The links between membrane composition, metabolic rate and lifespan

The acyl composition of tissue phospholipids varies in a systematic manner among species. Phospholipids, and thus membrane bilayers, from the tissues of small mammal and bird species have a high content of docosahexaenoic acid (DHA) compared to large species. A similar difference exists between the tissues of endothermic mammals and ectothermic reptiles. High DHA content in phospholipids is associated with high metabolic activity and this observation has led to the development of the "membrane pacemaker" theory of metabolism. This proposes that highly polyunsaturated acyl chains impart physical properties to membrane bilayers that enhance and speed up the molecular activity of membrane proteins and consequently the metabolic activity of cells, tissues and the whole animal. The brain has highly polyunsaturated membranes irrespective of body size and possible reasons for this are discussed. Highly polyunsaturated acyl chains are very susceptible to peroxidative damage. It is suggested that these chemical properties of highly polyunsaturated membrane acyl chains have important implications for understanding aging and the determination of longevity.     

The links between membrane composition, metabolic rate and lifespan.

The acyl composition of tissue phospholipids varies in a systematic manner among species. Phospholipids, and thus membrane bilayers, from the tissues of small mammal and bird species have a high content of docosahexaenoic acid (DHA) compared to large species. A similar difference exists between the tissues of endothermic mammals and ectothermic reptiles. High DHA content in phospholipids is associated with high metabolic activity and this observation has led to the development of the "membrane pacemaker" theory of metabolism. This proposes that highly polyunsaturated acyl chains impart physical properties to membrane bilayers that enhance and speed up the molecular activity of membrane proteins and consequently the metabolic activity of cells, tissues and the whole animal. The brain has highly polyunsaturated membranes irrespective of body size and possible reasons for this are discussed. Highly polyunsaturated acyl chains are very susceptible to peroxidative damage. It is suggested that these chemical properties of highly polyunsaturated membrane acyl chains have important implications for understanding aging and the determination of longevity.     

Transferrin and albumin excretion as a measure of glomerular function

Albumin and transferrin are relatively small protein molecules and highly negatively charged. Their levels in urine are a useful indicator of the integrity of membrane barriers of the kidney glomerular capillary wall. The present data shows that the excretion rates of albumin and transferrin and their kinetics of excretions are similar. Thus, their filtration mechanisms at the active site of the kidney membrane pores are similar. Total urinary protein/creatinine or albumin or transferrin/creatinine ratio were found to be linear and highly significant. Their measurement could indicate the degree of impaired glomerular permeability. Also, in the present study, a rapid biochemical method of measurement of the selectivity of proteinuria based on the transferrin/albumin ratios in random samples is reported. This method is particularly useful in the early stages of glomerular basement membrane damage.     

Greasing membrane fusion and fission machineries

Biological membrane fusion is a local-point event, extremely fast, and under strict control. Proteins are responsible for the mutual recognition of the fusion partners and for the initiation of biomembrane fusion, and thus determine where and when fusion occurs. However, the central event during membrane fusion is the merger of two membranes, which requires a transient reorganization of membrane lipids into highly curved fusion intermediates. This review focuses on the potential role of lipids in the generation of membrane curvature, and thus in the regulation of membrane fusion and fission.     

Two conserved residues are important for inducing highly ordered membrane domains by the transmembrane domain of influenza hemagglutinin

The interaction with lipids of a synthetic peptide corresponding to the transmembrane domain of influenza hemagglutinin was investigated by means of electron spin resonance. A detailed analysis of the electron spin resonance spectra from spin-labeled phospholipids revealed that the major effect of the peptide on the dynamic membrane structure is to induce highly ordered membrane domains that are associated with electrostatic interactions between the peptide and negatively charged lipids. Two highly conserved residues in the peptide were identified as being important for the membrane ordering effect. Aggregation of large unilamellar vesicles induced by the peptide was also found to be correlated with the membrane ordering effect of the peptide, indicating that an increase in membrane ordering, i.e., membrane dehydration, is important for vesicle aggregation. The possibility that hydrophobic interaction between the highly ordered membrane domains plays a role in vesicle aggregation and viral fusion is discussed.     

Haemoglobin and the red cell membrane.

The results presented here indicate that haemoglobin is an integral part of the red cell membrane. The haemoglobin content of the membrane is highly dependent on the Ca++ content of the membrane in health and disease. Changes in the red cell interior alter the whole organization of the membrane and are even reflected in the binding of immunoglobulins to the red cell surface. The preferential binding of Hb-s A2 and S to the membrane has been confirmed. This phenomenon cannot be explained by differences in the charge between these haemoglobins and Hb A.     

Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells

X-ray crystal structures of human membrane proteins, although potentially of extremely great impact, are highly underrepresented relative to those of prokaryotic membrane proteins. One key reason for this is that human membrane proteins can be difficult to express at a level, and at a quality, suitable for structural studies. This protocol describes the methods that we use to overexpress human membrane proteins from clonal human embryonic kidney 293 (HEK293S) cells lacking N-acetylglucosaminyltransferase I (GnTI(-)), and was recently used in our 2.1-? X-ray crystal structure determination of human RhCG. Upon identification of highly expressing cell lines, suspension cell cultures are scaled up in a facile manner either using spinner flasks or cellbag bioreactors, resulting in a final purified yield of ～0.5 mg of membrane protein per liter of medium. The protocol described here is reliable and cost effective, can be used to express proteins that would otherwise be toxic to mammalian cells and can be completed in 8-10 weeks.     

Phosphoproteins and protein kinases of the Golgi apparatus membrane

Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.     

Exocytotic and endocytotic membrane traffic in neurons

Because neurons are highly polarized and capable of various modes of neurosecretion the exocytotic and endocytotic membrane traffic in these cells is more complex than in other eukaryotic cells. Progress in our understanding of neuronal membrane traffic and organelle biogenesis has come from recently discovered analogies to epithelial and endocrine cells.     

Composition and expression of spectrin-based membrane skeletons in non-erythroid cells

Cellular differentiation is often accompanied by the expression of specialized plasma membrane proteins which accumulate in discrete regions. The biogenesis of these specialized membrane domains involves the assembly and co-localisation of a spectrin-based membrane skeleton. While the constituents of the membrane skeleton in non-erythroid cells are often immunologically related to erythroid spectrin, ankyrin, and protein 4.1, there are structural and functional differences between the isoforms of these membrane skeleton polypeptides, as well as highly variable patterns of expression during cellular differentiation. We consider this heterogeneity of structure and expression during development in the context of the hypothesis that non-erythroid spectrin, ankyrin, and protein 4.1 are involved in the formation of specialized membrane domains.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins

Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.     

Thermodynamics,the structure of integral membrane proteins,and transport

Membranes are structures whose lipid and protein components are at, or close to, equilibrium in the plane of the membrane, but are not at equilibrium across the membrane. The thermodynamic tendency of ionic and highly polar molecules to be in contact with water rather than with nonpolar media (hydrophilic interactions) is important in determining these equilibrium and nonequilibrium states. In this paper, we speculate about the structures and orientations of integral proteins in a membrane, and about how the equilibrium and nonequilibrium features of such structures and orientations might be influenced by the special mechanisms of biosynthesis, processing, and membrane insertion of these proteins. The relevance of these speculations to the mechanisms of the translocation event in membrane transport is discussed, and specific protein models of transport that have been proposed are analyzed.     

Predicting membrane protein type by functional domain composition and pseudo-amino acid composition

Given the sequence of a protein, how can we predict whether it is a membrane protein or non-membrane protein? If it is, what membrane protein type it belongs to? Since these questions are closely relevant to the function of an uncharacterized protein, their importance is self-evident. Particularly, with the explosion of protein sequences entering into databanks and the relatively much slower progress in using biochemical experiments to determine their functions, it is highly desired to develop an automated method that can be used to give a fast answers to these questions. By hybridizing the functional domain (FunD) and pseudo-amino acid composition (PseAA), a new strategy called FunD-PseAA predictor was introduced. To test the power of the predictor, a highly non-homologous data set was constructed where none of proteins has 25% sequence identity to any other. The overall success rates obtained with the FunD-PseAA predictor on such a data set by the jackknife cross-validation test was 85% for the case in identifying membrane protein and non-membrane protein, and 91% in identifying the membrane protein type among the following 5 categories: (1) type-1 membrane protein, (2) type-2 membrane protein, (3) multipass transmembrane protein, (4) lipid chain-anchored membrane protein, and (5) GPI-anchored membrane protein. These rates are much higher than those obtained by the other methods on the same stringent data set, indicating that the FunD-PseAA predictor may become a useful high throughput tool in bioinformatics and proteomics.     

Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process

The phagophore membrane is highly curved along the rim of the open cup, suggesting that the molecular mechanisms governing its formation and growth could rely on membrane curvature-dependent events. To this end, we recently reported that lipidation of the LC3 protein family is facilitated on highly curved membranes in vitro. We further showed that the conjugating enzyme ATG3 contains an amphipathic helix that is responsible for this membrane curvature dependency, and that the maintenance of this amphipathic structure is essential for ATG3 function in vivo.     

Gangliosides and the multiscale modulation of membrane structure

Cellular membranes are highly organized structures with multiple and multi-dimensional levels of order where lipid components are active players. The lipid role is especially evident in rafts, where lipid-driven collective interaction dictates the local structure of a membrane. However, lipids play as well other roles in many aspects of membrane mechanics and function.In this review, we would like to re-focus the attention of the readers on the importance of gangliosides in organizing the fine structure of cellular membranes, in lateral and transverse directions. Important biological events are likely to be affected such as the dynamic control of the shape of specialized plasma membrane areas and of the intracellular organelles, the in- and outward budding and fusion of membrane vesicles, the physical and functional coupling of the outer and the inner plasma membrane leaflet, involved in the transduction of signals across the membrane.     

Chlorophyll,glycerolipid and protein ratios in spinach chloroplast grana and stroma lamellae

The relative molar amounts of glycerolipids are similar in grana and stroma lamellae, as are the ratios of total glycerolipid to weight of membrane protein. However the chlorophyll content relative to protein of grana lamellae is about 40% higher than that of stroma lamellae from the same preparation. Previous reports of chemical composition or enzyme activity based on chlorophyll alone can be highly misleading. The large functional and conformational differences between these two membranes may be related to these differences in pigment content, but are likely to result primarily from qualitative protein differences. The data are in accord with a membrane model in which nonpolar regions of membrane protein bind lipid in fairly constant amounts.     

Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process

The phagophore membrane is highly curved along the rim of the open cup, suggesting that the molecular mechanisms governing its formation and growth could rely on membrane curvature-dependent events. To this end, we recently reported that lipidation of the LC3 protein family is facilitated on highly curved membranes in vitro. We further showed that the conjugating enzyme ATG3 contains an amphipathic helix that is responsible for this membrane curvature dependency, and that the maintenance of this amphipathic structure is essential for ATG3 function in vivo.     

Foreword: Protein secretion and the Golgi apparatus

Resonance and nonresonance Raman spectra have been obtained from neoplastically transformed and normal avian lymphocytes. The acyl chains of membrane phospholipids of neoplastic cells are more highly unsaturated than those of normal cells. The observation of prominent carotenoid bands in both cell populations indicates the availability of a sensitive, intrinsic probe of membrane potential and local membrane environment.