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PubMed1990

Lipid transport pathways in mammalian cells.

A major deficit in our understanding of membrane biogenesis in eukaryotes is the definition of mechanisms by which the lipid constituents of cell membranes are transported from their sites of intracellular synthesis to the multiplicity of membranes that constitute a typical cell. A variety of approaches have been used to examine the transport of lipids to different organelles. In many cases the development of new methods has been necessary to study the problem. These methods include cytological examination of cells labeled with fluorescent lipid analogs, improved methods of subcellular fractionation, in situ enzymology that demonstrates lipid translocation by changes in lipid structure, and cell-free reconstitution with isolated organelles. Several general patterns of lipid transport have emerged but there does not appear to be unifying mechanism by which lipids move among different organelles. Significant evidence now exists for vesicular and metabolic energy-dependent mechanisms as well as mechanisms that are clearly independent of cellular ATP content.

Animals↗
PubMed1989

Involvement of membrane lipids in protein export in Escherichia coli.

Several models for the transport of proteins across membranes predict a role for lipids. If these models are correct, then alterations in lipid metabolism may affect protein export and vice versa. We are investigating this possibility by studying Escherichia coli K-12 mutants with defects in protein export or phospholipid metabolism. A temperature-sensitive secA mutant, which is defective in protein export at 42 degrees C, exhibited severe pleiotropic effects on membrane biogenesis. Incubation of this strain at 42 degrees C resulted in the appearance of intracytoplasmic membranes, in alterations in lipopolysaccharide structure and in decreased cardiolipin and C18:1 fatty acid content. On the other hand, a pgsA mutant which is defective in the synthesis of acidic phospholipids, exhibited a protein export defect when studied in vivo or in vitro. These results are in agreement with a postulated role of membrane lipids in protein export.

Bacterial Proteins↗
PubMed1987

Phospholipid transfer activities in toad oocytes and developing embryos.

The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing 14C-labeled phospholipids and 3H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.

Acetates↗
PubMed1991

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.

Biological Transport, Active↗
PubMed2002

Mutants of Arabidopsis reveal many roles for membrane lipids.

Polyunsaturated acyl lipids constitute approximately 50% of the hydrophobic membrane barriers that delineate the compartments of cells. The composition of these lipids is critically important for many membrane functions and, thus, for proper growth and development of all living organisms. In the model plant Arabidopsis, the isolation of mutants with altered lipid compositions has facilitated biochemical and molecular approaches to understanding lipid metabolism and membrane biogenesis. Just as importantly, the availability of a series of plant lines with specific changes in membrane lipids have provided a new resource to study the structural and adaptive roles of lipids. Now, the sequencing of the Arabidopsis genome, and the development of reverse-genetics approaches provide the tools needed to make additional discoveries about the relationships between lipid structure and membrane function in plant cells.

Arabidopsis↗
PubMed2002

Phosphatidylserine transport to the mitochondria is regulated by ubiquitination.

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.

Biological Transport↗
PubMed2006

Identification of the sigmaE regulon of Salmonella enterica serovar Typhimurium.

The extracytoplasmic function sigma factor, sigma(E), has been shown to play a critical role in virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). The previously optimized two-plasmid system has been used to identify S. Typhimurium promoters recognized by RNA polymerase containing sigma(E). This method allowed identification of 34 sigma(E)-dependent promoters that direct expression of 62 genes in S. Typhimurium, 23 of which (including several specific for S. Typhimurium) have not been identified previously to be dependent upon sigma(E) in Escherichia coli. The promoters were confirmed in S. Typhimurium and transcriptional start points of the promoters were determined by S1-nuclease mapping. All the promoters contained sequences highly similar to the consensus sequence of sigma(E)-dependent promoters. The identified genes belonging to the S. Typhimurium sigma(E)-regulon encode proteins involved in primary metabolism, DNA repair systems and outer-membrane biogenesis, and regulatory proteins, periplasmic proteases and folding factors, proposed lipoproteins, and inner- and outer-membrane proteins with unknown functions. Several of these sigma(E)-dependent genes have been shown to play a role in virulence of S. Typhimurium.

Base Sequence↗
PubMed2005

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode.

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Animals↗
PubMed2003

Chloroplast biogenesis 87: Evidence of resonance excitation energy transfer between tetrapyrrole intermediates of the chlorophyll biosynthetic pathway and chlorophyll a.

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination of chlorophyll (Chl) and thylakoid apoprotein biosynthesis. As a working model for future investigations, we have proposed three Chl-thylakoid apoprotein biosynthesis models, namely, a single-branched Chl biosynthetic pathway (SBP) single-location model, an SBP multilocation model and a multibranched Chl biosynthetic pathway (MBP) sublocation model. Rejection or validation of these models can be probed by determination of resonance excitation energy transfer between various tetrapyrrole intermediates of the Chl biosynthetic pathway and various thylakoid Chl-protein complexes. In this study we describe the detection of resonance energy transfer between protoporphyrin IX (Proto), Mg-Proto and its monomethyl ester (Mp(e)) and divinyl and monovinyl protochlorophyllide a (Pchlide a) and several Chl-protein complexes. Induction of various amounts of tetrapyrrole accumulation in green photoperiodically grown cucumber cotyledons and barley leaves was achieved by dark incubation of excised tissues with delta-aminolevulinic acid (ALA) and various concentrations of 2,2'-dipyridyl for various periods of time. Controls were incubated in distilled water. After plastid isolation, treated and control plastids were diluted in buffered glycerol to the same Chl concentration. Excitation spectra were then recorded at 77 K at emission maxima of about 686, 694 and 738 nm. Resonance excitation energy transfer from Proto, Mp(e) and Pchlide a to Chl-protein complexes emitting at 686, 694 and 738 nm was observed by calculation of treated minus control difference excitation spectra. The occurrence of resonance excitation energy transfer between anabolic tetrapyrroles and Chl-protein complexes appeared as well-defined excitation bands with excitation maxima corresponding to those of Proto, Mp(e) and Pchlide a. Furthermore, it appeared that resonance excitation energy transfer from multiple short-wavelength, medium-wavelength and long-wavelength Proto, Mp(e) and Chlide a sites to various Chl-protein complexes took place. Because resonance excitation transfer from donors to acceptors cannot take place at distances larger than 100 A, it is proposed that the observed resonance excitation energy transfers are not compatible with the SBP single-location Chl biosynthesis thylakoid membrane biogenesis model. The latter assumes that a single-branched Chl biosynthetic pathway located in the center of a 450 x 130 A photosynthetic unit generates all of the Chl needed for the assembly of all Chl-protein complexes.

Chlorophyll↗
PubMed2002

Biochemistry and genetics of interorganelle aminoglycerophospholipid transport.

The organelle specific reactions that constitute the biosynthetic pathway for aminoglycerophospholipid synthesis provide an important means for examining the biochemistry and genetics of intracellular lipid transport. Biochemical studies with intact and permeabilized cells, and isolated organelles have defined some of the essential features of lipid transport between the endoplasmic reticulum and mitochondria and Golgi/vacuole. Genetic screens have now also identified mutations and genes that are involved in aminoglycerophospholipid traffic between different membranes in mammalian cells, yeast and bacteria. Increasingly, studies focused upon intermembrane lipid movement are revealing important new information about this essential aspect of membrane biogenesis.

Animals↗
PubMed1979

The study of biogenetic pathways using a perfusion technique containing perfluorochemicals.

A liver perfusion system was assembled and adapted for pulse labelling studies. The perfusion medium was prepared by emulsifying perfluorotributylamine and Pluronic F 68 in a CO2 atmosphere using a sonicator. Ribosome-rich and ribosome-poor rough microsomes, smooth microsomes and Golgi membranes could be prepared from perfused livers with a good purity and recovery as from non-perfused livers. The subfractionation technique used was simple and involved slight modifications of the methods described earlier by Eriksson (1973) and Ehrenreich et al. (1973). The specific activity of NADPH-cytochrome c reductase in microsomes and of UDP-galactosyltransferase in Golgi membranes from perfused and non-perfused livers were identical. The specific activity of glucose-6-phosphatase in microsomes was slightly decreased after perfusion, but the membrane permeability barrier to glucose-6-phosphate remained intact. The granulated microsomal fractions from perfused liver had a somewhat reduced number of membrane-bound ribosomes. The system developed proved useful in studies of the synthesis and intracellular transport of albumin. The technique should also be suitable for use in studies of membrane biogenesis.

Animals↗
PubMed1990

Biosynthesis and dynamics of lipids in Plasmodium-infected mature mammalian erythrocytes.

The asexual development of Plasmodium within the mature mammalian erythrocyte is associated with intense membrane biogenesis, notably to ensure the increase in the size of the parasite and of the parasitophorous vacuolar membranes PVM. A considerable increase in the content of most lipids except cholesterol [namely, phospholipids PL, neutral lipids, and fatty acids FA] occurs. The PL composition and the constitutive FAs of the parasite differ markedly from the original host cell membrane. Particularly notable is the absence of cholesterol and sphingomyelin SM from the parasite membranes. How can the parasite obtain such a quantity of new lipid molecules in a host cell totally devoid of any lipid biosynthetic activity? Like the normal erythrocyte, the infected cell is unable to synthesize cholesterol or FAs. In contrast, it exhibits an intense biosynthesis of neutral lipids and a bewildering variety of PL biosyntheses. Phosphatidylcholine PC is synthesized by a de novo pathway, and also by methylation of phosphatidylethanolamine PE, which itself originates from de novo biosynthesis or from decarboxylation of phosphatidylserine PS. Hence, interference with this intense and specific PL metabolism could provide the basis for a new malaria chemotherapy. Indeed, compounds that interfere with the entry of the plasmatic precursors (FAs or polar heads) or with their metabolism are lethal to the parasite. Lastly, we focus on the structural modifications of the host cell membrane with respect to lipids, including increased fluidity and enhanced transbilayer mobility of PLs. Possible modifications in the asymmetric distribution of PLs in the host cell membrane are discussed in light of the various methods used and their limits. The capacity of infected cells to take up and metabolize large quantities of exogenous vesicles of PLs accounts for the intense dynamics of lipids in the infected erythrocytes.

Animals↗
PubMed1980

Oversized flagellar membrane protein in paralyzed mutants of Chlamydomonas reinhardrii.

A mutant strain of Chlamydomonas reinhardtii is shown to possess an oversized flagellar membrane protein. The mutant has paralyzed flagella, is temperature sensitive for flagellar assembly, and has an abnormal axonemal protein composition. All phenotypes appear to derive from a single Mendelian mutation, and genetic analysis suggests that the mutation, which call ts222, is in the gene pfl. Because pf1 mutants are known to have radial-spoke defects (Piperno et al., 1977, Proc. Natl. Acad. Sci. U. S. A. 74:1600-1604; and Witman et al., 1978, J. Cell Biol. 76:729-797), a relation as yet undefined appears to exist between radial-spoke and flagellar membrane biogenesis.

Alleles↗
PubMed2005

A gene encoding a protein with a proline-rich domain (MtPPRD1), revealed by suppressive subtractive hybridization (SSH), is specifically expressed in the Medicago truncatula embryo axis during germination.

A gene MtPPRD1, encoding a protein of 132 amino acids containing a proline-rich domain (PRD), has been revealed by suppressive subtractive hybridization (SSH) with two mRNA populations of embryo axes harvested immediately before and after radicle emergence. Although at the protein level MtPPRD1 showed low homology with plant lipid transfer proteins (LTPs), it did exhibit the eight cysteine residues conserved in all plant LTPs, a characteristic signature that allows the formation of a hydrophobic cavity adapted for loading hydrophobic molecules. Expression studies of MtPPRD1 have been carried out by quantitative real time RT-PCR throughout germination and post-germination processes in control seeds and seeds in which germination was delayed by abscisic acid (ABA) or the glutamine synthetase inhibitor methionine sulphoximine (MSX) treatments. The results showed that MtPPRD1 expression is developmentally regulated, induced in the embryo axis immediately before radicle emergence, reaches its maximum expression and declines during the early post-germination phase. Organ specificity studies showed that, except for a low and probably constitutive expression in roots, MtPPRD1 is specifically expressed in the embryo axis. Based on both experimental and in silico studies several putative roles are proposed for MtPPRD1 in Medicago truncatula, this protein can intervene (i) as an LTP in membrane biogenesis and regulation of the intracellular fatty acid pool by binding and transferring fatty acids and phospholipids between membranes, (ii) in the control of a developmental process specific to late germination and to early phases of post-germination, and (iii) and/or pathogen defence.

Amino Acid Sequence↗
PubMed1993

Biogenesis of erythrocyte membrane skeleton in health and disease.

To study the biogenesis of red cell membrane skeleton at various stages of erythroid differentiation, we have chosen the following model systems: a) Rauscher erythroleukemia cell line representing the early stages of differentiation, b) Friend erythroleukemia cells, and c) in vitro cultured human erythroblasts. The latter two systems represent terminally differentiated erythroblasts. Using these model systems, we have shown asynchronous synthesis of membrane proteins during erythroid differentiation. At the early stages of erythroid development, the synthesis of spectrin, ankyrin and band 4.1 proteins is initiated before that of the band 3 protein. Following erythroid induction with erythropoietin and dimethylsulfoxide (DMSO), there is a dramatic increase in the synthesis of the band 3 protein without noticeable changes in the synthesis of other membrane proteins. This increase in band 3 synthesis is accompanied by increased stability and recruitment of the skeletal proteins into the membrane skeleton, leading to increased steady state levels. The progressive increase in band 3 synthesis continues during terminal maturation of erythroblasts. This is accompanied by increased stability and assembly of spectrin and ankyrin on the membrane, despite their reduced synthesis. These results point to a key role for the band 3 protein in anchoring and stabilizing these proteins into the permanent skeletal network. Finally, to detect defects of skeletal biosynthesis, we have extended these studies to a patient with severe hereditary spherocytosis characterized by a combined deficiency of spectrin and ankyrin. We have shown that this combined deficiency is a consequence of reduced ankyrin synthesis and mRNA content representing a thalassemia-like membrane protein mutation.

Animals↗
PubMed2005

Assay for the transbilayer movement of polyisoprenoid-linked saccharides based on the transport of water-soluble analogues.

Flippases are a class of membrane proteins that are proposed to facilitate the transbilayer movement of amphipathic polar lipids that are required for membrane biogenesis and the assembly of many diverse complex glycoconjugates in eukaryotic and prokaryotic cells. Despite their crucial roles in membrane biology, very little is known about their structures and the precise mechanism(s) by which they overcome the biophysical barriers of the hydrophobic core, and allow polar head groups to traverse membrane bilayers. This chapter presents methods based on the transport of water-soluble analogues that can be applied to investigate membrane proteins mediating the transverse diffusion of polyisoprenoid-linked glycolipid intermediates involved in the biosynthesis of N-linked glycoproteins, glycosylphosphatidylinositol anchors and bacterial polysaccharides.

Acyclic Monoterpenes↗
PubMed2001

Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences.

The molecular machinery underlying peroxisomal membrane biogenesis is not well understood. The observation that cells deficient in the peroxins Pex3p, Pex16p, and Pex19p lack peroxisomal membrane structures suggests that these molecules are involved in the initial stages of peroxisomal membrane formation. Pex19p, a predominantly cytosolic protein that can be farnesylated, binds multiple peroxisomal integral membrane proteins, and it has been suggested that it functions as a soluble receptor for the targeting of peroxisomal membrane proteins (PMPs) to the peroxisome. An alternative view proposes that Pex19p functions as a chaperone at the peroxisomal membrane. Here, we show that the peroxisomal sorting determinants and the Pex19p-binding domains of a number of PMPs are distinct entities. In addition, we extend the list of peroxins with which human Pex19p interacts to include the PMP Pex16p and show that Pex19p's CaaX prenylation motif is an important determinant in the affinity of Pex19p for Pex10p, Pex11pbeta, Pex12p, and Pex13p.

Amino Acid Sequence↗
PubMed1998

Effect of ethanol on intracellular vesicular transport from Golgi to the apical cell membrane: role of phosphatidylinositol 3-kinase and phospholipase A2 in Golgi transport vesicles association and fusion with the apical membrane.

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

Animals↗