Class Of Integral Membrane Proteins Research Articles

Tight junction proteins: a novel class of integral membrane proteins. Expression in human epidermis and in HaCaT keratinocytes.

Tight junction proteins comprise a novel group of integral membrane proteins necessary for cell-to-cell contacts and responsible for the barrier function in epithelial and endothelial cells in various tissues. The tight junction membrane domain contains at least three distinct proteins, named occludin, claudin and junctional adhesion molecule. Claudins are products of a gene family consisting of more than 20 members. We investigated mRNA expression of occludin and 13 different claudins in neonatal foreskin, adult skin and cultivated HaCaT keratinocytes by the Northern blot technique, and performed immunohistochemical staining of adult skin for occludin, claudin 1 and claudin 2. Occludin, claudin 1 and claudin 3 mRNAs were expressed in human neonatal and adult keratinocytes as well as in HaCaT keratinocytes. All other tested claudins were negative. Immunohistochemical staining of adult skin was positive for occludin in the intercellular space of the granular layer, and for claudin 1 in the inter-cellular space of the spinosum layer and basal layer, but negative for claudin 2 in all skin layers. Claudin 1 was also positive in the outer root sheath of hair follicles. Our results indicate that occludin, claudin 1 and claudin 3 are involved in cell-to-cell contacts between keratinocytes in human epidermis, although their functional importance remains unknown.

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.

Targeting of C-terminal (tail)-anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes.

A class of integral membrane proteins, referred to as 'tail-anchored proteins', are inserted into phospholipid bilayers via a single segment of hydrophobic amino acids at the C-terminus, thereby displaying a large functional domain in the cytosol. This membrane attachment strategy allows eukaryotic cells to position a wide range of cytoplasmic activities close to the surface of an intracellular membrane. Tail-anchored proteins often, but not always, demonstrate a selective distribution to specific intracellular organelles. This membrane-specific distribution is required for the large number of targeting proteins that are tail-anchored, but may or may not be critical for the numerous tail-anchored pro-apoptotic and anti-apoptotic proteins of the Bcl-2 family. Recent work has begun to address the mechanism for targeting tail-anchored proteins to their resident membranes, but questions remain. What targeting signals determine each protein's intracellular location? Are there receptors for these signals and, if so, how do they function? What steps are required to integrate tail-anchored proteins into the phospholipid bilayers? In this Traffic interchange, we summarise what is known about tail-anchored proteins, and outline the areas that are currently under study.

Recent advances in the understanding of membrane protein assembly and structure.

1. Introduction 2862. Membrane protein assembly inE. coli2862.1. Role of the SRP 2872.2. YidC – a translocon component devoted to membrane proteins? 2872.3. The TAT pathway 2882.4. ‘Spontaneous’ membrane protein insertion 2883. Membrane protein assembly in the ER 2893.1. How TM segments exit the translocon 2893.2. Proteins with multiple topologies 2903.3. Stop-transfer effector sequences 2913.4. Non-hydrophobic TM segments? 2913.5. ‘Frustrated’ topologies 2913.6. N-tail translocation across the ER 2924. Membrane protein assembly in mitochondria 2924.1. The Oxa1p pathway 2924.2. The TIM22/54 pathway 2935. Evolution of membrane protein topology 2935.1. RnfA/RnfE – two homologous proteins with opposite topologies 2935.2. YrbG – duplicating an odd number of TMs 2946. Genome-wide analysis of membrane proteins 2956.1. Prediction methods 2956.2. How many membrane proteins are there? 2956.3. The positive-inside rule 2966.4. Dominant classes of membrane proteins 2967. The structure of transmembrane α-helices 2967.1. What TM helices look like 2977.2. The ‘helical hairpin’ 2977.3. Prolines in TM helices 2977.4. Charged residues in TM helices: the ‘snorkel’ effect 2987.5. The ‘aromatic belt’ 2988. Helix–helix packing in a membrane environment 2988.1. Lessons learnt from glycophorin A 2988.2. Genetic screens for helix–helix interactions 2998.3. Statistical studies 2998.4. Membrane protein folding 2999. Recent 3D structures 3009.1. KcsA – the first ion channel 3009.2. MscL – sensing lateral pressure changes 3009.3. The cytochrome bc 1 complex 3009.4. Fumarate reductase 3019.5. Bacteriorhodopsin – watching a membrane protein at work 30110. Concluding remarks 30111. Acknowledgements 30212. References 302For a variety of reasons – not the least biomedical importance – integral membrane proteins are now very much in focus in many areas of molecular biology, biochemistry, biophysics, and cell biology. Our understanding of the basic processes of membrane protein assembly, folding, and structure has grown significantly in recent times, both as a result of new methodological developments, more high-resolution structure data, and the possibility to analyze membrane proteins on a genome-wide scale.So what is new in the membrane protein field? Various aspects of membrane protein assembly and structure have been reviewed over the past few years (Cowan & Rosenbusch, 1994; Hegde & Lingappa, 1997; Lanyi, 1997; von Heijne, 1997; Bernstein, 1998); here, I will try to bring together a number of exciting recent developments. Particularly noteworthy are the discoveries related to the mechanisms of membrane protein assembly into the inner membrane of E. coli, the inner membrane of mitochondria, and the way transmembrane segments are handled by the ER translocon.Other advances include detailed studies of the interaction between transmembrane helices and the lipid bilayer, and of helix–helix packing interactions in the membrane environment. The availability of full genomic sequences have made it possible to study membrane proteins on a genome-wide scale. Finally, a handful of new high-resolution 3D structures have appeared.This review will deal only with helix bundle proteins, i.e. integral membrane proteins where the transmembrane segments form α-helices. For reviews on the other major class of integral membrane proteins – the β-barrel proteins – see Schirmer (1998) and Buchanan (1999). For readers who prefer a more ‘literary’ introduction to the membrane protein field, may I suggest von Heijne (1999).

Carboxyl-terminal Targeting and Novel Post-translational Processing of JAW1, a Lymphoid Protein of the Endoplasmic Reticulum

Jaw1 is a lymphoid-restricted protein localized to the cytoplasmic face of the endoplasmic reticulum (ER) and is a member of a recently recognized class of integral membrane proteins that contain carboxyl-terminal membrane anchors. The carboxyl-terminal 71 amino acids of the Jaw1 protein, which contain a hydrophobic membrane spanning region, are sufficient to target a heterologous protein to the ER. By discontinuous sucrose gradient ultracentrifugation, differential sedimentation was noted for the four major Jaw1 protein isoforms, with two of the forms predominantly soluble and two microsome-bound. Pulse-chase immunoprecipitations suggest a post-translational modification of two major isoforms of the protein resulting in an increase in mobility on SDS-polyacrylamide gel electrophoresis. In vitro translation studies are compatible with a post-translational processing event that results in cleavage of a short 36 amino acid lumenal domain. These findings define a carboxyl-terminal domain of the Jaw1 protein that is both necessary and sufficient for ER localization. In addition, the processing of the small lumenal domain of Jaw1 represents a novel post-translational protein modification performed by the endoplasmic reticulum.

Bacterial porins: structure and function

Within the class of integral membrane proteins, the bacterial porins display a remarkable resistance to denaturants and proteases. This stability is probably crucial for the formation of highly ordered, three-dimensional crystals. Structural analysis of these crystals has been possible in atomic detail. This analysis has revealed interesting features, such as the aromatic girdles, and has helped to explain several observations, including the porins' ability to discriminate between polar and non-polar solutes. Recent research has thus improved our understanding of the porins in a qualitative fashion.

Relaxation kinetics of the Na+/glucose cotransporter.

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.

32] Surface charge effects on ion conduction in ion channels

Publisher Summary Ion channels belong to a class of integral membrane proteins, which when confronted with a stimulus, are able to change their conformation from a nonconductive to an ion-conductive one or vice versa. This chapter describes the surface charge effects on ion conduction in ion channels. A charged surface in the neighborhood of the ion conduction system influences the concentration of ions at the channel entrances and hence, its conductance. In biological membranes, fixed charges can originate in principle from three different sources: (1) the membrane lipids, (2) ionized groups in the channel-forming protein, and (3) nonprotein domains forming part of the molecular structure of the channel, such as acidic carbohydrate domains containing sialic acid. The most abundant charged phospholipid in biological membranes is phosphatidylserine (PS), which possesses a single negative charge from the carboxyl group in the serine residue of the lipid. Channel blockade by ions can be used to obtain an independent estimation of the surface charge of the vestibules.

Ion Channels in Paramecium, Yeast, and Escherichia coli

Ion channels in animal and plant cells transduce chemical, mechanical or other stimuli into ionic and electric information. The discovery of ion channels in microbes indicates an early origin for these transducers and the search is now on for their physiological roles. Ion channels and their regulation Animal behavior is largely governed by biological electricity. Ions, the carriers of electric currents, cannot pass through the hydrophobic lipid bilayer of the membrane. Instead they go through a special class of integral membrane proteins called ion channels. Whe~l open, channels allow ions to pass passively along the electrochemical gradients and therefore dissipate the gradients which are built with energy expenditure by ion pumps, a different class of membrane proteins. Because open channels drain energy, the channels are 'gated', and the gates are usually closed. The change of the channel protein to the open conformation depends on specific signals. Thus ion channels are switches that transduce signals into electric or ionic information, using the energy stored in the form of ion gradients. More than 60 ion channels are now recognized ~ and these can be classified by the signals that gate them. Some are gated by external ligands (e.g. the nicotinic acetylcholine receptor2), by internal ligands (e.g. the cGMP-gated Na + channel in phototransduction 3) or by other membrane proteins (e.g. the Gprotein-gated Ca 2+ channel4). Other channels are gated by transmembrane voltage (e.g. the depolarization-gated Na + channel of nerves 5) or mechanical forces (e.g. the ion channels 6 of the hair cells in the inner ear). Ion channels underlie smelling, tasting, hearing and vision, as well as responses to hormones and neurotransmitters.

On the insertion of proteins into membranes

Recent data concerning the primary structure and the interactions of proteins with membranes suggest the existence of two classes of integral membrane proteins. In the first class, the polypeptide chain crosses the membrane only once. The membrane penetrating fragment is markedly hydrophobic and contains several positive charges on its C-terminal border. In the second class, the protein is folded in a complex fashion within the membrane and the knowledge of its amino acid sequence is not sufficient to predict the manner in which the protein interacts with the membrane.

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.