This chapter reviews some characteristic features of membrane fusion activity for each virus and discusses the mechanisms of membrane fusion, especially low pH-induced membrane fusion. It concentrates on the interaction of the hydrophobic segment with the target cell membrane lipid bilayer and suggests the entrance of the segment into the lipid bilayer hydrophobic core as a key step in fusion. The envelope is a lipid bilayer membrane with the virus specific glycoproteins spanning it. The bilayer originates from the host cell membrane and has a lipid composition and transbilayer distribution quite similar to the host's. The viral glycoproteins have the functions of binding to the target cell surface and fusion with the cell membranes. The two functions are carried by a single glycoprotein in influenza virus (HA), vesicular stomatitis virus (VSV) G glycoprotein, and Semliki Forest virus SFV E glycoprotein. In Sendai virus (HVJ), the functions are carried by separate glycoproteins, hemagglutinin-neuraminidase (HN) for binding and fusion glycoprotein (F) for fusion. When viruses encounter target cells, they first bind to the cell surface through an interaction of the viral glycoprotein with receptors.
This chapter focuses on the recent information of the glycoprotein components of enveloped viruses and points out specific findings on viral envelopes. Although enveloped viruses of different major groups vary in size and shape, as well as in the molecular weight of their structural polypeptides, there are general similarities in the types of polypeptide components present in virions. The types of structural components found in viral membranes are summarized briefly in the chapter. All the enveloped viruses studied to date possess one or more glycoprotein species and lipid as a major structural component. The presence of carbohydrate covalently linked to proteins is demonstrated by the incorporation of a radioactive precursor, such as glucosamine or fucose, into viral polypeptides, which is resolved by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Enveloped viruses share many common features in the organization of their structural components, as indicated by several approaches, including electron microscopy, surface-labeling, and proteolytic digestion experiments, and the isolation of subviral components. The chapter summarizes the detailed structure of the glycoproteins of four virus groups: (1) influenza virus glycoproteins, (2) rhabdovirus G protein, (3) togavirus glycoprotein, and (4) paramyxovirus glycoproteins The information obtained includes the size and shape of viral glycoproteins, the number of polypeptide chains in the complete glycoprotein structure, and compositional data on the polypeptide and oligosaccharide portions of the molecules.
This chapter focuses on protein sorting in the secretory pathway. From primary and secondary biosynthetic sites in the cytosol and mitochondrial matrix, respectively, proteins and lipids are distributed to more than 30 final destinations in membranes or membrane-bound spaces, where they carry out their programmed function. Molecular sorting is defined, in its most general sense, as the sum of the mechanisms that determine the distribution of a given molecule from its site of synthesis to its site of function in the cell. The final site of residence of a protein in a eukaryotic cell is determined by a combination of various factors, acting in concert: (1) site of synthesis, (2) sorting signals or zip codes, (3) signal recognition or decoding mechanisms, (4) cotranslational or posttranslational mechanisms for translocation across membranes, (5) specific fusion-fission interactions between intracellular vesicular compartments, and (6) restrictions to the lateral mobility in the plane of the bilayer. Improvements in cell fractionation, protein separation, and immune precipitation procedures in the past decade have made them possible. Very little is known about the mechanisms that mediate the localization and concentration of specific proteins and lipids within organelles. Various experimental model systems have become available for their study. The advent of recombinant DNA technology has shortened the time needed for obtaining the primary structure of proteins to a few months.
Publisher Summary Protozoans, yeast, and even bacteria all have one class of portals, the ion channels. It, therefore, appears that all cellular forms of life have ion channels. Ion channels are gated pores. A certain stimulus can increase the probability of a given channel being open. Such a stimulus (gating principle) can be an external ligand (e.g., acetylcholine for the nicotinic acetylcholine receptor or channel), an internal second messenger (e.g., Ca 2+ for Ca 2+ -gated K + channel, cyclic guanosine monophosphate (cGMP) for the cGMP-gated channel in the rod outer segment), or cross-membrane voltage. These classes of channels have been extensively studied and reviewed. Other ion channels have subsequently been found to be gated by guanosine-5'-triphosphate (GTP)-binding proteins or by arachidonic acid. Last, but not least, is a class of channels that are gated by mechanical forces in the membrane. The activities of the last type of channels, those gated by mechanical forces, have been studied in the hair cells of the inner ear and in ciliated protozoans. The activities of individual stretch-activated channels were first demonstrated in chick skeletal muscle with a patch clamp. Since then, stretch-activated channels have been found in neurons, endothelial cells, blood cells, eggs, cultured plant cells, and guard cells, through patch-clamp examinations.
This chapter focuses on protein translocation in yeast. The yeast Saccharomyces cerevisiae carries out secretion and membrane biogenesis in a fashion analogous to mammalian cells and possesses all the relevant organelles: rough endoplasmic reticulum (ER), golgi apparatus, and secretory vesicles. It has a well-characterized, easily manipulated genome, and a convenient methodology exists for the isolation of genes of interest. The combination of in vivo and genetic analyses of yeast translocation mutants with the in vitro analysis of these mutants, using the yeast cell-free translocation assay, represents a powerful system for characterizing both membrane and cytosolic components involved in translocation. Several studies have examined the effect of precursor folding on translocation. Mitochondria1 membranes, which are able to translocate even large, branched molecules, show dramatically reduced translocation efficiencies when the preprotein is artificially held in a folded conformation. The use of denatured precursor proteins enhanced the efficiency of posttranslational translocation across mitochondrial, bacterial, and yeast ER membranes in vitro.
This chapter focuses on how proteins assemble into, or across, biological membranes––namely, mitochondria, endoplasmic reticulum, chloroplasts, and the bacterial cell surface. Microorganisms offer several major advantages for the study of this fundamental process: they have advanced genetics, can readily be grown in large culture for biochemistry, and their growth on minimal medium allows isotopic labeling for in vivo studies. Their relative roles in protein export require a genetic analysis; this should be facilitated by the recent isolation of the trigger factor gene. It is important to determine the basis of specificity of these and other chaperone proteins. Membrane vesicles have been solubilized in detergent and reconstituted to form proteoliposomes that support translocation ATPase. The detergent extract contains solubilized SecY protein, and this SecY is required for reconstitution.
Publisher Summary Neither cyclic adenosine monophosphate (cAMP) nor cyclic guanosine monophosphate (cGMP) are involved as “second messengers” in the antigen receptor-mediated initiation of lymphocyte activation. Increases in cAMP after mitogenic activation of T cells result from the additional agglutinating effects of multivalent lectins, whereas divalent ligands induce early cellular events and competence for the action of cytokines without cAMP elevation. Dramatic rises in cGMP levels within minutes after the addition of plant lectins or monoclonal antibodies directed against surface molecules to T cells have been reported, but these findings could not be reproduced by all investigators. The concept of cGMP being a positive activation signal in the early phase of lymphocyte activation is also questionable, because intracellular rises of cGMP through specific activation of the guanylate cyclase do not influence basal or mitogen-induced RNA synthesis. Only few data concerning changes in cyclic nucleotide levels after binding of interleukins to their newly expressed receptors in the late phase of lymphocyte activation have been reported. Further experiments will be necessary for clarifying the significance of these findings.
Publisher Summary The studies in this chapter suggest that there are multiple mechanisms for raising intracellular levels of cyclic adenosine monophosphate (cAMP) in neutrophils. While PGE1 and isoprotercnol act via a transmembrane guanine nucleotide regulatory protein, chemoattractants act via a calcium-dependent mechanism that may involve the inhibition of a native phosphodiestcrase. The ability of secretagogues, such as immune complexes and Ca2+ ionophores, to elicit an increase in cAMP raised the question of whether cAMP was an early intracellular messenger, activating neutrophils. The increase in cAMP elicited by secretagogues precedes cell activation, suggesting a role, at least temporally, for cAMP as an intracellular messenger. The cyclic nucleotides have been a source of investigation in neutrophils for over 20 years. A little progress from the initial studies proposed that cAMP was a negative effector and cyclic guanosine monophosphate (cGMP) was a positive one.
The calcium ion has an unusual importance in biological phenomena. Changes in the cytosolic free calcium concentration [Ca2+]i have evolved as a key intracellular messenger system to couple external stimuli to a variety of complex cellular responses. In phagocytic cells, (i.e., neutrophils and macrophages) changes in [Ca2+]i follow stimulation of several receptors and are thought to be involved in the regulation of adherence, chemotaxis, phagocytosis, degranulation, and production of toxic oxygen metabolites. The challenge for future research in phagocyte physiology will be the discrimination of those responses that are [Ca2+]i -activated and [Ca2+]i-dependent from those simply associated with a change in [Ca2+]i, the latter being due to the ligation of multifunctional receptors generating multiple signals and reactions. With the help of the new Ca2+ chelators, which allow the manipulation of [Ca2+]i in intact cells, answers to these questions are already becoming available. Yet nothing is known of the "missing messengers" that mediate phagocyte responses in those instances in which no generation of known intracellular signals has been documented.
The studies reviewed in this chapter illustrate that the measurement of proto-oncogene transcript abundance in stimulated lymphocytes is unlikely to shed light on the physiological control of lymphocyte activation. Lymphocytes interact with a large variety of cytokines and cell-associated regulatory molecules. The end result of the encounter of any individual T lymphocyte with an antigen-presenting cell probably depends on the sum of these modulatory signals as well as the specific interaction of the T cell antigen receptor with major histocompatibility complex (MHC)-associated ligand. Thus, the analyses of proto-oncogene expression during lymphocyte activation underestimate the complexity of lymphoid cell behavior. The chapter summarizes what is known about oncogene expression in lymphocytes exposed to a variety of activating stimuli and deduces general patterns from these studies, which have been pursued in very heterogeneous experimental systems. It discusses the hazards in directly interpreting changes in oncogene messenger RNA (mRNA) abundance by considering changes in Ick mRNA and p56lck protein expression in activated T cells.
The identification, cloning, and expression of the genes for lymphokines and their receptors have provided insight into their structure. Identification of the regulatory regions within the genome is also allowing the characterization of the processes that control the expression of these genes. The cloning of the receptor genes has so far provided little insight into how the lymphokine signal is transmitted across the cell membrane and eventually to the nucleus. Resting lymphocytes do not express significant numbers of high-affinity receptors for lymphokines. Following the activation of antigen receptors, cells are induced to express high-affinity receptors for an array of lymphokines. The eventual response is determined by the interaction of the lymphokines with their specific receptors on the surface of target cells. The response of the individual cell is determined by its particular pattern of lymphokine receptor expression and by its intracellular machinery. Because several different lymphokines can induce similar responses in a single cell, some lymphokine receptors may signal cells through conserved pathways.
This chapter examines the properties and regulation of the chloride (Cl) channels that contribute to the physiology of absorptive and secretory epithelial cells. Cl channels play a variety of functions in the physiology of cells. Cl channels contribute to the capacity of many cell types to regulate their intracellular composition and volume. In the response to cell swelling, Cl channel activation appears to be the rate-determining step in the ability of cells to reduce their volume toward normal values. In epithelial cells, Cl channels function in salt absorption and secretion. It is apparent that knowledge of Cl channels in secretory cells is more extensive than that for absorptive epithelia. The chapter categorizes the Cl channels of different cell types on the basis of their membrane location, biophysical properties, and mode of regulation. It mentions the relation of Cl channel properties to the physiology of absorptive and secretory cells. The chapter also compares epithelial cell Cl channels with those observed in a variety of other cell types.
This chapter discusses the experimental approaches to examine some of the propositions advanced in the two-stage model, focusing on experiments with bacteriorhodopsin. The idea that membrane protein folding can be considered in two distinct stages is considerably supported by the observations on bacteriorhodopsin. In the first stage, transbilayer helices are formed as independently stable entities. The principal energies contributing to helical stability are the hydrogen bonding of the main chain and the hydrophobic effect arising from the presence of largely nonpolar side chains. In stage II, the helices interact with each other to form a complete, functional tertiary structure. Some folding of the loop regions also occurs. Stage II is thought to be driven largely by the preferred packing of helices against each other compared with the packing of helices against lipid. Other important contributions to stability arise from the tendency of lipid to self-associate compared with associations with helices, contributions from the polypeptide links among helices, polar interactions, and interactions with prosthetic groups if they are present. Experiments with bacteriorhodopsin have provided support for this model.
This chapter focuses on the mechanisms by which arachidonic acid (AA) metabolites affect lymphocyte activation and function and evaluates their immunoregulatory potential. The potential in vivo effects of AA metabolites on lymphocytes have not been adequately studied; this is in part due to the rapid clearance of these molecules as well as uncertainty concerning their cellular source, rates of synthesis, and physiologic levels at sites of immunologically initiated inflammation. These factors should be borne in mind when the pharmacologic properties and effects of AA metabolites on in vitro lymphocyte function are described. Measurement of AA metabolite levels in the in vivo inflammatory milieu together with the functional responses of lymphocytes in this environment help to address this. The specific role assumed by AA metabolites in the process of lymphocyte activation is still unclear. These mediators may exert positive and negative effects on lymphocyte function, depending upon the source of the mediator, the nature of the activating lymphocyte agent, and the particular lymphocyte subset in question.
Recently, the patch-clamp technique has been used to study ion transport in epithelial tissues. The ability of an epithelial tissue to perform net ion transport is a consequence of the asymmetric distribution of ion channels and transporters in the apical and basolateral membranes of the epithelial cells. Application of patch-clamp techniques to the study of ion channels in epithelia is complicated by this assymmetric distribution of channel populations. This chapter discusses recent advances in the application of the patch-clamp technique to the study of cation channels in epithelia tissue. It defines a cation channel as a proteinaccous pore that spans a lipid bilayer, and allows cations to flow through this membrane-spanning protein as opposed to the lipid bilayer. Indeed, this would be an adequate definition if cations could not permeate anion-selective channels. The chapter reviews the technical approaches used by investigators in the study of cation channels in epithelia. It also outlines the experimental approaches used to study cation channels in epithelia. The physiological relevance of the patch-clamp data obtained from three different epithelial tissues are discussed.
This chapter focuses on the models of membrane organization. The most widely cited model of the organization of cell membranes is termed "fluid mosaic." It emphasizes the mobility and autonomy of membrane lipids and proteins and implies a role for molecular mobility in membrane function. Indeed, this aspect of the model has been the basis for proposals that reactions among membrane proteins may be coupled by a collision of diffusing species and that signaling from the cell surface may require clustering of mobile receptors by antigens or hormones. This chapter discusses results with a technique, fluorescence photobleaching and recovery (FPR) that was developed to quantitate Dlat in membranes and how this technique for measuring molecular mobility on a scale of micrometers per minute can be used to demonstrate molecular associations and molecular immobilization––that is, the way in which FPR can be used to investigate the spatial and temporal organization of cell surfaces. The FPR technique, which measures lateral mobility of membrane lipids and proteins, can be readily used to show that the cell surface is "grainy," differentiated into regions of specialized composition and function. Examples show that FPR can be used as a qualitative tool for detecting molecular associations in surfaces. A minimum of two cross-linking antibodies are required, and ideally a third, irrelevant monoclonal antibody should be available to show that the effects of surface cross-linking are specific for the pair of molecules of interest.
This chapter focuses on the current state of knowledge of the structure and function of perforin. The properties of other granule proteins, granzymes and proteoglycan, are reviewed in this chapter. Perforin is localized in the cytoplasmic granules of cytolytic T cells and NK cells, where it is associated with the granule proteoglycan chondroitin sulfate A. Because they contain perforin, isolated cytoplasmic granules are highly cytolytically active in the presence of Ca. Perforin has recently been isolated and characterized, including sequence determination by complementary (cDNA) cloning. The tools generated by this work are being used for a critical assessment of the role of perforin as opposed to other molecules in lymphocyte-mediated cytolysis. Lymphocyte-mediated cytotoxicity by perforin has three effects. The direct membrane damage by transmembrane channel formation can lead to target cell lysis by the effects of osmotic imbalance and the loss of the transmembrane potential.
