Poly(DL-proline), a synthetic polypeptide behaving as an ion channel across bilayer membranes

The synthesis and characterization of poly(DL-proline) are reported in relation with its predicted property of forming ion channels across membranes. The analysis of the conductance induced in synthetic bilayer membranes doped with poly(DL-proline) shows ionic permeoselectivity and the characteristic time course of fluctuations of ion channels, according to the similarity with the active structure of gramicidin A in membranes during the ion passage. An alternative mechanism of ion transport across bilayer membranes is also advanced.     

Ca2+ transport mediated by a synthetic neutral Ca2+ -ionophore in biological membranes.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

Engineering the bilayer: Emerging genetic tool kits for mechanistic lipid biology

The structural diversity of lipids underpins the biophysical properties of cellular membranes, which vary across all scales of biological organization. Because lipid composition results from complex metabolic and transport pathways, its experimental control has been a major goal of mechanistic membrane biology. Here, we argue that in the wake of synthetic biology, similar metabolic engineering strategies can be applied to control the composition, physicochemical properties, and function of cell membranes. In one emerging area, titratable expression platforms allow for specific and genome-wide alterations in lipid biosynthetic genes, providing analog control over lipidome stoichiometry in membranes. Simultaneously, heterologous expression of biosynthetic genes and pathways has allowed for gain-of-function experiments with diverse lipids in non-native systems. Finally, we highlight future directions for tool development, including recently discovered lipid transport pathways to intracellular lipid pools. Further tool development providing synthetic control of membrane properties can allow biologists to untangle membrane lipid structure-associated functions.     

Ca transport mediated by a synthetic neutral Ca-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

The structures of BtuCD and MscS and their implications for transporter and channel function

The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch-activated mechanosensitive channel of small conductance (MscS) and the ATP-dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.     

Comparison of the effects of dDAVP and AVP on the sodium transport in the frog skin

The effect of 1-deamino-8-D-arginine-vasopressin, dDAVP, the synthetic analogue of vasopressin, upon the active sodium transport across the frog skin was studied using standard microelectrode technique and compared with the effect of synthetic arginine-vasopressin, AVP. dDAVP applied to the basolateral side of the epithelium stimulated the active sodium transport as reflected by the increase of short-circuit current, Isc, and transepithelial electrical potential difference, Voc. Potential difference across both the apical, Vo, and the basolateral, Vi, cell membranes decreased. The driving force of transepithelial sodium transport, ENa, did not change. The transepithelial electrical resistance, Rt, ohmic resistance of the active sodium transport, RNa, and apical cell membrane resistance, Ro, rapidly decreased, while the resistance of the basolateral cell membrane, Ri, and the resistance of the shunt pathway, Rs, remained unchanged. It is concluded that dDAVP primarily increases sodium permeability of the apical cell membrane which subsequently stimulates sodium pump activity. This action is similar to that of AVP.     

Isolation of silicate ionophore(s) from the apochlorotic diatom Nitzschia alba

An organic extract of Nitzschia alba cells possesses ionophoretic activity towards silicate, as it induces silicate transport across an organic phase or across synthetic lipid membranes. The activity is dependent upon Na+ and prefers silicon to germanium, a congener. The activity can be resolved into two apparently pure fractions by a combination of high performance liquid chromatography and thin-layer chromatography. Preliminary characterization indicates that the compound(s) contains vicinal hydroxyl groups but is devoid of amino, sugar or phosphate groups.     

Ca2+ transport mediated by a synthetic neutral Ca2+-ionophore in biological membranes

The effect of a synthetic neutral ligand on the Ca²⁺ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca²⁺-ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca²⁺ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate or carbonylcyanide $\text{p-}\text{trifluoromethoxyphenylhydrazone}$ (FCCP) are present, presumably to facilitate the diffusion of the charged Ca²⁺-ionophore complex across the hydrophobic core of the membrane.In mitochondria, the neutral ionophore promotes the active transport of Ca²⁺ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.     

The yeast CLC chloride channel is proteolytically processed by the furin-like protease Kex2p in the first extracellular loop

CLC chloride channels are a family of channel proteins mediating chloride transport across the plasma membrane and intracellular membranes. The single yeast CLC protein Gef1p is localized to the Golgi and endosomal system. Investigating epitope-tagged variants of Gef1p, we found that the channel is proteolytically processed in the secretory pathway. Proteolytic cleavage occurs in the first extracellular loop of the protein at residues KR136/137 and is carried out by the Kex2p protease. Fragments mimicking the N- and C-terminal products of the cleavage reaction are non-functional when expressed alone. However, functional channels can assemble when the two fragments are co-expressed.     

Lipid Phase Fatty Acid Flip-Flop, Is It Fast Enough for Cellular Transport?

The mechanism by which fatty acids are transported across cell membranes is controversial. The essence of the controversy is whether transport requires membrane protein mediation or whether the membrane's lipid phase provides a pathway so rapid that a protein is not needed. This review focuses on the mechanisms of fatty acid transport across lipid bilayer membranes. These results for lipid membranes are used to help evaluate transport across cell membranes. Within the context of this analysis, a lipid phase mediated process is consistent with results for the transport of fatty acids across erythrocytes but provides a less adequate explanation for fatty acid transport across more complex cells. Received: 16 June 1999/Revised: 21 January 2000     

Biomimetic polyesters and their role in ion transport across cell membranes

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.     

The translocation of Ca2+ across phospholipid bilayers induced by a synthetic neutral Ca2+ -ionophore.

The effect of a neutral synthetic Ca2+ -ligand, which induces selective Ca2+ transport in electrodialysis experiments in bulk membranes, on the Ca2+ permeability of phospholipid bilayers has been investigated. The ligand is able to promote the transport of Ca2+ across synthetic phospholipid bilayers and can therefore be classified as a Ca2+ -ionophore. Its activity is enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The efficiency of the neutral carrier-mediated Ca2+ transport is rather low as compared with that of the charged Ca2+ -ionophore X537A. The Ca2+ selectivity of the nuetral ionophore is decreased by its incorporation in the low dielectric ambient of the phospholipid bilayer.     

Protein translocation across membranes.

Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.     

Permeability properties of unilamellar vesicles containing choline plasmalogens and comparison with other choline glycerophospholipid species

The rates of non-electrolyte and ion diffusion across bilayer membranes consisting of choline plasmologens or of their alkyl and acyl analogs were studied. The influx of [¹⁴C]glucose, ⁸⁶Rb⁺ and ³⁶Cl^? into small unilamellar vesicles made from a semisynthetic choline plasmalogen and from synthetic diacyl, alkylacyl and dialkyl analogs with comparable side chain compositions were measured. Rates of glucose and Rb⁺ diffusion are about equal in alkenylacyl- and diacyl-glycerophosphocholine (GPC) bilayers, but are reduced in dialkyl-GPC membranes; the permeability coefficients correlate with the packing densities of the respective choline glycerophospholipids in monolayers at the air water interface. Rates of chloride diffusion are consistently higher in membranes formed from phospholipids containing alkenyl or alkyl other bonds as compared to the diacyl analogs. Highest rates of Cl^? diffusion are observed with choline plasmalogen vesicles. The phospholipid side chain composition has little influence on Cl^? permeation, but glucose and Rb⁺ diffusion are markedly affected. Incorporation of cholesterol (30 mol%) into choline plasmalogen membranes reduces their solute permeability by approximately 70%. A similar effect is found with the other choline phospholipid analogs. Thus, the choline phospholipid—cholesterol interaction, as far as it is reflected in reduced bilayer permeability, is not influenced by the presence of the alkenylether bond of plasmalogens.     

Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.     

Protein Targeting into Secondary Plastids1

ABSTRACT. Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus‐encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus‐encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre‐existing ones to new requirements in secondary plastids.     

Fluctuation of surface charge in membrane pores

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3'-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.     

Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B₁₂ across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.