in yeast cells lacking porin and the second by the occurrence of residual pore activity in mitochondrial outer membrane purified from porinless mutant cells.     

Tetraspanins and cell membrane tubular structures

Tetraspanins regulate a variety of cellular functions. However, the general cellular mechanisms by which tetraspanins regulate these functions remain poorly understood. In this article we collected the observations that tetraspanins regulate the formation and/or development of various tubular structures of cell membrane. Because tetraspanins and their associated proteins (1) are localized at the tubular structures, such as the microvilli, adhesion zipper, foot processes, and penetration peg, and/or (2) regulate the morphogenesis of these membrane tubular structures, tetraspanins probably modulate various cellular functions through these membrane tubular structures. Some tetraspanins inhibit membrane tubule formation and/or extension, while others promote them. We predict that tetraspanins regulate the formation and/or development of various membrane tubular structures: (1) microvilli or nanovilli at the plasma membranes free of cell and matrix contacts, (2) membrane tubules at the plasma membrane of cell-matrix and cell-cell interfaces, and (3) membrane tubules at the intracellular membrane compartments. These different membrane tubular structures likely share a common morphogenetic mechanism that involves tetraspanins. Tetraspanins probably regulate the morphogenesis of membrane tubular structures by altering (1) the biophysical properties of the cell membrane such as curvature and/or (2) the membrane connections of cytoskeleton. Since membrane tubular structures are associated with cell functions such as adhesion, migration, and intercellular communication, in all of which tetraspanins are involved, the differential effects of tetraspanins on membrane tubular structures likely lead to the functional difference of tetraspanins.     

Possible regulation of membrane-associated cyclic AMP phosphodiesterase in rat cerebral cortex by lipids.

Cyclic AMP phosphodiesterase (PDE) in membrane fraction from rat cerebral cortex was activated by Triton X-100, and treatment at alkaline pH and with phospholipase C. These results suggest that membrane PDE exists in a latent form and is influenced by microenvironmental changes within the membrane. Furthermore, the PDE, unlike soluble enzyme, is not influenced by a protein activator and Ca++.     

Possible regulation of membrane-associated cyclic AMP phosphodiesterase in rat cerebral cortex by lipids

Summary Cyclic AMP phosphodiesterase (PDE) in membrane fraction from rat cerebral cortex was activated by Triton X-100, and treatment at alkaline pH and with phospholipase C. These results suggest that membrane PDE exists in a latent form and is influenced by microenvironmental changes within the membrane. Furthermore, the PDE, unlike soluble enzyme, is not influenced by a protein activator and Ca⁺⁺.     

Membrane traffic in the secretory pathway

SNARE (SNAP receptor) proteins drive intracellular membrane fusion and contribute specificity to membrane trafficking. The formation of SNAREpins between membranes is spatially and temporally controlled by a network of sequentially acting accessory components. These regulators add an additional layer of specificity, arrest SNAREpin intermediates, lower the energy required for fusion, and couple membrane fusion to triggering signals. The functional activity of some of these regulators determines the plasticity of regulated exocytosis. (Part of a Multi-author Review)     

Hormone-induced subcellular redistribution of trimeric G proteins

Trimeric guanine nucleotide-binding proteins (G proteins) function as the key regulatory elements in a number of transmembrane signaling cascades where they convey information from agonist-activated receptors to effector molecules. The subcellular localization of G proteins is directly related to their functional role, i.e., the dominant portion of the cellular pool of G proteins resides in the plasma membrane. An intimate association of G protein subunits with the plasma membrane has been well known for a long time. However, results of a number of independent studies published in the past decade have indicated clearly that exposure of intact target cells to agonists results in subcellular redistribution of the cognate G proteins from plasma membranes to the light-vesicular membrane fractions, in internalization from the cell surface into the cell interior and in transfer from the membrane to the soluble cell fraction (high-speed supernatant), i.e., solubilization. Solubilization of G protein α subunits as a consequence of stimulation of G protein-coupled receptors (GPCRs) with agonists has also been observed in isolated membrane preparations. The membrane-cytosol shift of G proteins was detected even after direct activation of these proteins by non-hydrolyzable analogues of GTP or by cholera toxin-induced ADP-ribosylation. In addition, prolonged stimulation of GPCRs with agonists has been shown to lead to down-regulation of the relevant G proteins. Together, these data suggest that G proteins might potentially participate in a highly complex set of events, which are generally termed desensitization of the hormone response. Internalization, subcellular redistribution, solubilization, and down-regulation of trimeric G proteins may thus provide an additional means (i.e., beside receptor-based mechanisms) to dampen the hormone or neurotransmitter response after sustained (long-term) exposure. Received 31 August 2001; received after revision 31 October 2001; accepted 7 November 2001     

Ca<Superscript>2+</Superscript> signals,cell membrane disintegration,and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca²⁺. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNFα, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT₂₉, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca²⁺, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca²⁺, which was paralleled by the activation of outwardly rectifying Cl^? currents, which were typical for TMEM16F/ANO6. Ca²⁺ oscillations were due to Ca²⁺ release from endoplasmic reticulum, and were independent of extracellular Ca²⁺. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl^? currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death.     

Pathophysiology of mitochondrial cell death control

Mitochondria have been recently recognized to play a major role in the control of apoptosis or programmed cell death. Permeabilization of mitochondrial membranes, a decisive feature of early cell death, is regulated by members of the Bcl-2 family which interact with the permeability transition pore complex (PTPC). Thus, the cytoprotective oncoprotein Bcl-2 stabilizes the mitochondrial membrane barrier function, whereas the tumor suppressor protein Bax permeabilizes mitochondrial membranes. The regulation of membrane permeabilization is intertwined with that of the bioenergetic and redox functions of mitochondria. The implications of alterations in the composition of the PTPC and in mitochondrial function for the pathophysiology of cancer (reduced apoptosis) and neurodegeneration (enhanced apoptosis) are discussed.