Ceramide-enriched membrane domains—Structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Ceramide-enriched membrane domains

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.     

Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold hemolysis

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Arguello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.     

Structure and function of membrane fusion peptides

Membrane fusion peptides are highly conserved hydrophobic domains of fusion proteins that insert into membranes during membrane fusion. Recent success with solving the structures of the influenza hemagglutinin fusion peptide and some critical mutants of this peptide in membrane environments at high resolution has led to a new understanding of the mechanism of membrane fusion. This review highlights the structures that have been solved and summarizes recent thermodynamic and spectroscopic studies on the interactions of this interesting class of peptides with lipid bilayers.     

Sorting and function of peroxisomal membrane proteins

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.     

Properties and function of clostridial membrane ATPase

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg²⁺ dependent; Co²⁺ and Mn²⁺ but not Ca²⁺ could replace Mg²⁺ to some extent; the activation by Mg²⁺ was slightly antagonized by Ca²⁺. Even in the presence of Mg²⁺, Na⁺ or K⁺ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]_{0.5 V} = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg²⁺. Solubilization, however, led to instability of the enzyme.

The clostridial solubilized and membrane-bound ATPase showed different properties similar to the “allotopic” properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10^-4 M, led to 80% inhibition of the membrane-bound enzyme; oligomycin, ouabain, or NaN₃ had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment.

Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H⁺ translocation. A H⁺-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prokaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H⁺-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Toxic substances and cell membrane function.

The exposed location and functional importance of cell membranes make them particularly susceptible to the toxic effects of many chemicals. The likelihood of such effects has been appreciated for many years. However, the recent advent of new techniques has greatly increased our understanding of the complexities of membrane structure and function. These data make it quite clear that the interaction of toxic compounds with either the protein or the lipid component of cell membranes may substantially alter membrane function. This paper summarizes the current concepts of membrane structure and function and discusses the techniques currently in use to study cell membranes. Several examples are presented in which xenobiotics significantly alter membrane function. These include effects of heavy metals on passive ion permeability, impairment of osmoregulation and calcium transport by organochlorine pesticides, inhibition of the transport of neurotransmitter metabolites by phenoxyacetic acid herbicides in choroid plexus, and reduction in intestinal nutrient transport by heavy metals. Hence the study of the interactions of foreign compounds with membrane function may enhance our understanding of mechanisms both of toxicity and of basic membrane function.     

Dietary fatty acids and membrane protein function

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.     

Advances in membrane mimetics and mass spectrometry for understanding membrane structure and function

Membrane proteins and lipids play roles in regulating biological functions of cells. However, the analysis of interactions between membrane proteins and lipids in biological membranes remains challenging. Native membranes typically contain heterogenous lipid mixtures and low amounts of membrane proteins. This review presents recent developments in membrane mimetics and complementary mass spectrometry approaches for the investigation of membrane protein–lipid interactions after protein expression and purification. Furthermore, it is exemplified how delipidation knowledge on membrane mimetics can be used to gain insights into the role of lipids for protein structure and function. Because every technology has its strengths and weaknesses, it becomes apparent that integrated research approaches will facilitate the investigation of complex membrane environments in the future.     

Microscopic assessment of membrane protein structure and function

Membrane proteins represent an important class of proteins that are encoded by about 40% of all genes, but compared to soluble proteins structural information is sparse. Most of the atomic coordinates currently available are from bacterial membrane proteins and have been obtained by X-ray crystallography. Recent results demonstrate the imaging power of the atomic force microscope and the accuracy of electron crystallography. These methods allow membrane proteins to be studied while embedded in the bilayer, and thus in a functional state. The low signal-to-noise ratio of cryoelectron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review discusses examples of microscopic membrane protein structure determination and illuminates recent progress.     

Structure and function of human erythrocyte membrane skeletons

On the basis of extensive studies of the literature and of own results the present knowledge about the structure of the membrane skeleton of human erythrocytes is summarized and functional and clinical aspects are described. The spectrins are the centre of interest. Their interconnections, spatial arrangement and association with other components of the membrane are explained in greater detail. With regard to the membrane skeleton questions of erythrocyte shape, membrane integrity, phospholipid asymmetry, distribution of transmembrane proteins and cell deformation are discussed.     

Properties and function of clostridial membrane ATPase.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.     

Lipid regulation of cell membrane structure and function

Recent studies of structure-function relationships in biological membranes have revealed fundamental concepts concerning the regulation of cellular membrane function by membrane lipids. Considerable progress has been made in understanding the roles played by two membrane lipids: cholesterol and phosphatidyl-ethanolamine. Cholesterol has been shown to regulate ion pumps, which in some cases show an absolute dependence on cholesterol for activity. These studies suggest that an essential role that cholesterol plays in mammalian cell biology is to enable crucial membrane enzymes to provide function necessary for cell survival. Studies of phosphatidylethanolamine regulation of membrane protein activity and regulation of membrane morphology led to hypotheses concerning the roles for this particular lipid in biological membranes. New information on lipid-protein interactions and on the nature of the lipid head groups has permitted the development of mechanistic hypotheses for the regulation of membrane protein activity by phosphatidyl-ethanolamine. In addition, intermediates in the lamellar-nonlamellar phase transitions of membrane systems containing phosphatidylethanolamine, or other lipids with similar properties, have recently been implicated in facilitating membrane fusion. Finally, studies of transmembrane movement of lipids have provided new insight into the regulation of membrane lipid asymmetry and the biogenesis of cell membranes. These kinds of studies are harbingers of a new generation of progress in the field of cell membranes.