Pathways of proton transfer in the light-driven pump bacteriorhodopsin

The mechanism of proton transport in the light-driven pump bacteriorhodopsin is beginning to be understood. Light causes the all-trans to 13-cis isomerization of the retinal chromophore. This sets off a sequential and directed series of transient decreases in the pK_a's of a) the retinal Schiff base, b) an extracellular proton release complex which includes asp-85, and c) a cytoplasmic proton uptake complex which includes asp-96. The timing of these pK changes during the photoreaction cycle causes sequential proton transfers which result in the net movement of a proton across the protein, from the cytoplasmic to the extracellular surface.     

The bacterial translocase: a dynamic protein channel complex

The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.Received 10 January 2003; received after revision 2 April 2003; accepted 4 April 2003     

Reprogramming the metabolome rescues retinal degeneration

Metabolomics studies in the context of ophthalmology have largely focused on identifying metabolite concentrations that characterize specific retinal diseases. Studies involving mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have shown that individuals suffering from retinal diseases exhibit metabolic profiles that markedly differ from those of control individuals, supporting the notion that metabolites may serve as easily identifiable biomarkers for specific conditions. An emerging branch of metabolomics resulting from biomarker studies, however, involves the study of retinal metabolic dysfunction as causes of degeneration. Recent publications have identified a number of metabolic processes—including but not limited to glucose and oxygen metabolism—that, when perturbed, play a role in the degeneration of photoreceptor cells. As a result, such studies have led to further research elucidating methods for prolonging photoreceptor survival in an effort to halt degeneration in its early stages. This review will explore the ways in which metabolomics has deepened our understanding of the causes of retinal degeneration and discuss how metabolomics can be used to prevent retinal degeneration from progressing to its later disease stages.     

Amyloid precursor protein and its homologues: a family of proteolysis-dependent receptors

The Alzheimer’s amyloid precursor protein (APP) belongs to a conserved gene family that also includes the mammalian APLP1 and APLP2, the Drosophila APPL, and the C. elegans APL-1. The biological function of APP is still not fully clear. However, it is known that the APP family proteins have redundant and partly overlapping functions, which demonstrates the importance of studying all APP family members to gain a more complete picture. When APP was first cloned, it was speculated that it could function as a receptor. This theory has been further substantiated by studies showing that APP and its homologues bind both extracellular ligands and intracellular adaptor proteins. The APP family proteins undergo regulated intramembrane proteolysis (RIP), generating secreted and cytoplasmic fragments that have been ascribed different functions. In this review, we will discuss the APP family with focus on biological functions, binding partners, and regulated processing.     

Biochemical events associated with rapid cellular damage during the oxygen- and calcium-paradoxes of the mammalian heart

The O2- and Ca2(+)-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e- and also oxygen radicals or redox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised conditions, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2(+)-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2(+)-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Xanthorhodopsin: Proton pump with a carotenoid antenna

Retinal proteins function as photoreceptors and ion pumps. Xanthorhodopsin of Salinibacter ruber is a recent addition to this diverse family. Its novel and distinctive feature is a second chromophore, a carotenoid, which serves as light-harvesting antenna. Here we discuss the properties of this carotenoid/retinal complex most relevant to its function (such as the specific binding site controlled by the retinal) and its relationship to other retinal proteins (bacteriorhodopsin, archaerhodopsin, proteorhodopsin and retinal photoreceptors of archaea and eukaryotes). Antenna addition to a retinal protein has not been observed among the archaea and emerged in bacteria apparently in response to environmental conditions where light-harvesting becomes a limiting factor in retinal protein functioning. Received 2 April 2007; received after revision 14 May 2007; accepted 16 May 2007     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP

LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.     

Sulfatide-tenascin interaction mediates binding to the extracellular matrix and endocytic uptake of liposomes in glioma cells

Tenascin-C is an extracellular matrix glycoprotein, whose expression is highly restricted in normal adult tissues, but markedly up-regulated in a range of tumors, and therefore serves as a potential receptor for targeted anticancer drug or gene delivery. We describe here a liposomal carrier system in which the targeting ligand is sulfatide. Experiments with tenascin-C-expressing glioma cells demonstrated that binding of liposomes to the extracellular matrix relied essentially on the sulfatide-tenascin-C interaction. Following binding to the extracellular matrix, the sulfatide-containing liposomes were internalized via both caveolae/lipid raft- and clathrin-dependent pathways, which would ensure direct cytoplasmic release of the cargoes carried in the liposomes. Such natural lipid-guided intracellular delivery targeting at the extracellular matrix glycoproteins of tumor cells thus opens a new direction for development of more effective anticancer chemotherapeutics in future. K. Shao & Q. Hou: These authors contributed equally to this work. Received 22 September 2006; received after revision 5 December 2006; accepted 9 January 2007     

Myelin architecture: zippering membranes tightly together

Rapid nerve conduction requires the coating of axons by a tightly packed multilayered myelin membrane. In the central nervous system, myelin is formed from cellular processes that extend from oligodendrocytes and wrap in a spiral fashion around an axon, resulting in the close apposition of adjacent myelin membrane bilayers. In this review, we discuss the physical principles underlying the zippering of the plasma membrane of oligodendrocytes at the cytoplasmic and extracellular leaflet. We propose that the interaction of the myelin basic protein with the cytoplasmic leaflet of the myelin bilayer triggers its polymerization into a fibrous network that drives membrane zippering and protein extrusion. In contrast, the adhesion of the extracellular surfaces of myelin requires the down-regulation of repulsive components of the glycocalyx, in order to uncover weak and unspecific attractive forces that bring the extracellular surfaces into close contact. Unveiling the mechanisms of myelin membrane assembly at the cytoplasmic and extracelluar sites may help to understand how the myelin bilayers are disrupted and destabilized in the different demyelinating diseases.     

The ATP synthase (F0-F1) complex in oxidative phosphorylation.

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P(i). The bacterial and mitochondrial H(+)-ATP synthases both consist of a membranous sector, F0, which forms a H(+)-channel, and an extramembranous sector, F1, which is responsible for catalysis. When detached from the membrane, the purified F1 sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H(+)-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Transport of a low molecular weight extracellular esterase into membrane vesicles of Candida lipolytica

The low mol. wt extracellular esterase of Candida lipolytica is actively transported into membrane vesicles. In the absence of metabolic energy, a proton gradient can drive the transport process. The transport system does not accumulate the enzyme at peak levels due to the presence of a leak pathway.     

Nitrate and nitrite transport in bacteria

The topological arrangements of nitrate and nitrite reductases in bacteria necessitate the synthesis of transporter proteins that carry the nitrogen oxyanions across the cytoplasmic membrane. For assimilation of nitrate (and nitrite) there are two types of uptake system known: ABC transporters that are driven by ATP hydrolysis, and secondary transporters reliant on a proton motive force. Proteins homologous to the latter type of transporter are also involved in nitrate and nitrite transport in dissimilatory processes such as denitrification. These proteins belong to the NarK family, which is a branch of the Major Facilitator Superfamily. The mechanism and substrate specificity of transport via these proteins is unknown, but is discussed in the light of sequence analysis of members of the NarK family. A hypothesis for nitrate and nitrite transport is proposed based on the finding that there are two distinct types of NarK.     

Transport of a low molecular weight extracellular esterase into membrane vesicles ofCandida lipolytica

Summary The low mol. wt extracellular esterase ofCandida lipolytica is actively transported into membrane vesicles. In the absence of metabolic energy, a proton gradient can drive the transport process. The transport system does not accumulate the enzyme at peak levels due to the presence of a leak pathway.     

Biochemical events associated with rapid cellular damage during the oxygen-and calcium-paradoxes of the mammalian heart

Summary The O_2– and Ca²⁺-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H⁺ and e^– and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca²⁺ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca²⁺ or by raising [Ca²⁺]_i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca²⁺-paradox by the membrane perturbation associated with removal of extracellular Ca²⁺; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca²⁺]_i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca²⁺-paradox which continues under anoxia. Massive entry of Ca²⁺ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Functional, biochemical and genetic diversity of prokaryotic nitrate reductases

Prokaryotic nitrate reduction can serve a number of physiological roles and can be catalysed by a number of biochemically distinct nitrate reductases. Three distinct nitrate reductase classes can be indentified in prokaryotes, NAS, NAR and NAP. NAS is located in the cytoplasmic compartment and participates in nitrogen assimilation. NAR is usually a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment and is involved in anaerobic nitrate respiration. NAP is a two-subunit complex, located in the periplasmic compartment, that is coupled to quinol oxidation via a membrane anchored tetraheme cytochrome. It shows considerable functional flexibility by participating in anaerobic respiration or redox energy dissipation depending on the organism in which it is found. The members of all three classes of enzymes bind the bis-molybdopterin guanine dinucleotide cofactor at the active site, but they differ markedly in the number and nature of cofactors used to transfer electrons to this site. Analysis of prokaryotic genome sequences available at the time of writing reveals that the different nitrate reductases are phylogenetically widespread.     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.     

Lecticans: organizers of the brain extracellular matrix

Lecticans are a family of chondroitin sulfate proteoglycans, encompassing aggrecan, versican, neurocan and brevican. These proteoglycans are characterized by the presence of ahyaluronan-binding domain and a C-type lectin domain in their core proteins. Through these domains, lecticans interact with carbohydrate and protein ligands in the extracellular matrix and act as linkers of these extracellular matrix molecules. In adult brain, lecticans are thought to interact with hyaluronan and tenascin-R to form a ternary complex. We propose that the hyaluronan-lectican-tenascin-R complex constitutes the core assembly of the adult brain extracellular matrix, which is found mainly in pericellular spaces of neurons as ‘perineuronal nets’. Received 27 September 1999; accepted 26 October 1999