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PubMed2006

Very long-chain fatty acid-containing lipids rather than sphingolipids per se are required for raft association and stable surface transport of newly synthesized plasma membrane ATPase in yeast.

The proton-pumping H+-ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid "rafts") already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide.

Cell Membrane↗
PubMed2006

The role of lipids in the biogenesis of integral membrane proteins.

Most integral membrane proteins are cotranslationally inserted into the lipid bilayer. In prokaryotes, membrane insertion of the nascent chain takes place at the plasma membrane, whereas in eukaryotes insertion takes place into the endoplasmatic reticulum. In both kingdoms of life, however, the same membrane that acquaints the newly born membrane protein also synthesizes the bilayer lipids and thus ensures the balanced growth of the membrane as a whole. Recent evidence indicates that the lipid composition of the host membrane can determine the fate of the newborn membrane protein, as it can affect (1) the efficiency of translocation, (2) the topology of the resulting membrane protein, (3) its stability, (4) its assembly into oligomeric complexes, (5) its transport and sorting along the secretory pathway, and (6) its enzymatic activity. The lipid composition of the membrane thus can affect the biogenesis and function of integral membrane proteins at multiple steps along its biogenetic pathway. While understanding this interdependence between bilayer lipids and protein biogenesis is interesting in its own right, careful consideration of a potential host's membrane lipid composition may also allow optimization of the yield and activity of membrane proteins that are expressed in a heterologous organism. Here, we review and discuss some examples that illustrate the interdependence between bilayer lipids and the biogenesis of integral membrane proteins.

Membrane Lipids↗
PubMed1985

Phospholipid transfer proteins from lung, properties and possible physiological functions.

Phospholipid transfer proteins have been found in lung just as they have in tissues throughout the body. There is speculation that the proteins are involved in membrane biogenesis and in determining the phospholipid composition of membranes. For this reason the lung, which contains subcellular organelles of distinct phospholipid composition, is of interest in terms of its complement of phospholipid transfer proteins. The lamellar bodies of pulmonary type II alveolar cells have a phospholipid composition unique in terms of the proportions of dipalmitoyl phosphatidylcholine and phosphatidylglycerol. Studies of the phospholipid transfer proteins in lung have demonstrated two molecular species of the transfer proteins that differ significantly from those found in liver and other tissues. These proteins show specificity for the transfer of dipalmitoyl phosphatidylcholine and phosphatidylglycerol.

Animals↗
PubMedDate not supplied

Localization of interferon-induced lupus inclusions demonstrated by computer image reconstruction of monensin-treated Daudi cells.

A structural analysis of cells that contained the interferon-alpha-induced lupus inclusions (LI) was performed using a high-voltage electron microscope to determine the exact cellular location of LI and their association with normal cell organelles. LI were induced in the human B lymphoblastoid cell line, Daudi, by culturing with the pure recombinant human leukocyte interferon, IFLrA. Just prior to harvesting, a portion of the cells was treated with monensin to selectively swell the Golgi apparatus, and thereby simplify their identification using the electron microscope. Organellar associations between LI and the outer nuclear envelope and Golgi apparatus were identified in stereopairs of 1-micron sections prepared from both cells that were not treated with monensin and those that were treated with monensin. Serial 0.25-micron sections of the monensin-treated cells were prepared, and seven arbitrarily chosen cells were examined. Each of these cells contained a single LI, and it formed throughout an endoplasmic-reticulum region that made contact with both the outer nuclear envelope and the Golgi vesicles. Reconstruction of a cell by computer from the digitized negatives of serial sections clearly illustrated these relationships. This study reports the first determination of the association between LI and the Golgi apparatus. It also identifies the presence of only one LI in every cell, and the routine association of the LI with both the outer nuclear envelope and the Golgi apparatus. The unique cell location of LI formation suggests their functioning in membrane biogenesis, the trafficking of proteins to the plasma membrane or to cytoplasmic vesicles, or the processing of proteins for secretion.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Molecular machinery for non-vesicular trafficking of ceramide.

Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.

Alternative Splicing↗
PubMed2005

Synthesis of sphingolipids with very long chain fatty acids but not ergosterol is required for routing of newly synthesized plasma membrane ATPase to the cell surface of yeast.

The proton pumping H(+)-ATPase, Pma1p, is an abundant and very long-lived polytopic protein of the Saccharomyces cerevisiae plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as an excellent model to study plasma membrane biogenesis. We have previously shown that newly synthesized Pma1p is mistargeted to the vacuole in an elo3Delta mutant that affects the synthesis of the ceramide-bound C26 very long chain fatty acid (Eisenkolb, M., Zenzmaier, C., Leitner, E., and Schneiter, R. (2002) Mol. Biol. Cell 13, 4414-4428) and now describe a more detailed analysis of the role of lipids in Pma1p biogenesis. Remarkably, a block at various steps of sterol biosynthesis, a complete block in sterol synthesis, or the substitution of internally synthesized ergosterol by externally supplied ergosterol or even by cholesterol does not affect Pma1p biogenesis or its association with detergent-resistant membrane domains (lipid "rafts"). However, a block in sphingolipid synthesis or any perturbation in the synthesis of the ceramide-bound C26 very long chain fatty acid results in mistargeting of newly synthesized Pma1p to the vacuole. Mistargeting correlates with a lack of newly synthesized Pma1p to acquire detergent resistance, suggesting that sphingolipids with very long acyl chains affect sorting of Pma1p to the cell surface.

Calcium-Transporting ATPases↗
PubMed1993

Morphogenesis of the protein secretory system in PC12 cells infected with Japanese encephalitis virus.

Infection of PC12 cells with Japanese encephalitis (JE) virus caused marked proliferation of the protein secretory system. Accordingly, in this study the morphogenesis of the secretory organelles, i.e., rough endoplasmic reticulum (RER) and the Golgi apparatus, in JE virus-infected PC12 cells was analyzed by electron microscopical observation. Starting 24 h postinoculation (p.i.), a structure that represented nascent RER appeared in the cytoplasm in the form of rows of ribosomes which surrounded membrane-unbounded, electron-lucent lacunae in a reticular, honey-comb pattern (reticular RER). Although the reticular RER lacked membrane components, its lacunae contained progeny virions, indicating that the rows of ribosomes synthesized the viral proteins and discharged them into the lacunae for the viral assembly. The reticular RER apparently transformed into the familiar lamellar RER during the RER morphogenesis as the lacunae coalesced to form flat cisternae and RER membrane assembled to border the cisternae. These findings indicated that the proliferating RER was the site of not only active protein synthesis but also active membrane biogenesis. The proliferating RER released a large number of membrane vesicles including virion-carrying vesicles into the cytoplasm. These vesicles congregated in the juxtanuclear region, especially around the centrioles, and fused to existing Golgi complexes for enlargement or fused among themselves to form new Golgi complexes. The present study, therefore, indicated that (a) nascent RER was formed by polysomes that arranged themselves in rows of ribosomes without participation of a preexisting membrane framework of endoplasmic reticulum (ER), (b) membrane components of RER were assembled de novo within the structure during the RER morphogenesis, and (c) RER released membrane vesicles that moved to the Golgi apparatus and contributed to the morphogenesis of the Golgi apparatus. Possible causative mechanisms involved in the proliferation of the secretory system in JE virus-infected PC12 cells are discussed.

Animals↗
PubMed2005

New fluorescent probes reveal that flippase-mediated flip-flop of phosphatidylinositol across the endoplasmic reticulum membrane does not depend on the stereochemistry of the lipid.

Glycerophospholipid flip-flop across biogenic membranes such as the endoplasmic reticulum (ER) is a fundamental feature of membrane biogenesis. Flip-flop requires the activity of specific membrane proteins called flippases. These proteins have yet to be identified in biogenic membranes and the molecular basis of their action is unknown. It is generally believed that flippase-facilitated glycerophospholipid flip-flop across the ER is governed by the stereochemistry of the glycerolipid, but this important issue has not been resolved. Here we investigate whether the ER flippase stereochemically recognizes the glycerophospholipids that it transports. To address this question we selected phosphatidylinositol (PI), a biologically important molecule with chiral centres in both its myo-inositol headgroup and its glycerol-lipid tail. The flip-flop of PI across the ER has not been previously reported. We synthesized fluorescence-labeled forms of all four diastereoisomers of PI and evaluated their flipping in rat liver ER vesicles, as well as in flippase-containing proteoliposomes reconstituted from a detergent extract of ER. Our results show that the flippase is able to translocate all four PI isomers and that both glycerol isomers of PI flip-flop across the ER membrane at rates similar to that measured for fluorescence-labeled phosphatidylcholine. Our data have important implications for recent hypotheses concerning the evolution of distinct homochiral glycerophospholipid membranes during the speciation of archaea and bacteria/eukarya from a common cellular ancestor.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Binding of cerebrosides and sulfatides to saposins A-D.

Saposins are a family of four small glycoproteins, all of which are derived from prosaposin, and are involved in the lysosomal hydrolysis of various sphingolipids. Results from this investigation demonstrate that saposins A-D bind to galactosyl- and glucosylceramide. The binding was highly dependent on the solution pH; maximum binding of glucosylceramide to all saposins occurred at pH 7. Maximum binding of galactosylceramide to saposins B and D occurred at a more basic pH (8.5). The binding of glucosylceramide to saposins was significantly inhibited by Mg2+, Ca2+, or Zn2+. Although maximum binding of sulfatide to saposins A, C, and D occurred at acidic pH, the binding to saposin B was maximum at pH 8.5. Saposin A also bound sphingomyelin or phosphatidylcholine at neutral pH. No significant binding was evident between these lipids and saposins B-D at any pH value. The existence of saposin-lipid complexes was further confirmed in selected samples by gel filtration, isoelectric focusing, and a TLC binding assay. We have also shown that galactosylceramide bound to saposins A-D was efficiently transported to a rat brain microsomal fraction. This result suggests that saposins and possibly their precursor, prosaposin, may be involved in membrane biogenesis such as the assembly of myelin and plasma membranes.

Animals↗
PubMed1981

Cellular and thylakoid-membrane glycolipids of Chlamydomonas reinhardtii 137+.

The glycolipids of the green alga Chlamydomonas reinhardtii 137+ have been quantitated in the phototrophically-cultured cell and in its thylakoid membrane. Three lipids, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the sulfated glycolipid sulfoquinovosyldiacylglycerol (SL), constitute the total glycolipid complement and some 70-80% of the total polar lipid at both levels. About 70% of the alga's sulfolipid, but only about half of its galactolipid, is localized in the thylakoid. The three cellular and thylakoid glycolipids all contain prominent hexadecanoic and octadecanoic fatty acids. Quantitatively, each lipid has a distinctive acyl profile, making SL the most highly saturated and MGDG the least saturated glycolipid. Differences between the fatty acid profiles of each corresponding cellular and thylakoid glycolipid indicate that discrimination of the glycolipid species assembled into photosynthetic membrane takes place during thylakoid membrane biogenesis.

Cell Membrane↗
PubMed1996

Overproduction of a foreign membrane protein in Escherichia coli stimulates and depends on phospholipid synthesis.

When the Pseudomonas oleovorans alk system, consisting of the alkBFGHJKL and alkST genes, is expressed in Escherichia coli W3110, significant changes in phospholipid metabolism of the host are observed. A major role seems to be played by the cytoplasmic membrane protein alkane hydroxylase (AlkB), which is synthesized as up to 10-15% of the total protein in this strain [Nieboer, M., Kingma, J. & Witholt, B. (1993) The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110[pGEc47] affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subfraction, Mol. Microbiol. 8, 1039-1051]. In the present paper, we have studied the link between synthesis of the membrane protein and the synthesis of phospholipids and fatty acids by examining the kinetics of these processes. Using [35S]methionine labeling, it was shown that induction of AlkB was maximal within 30-60 min after addition of inducer, when up to 15% of all newly synthesized protein is AlkB. Phospholipid synthesis was followed by measuring the incorporation of 14C-labeled acetate and 33P-labeled phosphoric acid into phospholipids. Despite a negative effect of the inducer on the growth rate of W3110[pGEc47], net phospholipid synthesis was significantly enhanced as a result of the expression of alkB. Synthesis of all three major phospholipids were stimulated to comparable extents by the induction of alkB. Induction did not increase 33P incorporation into lipids in the control recombinant alk+ strain which lacked alkB. Simultaneous with AlkB synthesis, the conversion of unsaturated 9-hexadecenoic acid (C16:1) into 9,10-methylene hexadecanoic acid (C17:ocyc) was reduced in the alk+ recombinant. Overall, these data show that the production of a foreign membrane protein in E. coli can engender a response of the phospholipid-synthesizing system of the host. In the absence of such a response, induction of the alk system would be much more toxic to the cells. Apparently, the increased phospholipid synthesis plays an important role in enabling the AlkB overproducing strain to grow.

Bacterial Proteins↗
PubMed1981

Leader peptidase is found in both the inner and outer membranes of Escherichia coli.

Many membrane proteins are synthesized as transient precursors with an NH2-terminal leader (or signal) peptide. During insertion of these proteins into the membrane, leader peptides are removed by leader peptidase. One such enzyme has been detected in detergent extracts of Escherichia coli membranes and extensively purified using as an assay the removal of the leader sequence of procoat, the precursor of the major coat protein of bacteriophage M13. We now report that this leader peptidase is found in equal abundance in the inner and outer membranes of E. coli. Enzyme from each membrane accurately cleaves procoat to mature M13 coat protein. The salt, pH, and Mg2+ optima and inhibitor sensitivities of enzyme from each membrane are identical. Furthermore, the activities are indistinguishable upon ion exchange chromatography and nondenaturing gel electrophoresis. Finally, a strain of E. coli with a plasmid which causes overproduction of leader peptidase has elevated levels of enzyme in both the inner and outer membranes. Leader peptidase is the only known enzyme which is found in both inner and outer membrane fractions of E. coli; this may reflect its role in membrane biogenesis.

Cell Membrane↗
PubMed1989

Spontaneous phosphatidylcholine transfer by collision between vesicles at high lipid concentration.

The transfer kinetics of [3H]-1-palmitoyl-2-oleoylphosphatidylcholine ([3H]POPC) and 1-palmitoyl-2-(pyrenyldecanoyl)phosphatidylcholine (PyrPC) from POPC small unilamellar vesicles were examined at 37 degrees C with lipid concentrations ranging from 0.1 to 40 mM. The rate of [3H]POPC transfer was determined by analyzing the movement of this lipid from charged donor to neutral acceptor vesicles. The rate of decay of the ratio of the intensity of pyrene excimer fluorescence to that from the pyrene monomer (E/M) upon addition of an unlabeled vesicle population to a population containing PyrPC was used to evaluate PyrPC transfer. For both lipids, the kinetic data are best described by a model which assumes that transfer occurs by vesicle collisions as well as by desorption from the bilayer. For [3H]POPC, the off-rate constant is 0.014 h-1 while the collisional rate constant is 0.0016 mM-1 h-1. PyrPC has an off-rate constant of 0.023 h-1 and a collisional constant of 0.0015 mM-1 h-1. These numbers were calculated by assuming the rate of interbilayer transfer to be negligible relative to that of intervesicular transfer. The large transfer fluxes in the high vesicle concentration range where the collisional process dominates suggest that spontaneous transfer may be of importance in membrane biogenesis.

Kinetics↗
PubMed2004

Domain architecture and activity of human Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane proteins.

Pex19p is a peroxin involved in peroxisomal membrane biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pex19p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pex19p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex.

ATP-Binding Cassette Transporters↗
PubMed1985

A large intracellular pool of inactive Na channel alpha subunits in developing rat brain.

An intracellular pool of Na channel alpha subunits has been detected in developing brain cells in vivo and in vitro by phosphorylation with cAMP-dependent protein kinase, immunoprecipitation with specific antiserum, and NaDodSO4 gel electrophoresis or by radioimmunoassay. These alpha subunits are membrane-bound, contain complex carbohydrate chains, and have an apparent molecular weight of 260,000 like mature alpha subunits. In contrast to mature alpha subunits, the intracellular subunits are not covalently attached to a beta 2 subunit, and they do not bind saxitoxin with high affinity. They comprise 67-77% of the total immunoreactive alpha subunit in developing rat brain cells but are not a prominent component in the adult brain. It is proposed that this intracellular pool of alpha subunits forms a ready reserve of preformed subunits for incorporation into the surface membrane during periods of active membrane biogenesis. The results suggest that disulfide linkage of the alpha and beta 2 subunits, insertion into the cell surface membrane, and attainment of a functional conformation are closely related late events in the biogenesis of the Na channel. These processes may regulate the number of functional Na channels in the developing brain.

Animals↗
PubMed1977

[Incorporation of 32P into membrane phospholipids during chloroplast biogenesis].

The composition of membrane phospholipids during chloroplast biogenesis was studied. The maximal level of phosphatidic acid was observed in the membrane fraction of proplastids. Phosphatidylglycerol was found to be the most abundant phospholipid component of grana thylakoids. The evidence from the in vivo experiments indicates that phosphatidic acid and phosphatidylglycerol incorporate the 32P label at a high rate at all stages of the chloroplast biogenesis. It is concluded that plastids are the site of the phosphatidylglycerol biosynthesis in the plant cell.

Chloroplasts↗
PubMed2001

Biogenesis of inner membrane proteins in Escherichia coli.

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.

Bacterial Proteins↗