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The enterohepatic recirculation of bile acids (BAs) is important in several physiological processes. Although there has been considerable research on liver regeneration after two-thirds partial hepatectomy (PHx), little is known about how the liver protects itself against BA toxicity during regeneration. In this study, various BAs in plasma and liver, the composition of micelle-forming bile constituents, as well as gene expression of the main hepatobiliary transporters were quantified in sham-operated and PHx mice 24 and 48 h after surgery. PHx did not influence the hepatic concentrations of taurine-conjugated BAs (T-BA) but increased the concentration of glycine-conjugated (G-BA) and unconjugated BAs. Total BA excretion (μg·min−1·g liver wt−1) increased 2.4-fold and was accompanied by a 55% increase in bile flow after PHx. The plasma concentrations of T-BAs (402-fold), G-BAs (17-fold), and unconjugated BAs (500-fold) increased. The mRNA and protein levels of the BA uptake transporter Ntcp were unchanged after PHx, whereas the canalicular Bsep protein increased twofold at 48 h. The basolateral efflux transporter Mrp3 was induced at the mRNA (2.6-fold) and protein (3.1-fold) levels after PHx, which may contribute to elevated plasma BA and bilirubin levels. Biliary phospholipid excretion was nearly doubled in PHx mice, most likely owing to increased mRNA expression of the phospholipid transporter, Mdr2. In conclusion, the remnant liver after PHx excretes 2.5-fold more BAs and three times more phospholipids per gram liver than the sham-operated mouse liver. Upregulation of phospholipid transport may be important in protecting the biliary tract from BA toxicity during PHx.

P4-ATPases comprise a family of P-type ATPases that actively transport or flip phospholipids across cell membranes. This generates and maintains membrane lipid asymmetry, a property essential for a wide variety of cellular processes such as vesicle budding and trafficking, cell signaling, blood coagulation, apoptosis, bile and cholesterol homeostasis, and neuronal cell survival. Some P4-ATPases transport phosphatidylserine and phosphatidylethanolamine across the plasma membrane or intracellular membranes whereas other P4-ATPases are specific for phosphatidylcholine. The importance of P4-ATPases is highlighted by the finding that genetic defects in two P4-ATPases ATP8A2 and ATP8B1 are associated with severe human disorders. Recent studies have provided insight into how P4-ATPases translocate phospholipids across membranes. P4-ATPases form a phosphorylated intermediate at the aspartate of the P-type ATPase signature sequence, and dephosphorylation is activated by the lipid substrate being flipped from the exoplasmic to the cytoplasmic leaflet similar to the activation of dephosphorylation of Na(+)/K(+)-ATPase by exoplasmic K(+). How the phospholipid is translocated can be understood in terms of a peripheral hydrophobic gate pathway between transmembrane helices M1, M3, M4, and M6. This pathway, which partially overlaps with the suggested pathway for migration of Ca(2+) in the opposite direction in the Ca(2+)-ATPase, is wider than the latter, thereby accommodating the phospholipid head group. The head group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca(2+)- and Na(+)/K(+)-ATPases. Hence, the P4-ATPase mechanism is quite similar to the mechanism of these ion pumps, where the glutamate translocates the ions by moving like a pump rod. The accessory subunit CDC50 may be located in close association with the exoplasmic entrance of the suggested pathway, and possibly promotes the binding of the lipid substrate. This review focuses on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms.

In this work we use an Asynchronous Differential Evolution (ADE) method to estimate parameters of the Separated Form Factor (SFF) model which is used to investigate a structure of drug delivery Phospholipid Transport Nano System (PTNS) unilamellar vesicles by experimental small angle synchrotron X-ray scattering spectra (SAXS). We compare the efficiency of different optimizing procedures (OP) for the search for the SFF-model parameters. It is shown that the probability to find the global solution of this problem by ADE-methods is significantly higher than that by either Nelder-Mead method or a Quasi-Newton method with Davidon-Fletcher-Powell formula. The parallel realization of ADE accelerates the calculations significantly. The speed-up obtained by the parallel realization of ADE and results of the model are presented.

The ATP binding cassette transporter A1 (ABCA1) mediates cellular cholesterol and phospholipid efflux, and is implicated in phosphatidylserine translocation and apoptosis. Loss of functional ABCA1 in null mice results in severe placental malformation. This study aimed to establish the placental localisation of ABCA1 and to investigate whether ABCA1 expression is altered in placentas from pregnancies complicated by pre-eclampsia and antiphospholipid syndrome. ABCA1 mRNA and protein localisation studies were carried out using in situ hybridization and immunohistochemistry. Comparisons of gene expression were performed using real-time PCR and immunoblotting. ABCA1 mRNA and protein was localised to the apical syncytium of placental villi and endothelia of fetal blood vessels within the villi. ABCA1 mRNA expression was reduced in placentas from women with APS when compared to controls (p

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.Issledovali belkovyĭ profil' émbrionov ryb vida Danio rerio, vyrashchennykh v srede s doksorubitsinom, vkliuchennym v fosfolipidnuiu transportnuiu nanosistemu (doksolip), ispol'zuia sochetanie odnomernogo élektroforeza s posleduiushcheĭ vremiaproletnoĭ mass-spektrometrieĭ s lazernoĭ desorbtsieĭ i ionizatsieĭ (MALDI-TOF-PMF). Pri vyrashchivanii émbrionov D. rerio v srede s doksolipom otmecheno sushchestvennoe uvelichenie kolichestva tsitoskeletnykh belkov, snizhenie kolichestva iadernykh belkov, uchastvuiushchikh v sinteze DNK i RNK, i ischeznovenie vitellogenina 2 po sravneniiu s kontrolem (sreda s fosfolipidnoĭ transportnoĭ nanosistemoĭ). Analiz proteomnykh profileĭ émbrionov, podvergnutykh obrabotke doksolipom, svidetel'stvuet o snizhenii émbriotoksichnosti doksorubitsina pri vkliuchenii ego v fosfolipidnuiu nanosistemu.

Phospholipid transport nanosystem (PTNS) for drug delivery is based on soybean phosphatidylcholine. The morphology of PTNS is investigated by means of small-angle X-ray scattering. The obtained results allow one to answer the key question from the viewpoint of organization of drug incorporation whether the PTNS nanoparticles have a structure of micelles or vesicles. It is demonstrated that PTNS is a vesicular system with an average vesicle radius of 160 ± 2A.

Background— Extrahepatic tissues have long been considered critical contributors of cholesterol to nascent HDL particles in the reverse cholesterol transport pathway, in which ABCA1 plays the crucial role. Recent studies, however, including both overexpression and deletion of ABCA1 selectively in the liver, have highlighted the primary role of the liver in the maintenance of HDL levels in vivo. Methods and Results— The availability of mice with complete deletion of ABCA1 (total knockout [TKO]) and with liver-specific deletion of ABCA1 (LSKO) has enabled us to dissect the discrete roles of hepatic relative to extrahepatic ABCA1 in HDL biogenesis. Delivery of adenoviral ABCA1 resulted in selective expression of physiological levels of ABCA1 in the livers of both LSKO and TKO mice, resulting in increased HDL cholesterol (HDL-C). Expression of ABCA1 in the liver of LSKO mice resulted in plasma HDL-C levels that were similar to those in wild-type mice and significantly above those seen in similarly treated TKO mice. HDL particles from ABCA1-expressing LSKO mice were larger and contained significantly increased cholesterol compared with TKO mice. Infusion of human apolipoprotein A-I/phospholipid reconstituted HDL particles normalized plasma HDL-C levels in LSKO mice but had no effect on HDL-C levels in TKO mice. Conclusions— Although hepatic ABCA1 appears crucial for phospholipid transport, extrahepatic tissues play an important role in cholesterol transfer to nascent HDL particles. These data highlight the discrete and specific roles of both liver and extrahepatic ABCA1 in HDL biogenesis in vivo and indicate that ABCA1 shows lipid cargo selectivity depending on its site of expression.

PtdSer (phosphatidylserine) is synthesized in the endoplasmic reticulum and the related MAM (mitochondria-associated membrane), and transported to the PtdSer decarboxylases, Pds1p in the mitochondria, and Psd2p in the Golgi. Genetic and biochemical analyses of PtdSer transport are now revealing the role of specific protein and lipid assemblies on different organelles that regulate non-vesicular PtdSer transport. The transport of PtdSer from MAM to mitochondria is regulated by at least three genes: MET30 (encoding a ubiquitin ligase), MET4 (encoding a transcription factor), and one or more unknown genes whose transcription is regulated by MET4. MET30-dependent ubiquitination is required for the MAM to function as a competent donor membrane and for the mitochondria to function as a competent acceptor membrane. Non-vesicular transport of PtdSer to the locus of Psd2p is under the control of at least three genes, STT4 [encoding Stt4p (phosphatidylinositol 4-kinase)], PSTB2 (encoding the lipid-binding protein PstB2p) and PSD2 (encoding Psd2p). Stt4p is proposed to produce a pool of PtdIns4P that is necessary for lipid transport. PstB2p and Psd2p must be present on the acceptor membrane for PtdSer transport to occur. Psd2p contains a C2 (Ca2+ and phospholipid binding sequence) domain that is required for lipid transport. Reconstitution studies with chemically defined donor membranes demonstrate that membrane domains rich in the anionic lipids, PtdSer, PtdIns4P and phosphatidic acid function as the most efficient donors of PtdSer to Psd2p. The emerging view is that macromolecular complexes dependent on protein–protein and protein–lipid interactions form between donor and acceptor membranes and serve to dock the compartments and facilitate phospholipid transport.

Background— ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr−/−) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown. Methods and Results— Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr−/− mice. Peritoneal macrophages isolated from the ABCA1 BAC → LDLr−/− chimeras exhibited a 60% ( P =0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245±36×10 3 μm 2 in ABCA1 BAC → LDLr−/− mice versus 210±20×10 3 μm 2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC → LDLr−/− mice remained only 164±15×10 3 μm 2 ( P =0.0008) compared with 513±56×10 3 μm 2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393±27×10 3 μm 2 compared with 640±59×10 3 μm 2 in control transplanted mice; P =0.0015). Conclusion— ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.

Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.

Phospholipid transport between membranes is a fundamental aspect of organelle biogenesis in eukaryotes; however, little is know about this process. A significant body of data demonstrates that newly synthesized phospholipids can move between membranes by routes that are independent of the vesicular traffic that carries membrane proteins. Evidence continues to accumulate in support of a system for phospholipid transport that occurs at zones of apposition and contact between donor membranes - the source of specific phospholipids - and acceptor membranes that are unable to synthesize the necessary lipids. Recent findings identify some of the lipids and proteins that must be present on membranes for inter-organelle phospholipid transport to occur between the endoplasmic reticulum and mitochondria or Golgi. These data suggest that protein and lipid assemblies on donors and acceptors promote membrane docking and facilitate lipid movement.

Recent epidemiological, biological and genetic data indicate a relationship between cholesterol and Alzheimer's disease (AD) including the association of polymorphisms of ABCA1 (a gene that is known to participate in cholesterol and phospholipid transport) with AD prevalence. Based on these data, we postulated that genetic variation in the related and brain-specific ABCA2 gene leads to increase risk of AD. A large case-control study was conducted where the sample was randomly divided into a hypothesis-testing sample (230 cases/286 controls) and a validation sample (210 cases/233 controls). Among the 45 SNPs we tested, one synonymous SNP (rs908832) was found significantly associated with AD in both samples. Additional analyses performed on the whole sample showed a very strong association between this marker and early-onset AD (OR = 3.82, 95% C.I. = [2.00 - 7.30], P = 5 x 10(-5)). Further research is needed to understand the functional role of this polymorphism. However, together with the reported associations of AD with APOE, CYP46A1 and ABCA1, the present result adds a very significant support for the role of cholesterol and phospholipid homeostasis in AD and a rationale for testing novel cholesterol homeostasis-related therapeutic strategies in AD.

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1-Mdm35-PA complex and the functional characterization of Ups1-Mdm35 in PA binding and transfer. Ups1 features a barrel-like structure consisting of an antiparallel β-sheet and three α-helices. Mdm35 adopts a three-helical clamp-like structure to wrap around Ups1 to form a stable complex. The β-sheet and α-helices of Ups1 form a long tunnel-like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate-binding site also plays an important role in the function of Ups1-Mdm35. Our study reveals the molecular basis of the function of Ups1-Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins.

In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. Since cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial outer and inner membranes. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na+,K+-ATPase and Ca2+-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase. P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPalpha was achieved in the presence of DiC7:0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TPalpha, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPalpha required a low temperature (4 degrees C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 microg ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TPalpha per liter of E. coli culture. Conditions for full activation of PI-TPbeta were similar to those for PI-TPalpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 microg ml(-1)) were required. In contrast to PI-TPalpha, PI-TPbeta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta-alanine. In summary, despite 94% sequence similarity, PI-TPalpha and PI-TPbeta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.

Soluble CD14 (sCD14), a 55-kDa glycoprotein found in plasma, has been shown to act as a shuttle for bacterial LPS and phospholipids, transporting LPS and phospholipid monomers from LPS aggregates or liposomes to high density lipoprotein particles. sCD14 has also been shown to mediate the transport of LPS and phosphatidylinositol into cells. Here we show that sCD14 mediates not only the influx but also the efflux of cellular phospholipids. Addition of sCD14 enhanced efflux of cellular phospholipids labeled with [3H]palmitic acid, [3H]oleic acid, or [3H]choline chloride from differentiated THP-1 monocytic cells. Efflux was dependent on the concentration of sCD14 added and was essentially complete in 30 min. The role of membrane-bound CD14 (mCD14) in lipid efflux was assessed using matched pairs of cell lines that express or fail to express this protein. While efflux was very dependent on mCD14 in U373 cells, it was not dependent on mCD14 in Chinese hamster ovary cells, suggesting a role for additional cellular proteins in determining the pathway of phospholipid efflux. A deletion mutant of sCD14 lacking the LPS binding site had less ability to efflux phospholipids than intact sCD14, suggesting that this site is needed for CD14 to serve in phospholipid transport. [3H]Palmitate-labeled lipids released by sCD14 were precipitated with anti-CD14 then analyzed by HPLC. Phosphatidylcholine was the dominant phospholipid exported and bound to sCD14. These results demonstrate that sCD14 mediates efflux of phospholipids from cells and suggest that sCD14 contributes to phospholipid transport in blood.

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5′ flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon–intron boundaries display the canonical GT/AG sequences. Using promoter–luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.

Transbilayer phospholipid asymmetry is a common structural feature of most biological membranes. This organization of lipids is generated and maintained by a number of phospholipid transporters that vary in lipid specificity, energy requirements and direction of transport. These transporters can be divided into three classes: (1) bidirectional, non-energy dependent 'scramblases', and energy-dependent transporters that move lipids (2) toward ('flippases') or (3) away from ('floppases') the cytofacial surface of the membrane. One of the more elusive members of this family is the plasma membrane aminophospholipid flippase, which selectively transports phosphatidylserine from the external to the cytofacial monolayer of the plasma membrane. This review summarizes the characteristics of aminophospholipid flippase activity in intact cells and describes current strategies to identify and isolate this protein. The biochemical characteristics of candidate flippases are critically compared and their potential role in flippase activity is evaluated.

Background : The discovery that the apolipoprotein E4 (apoE4) allele is strongly linked to both sporadic and familial late-onset Alzheimer's disease (AD) raises the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain. We recently proposed that the abnormally low concentrations of apoE observed in the brains of apoE4 AD subjects could compromise cholesterol, fatty acid, and phospholipid transport in the central nervous system. This, in turn, would indirectly impair the cholinergic system, which, in contrast to other neurotransmitters in the central nervous system, relies heavily on lipids to synthesize acetylcholine. Several independent investigators have now confirmed the original observation of an inverse relationship between apoE4 allele copy number and residual brain choline acetyltransferase activity and nicotinic-receptor binding sites in the brains of subjects with AD. More importantly, it has been shown that the presence of the apoE4 allele differentially affects the quality and size of drug responsiveness in subjects with AD treated with cholinomimetic and noncholinomimetic agents. We also examine the role of apoE as a potent therapeutic target for AD.

Human MDR1 P-glycoprotein (P-gp) is a membrane efflux pump for cytotoxics, whereas MDR3 P-gp is a phosphatidylcholine transporter. We have examined a role for MDR1 P-gp in phosphatidylcholine transport in MDR3-negative epithelial cells that have been induced to express the MDR1 P-gp by exposure to cytotoxics. The accumulation and retention of the fluorescently labelled phosphatidylcholine analogue, C12-NBD-PC, was studied in resistant, KBV1 and MCFadr, and sensitive, KB3-1 and MCF7, cells. Lower accumulation and decreased retention of C12-NBD-PC was evident in resistant cells, e.g., KBV1 accumulated 56%, and MCFadr accumulated 60%, of C12-NBD-PC levels in KB3-1 and MCF7, respectively. Treatment with the MDR1 P-gp inhibitor, verapamil, altered the kinetics of C12-NBD-PC in the resistant cells to more closely follow the pattern of C12-NBD-PC handling by sensitive cells. Comparison of C12-NBD-PC to that of the model MDR1 P-gp substrate, rhodamine-123, indicated phosphatidylcholine turnover kinetics by MDR1 P-gp to be relatively low. The transport by MDR1 P-gp of phosphatidylcholine from inner to outer membrane leaflet may regulate P-gp function and fulfill a role in the MDR1 multidrug-resistant phenotype.

The yeast DRS2 gene, which is required for growth at 23 degreesC or below, encodes a member of a P-type ATPase subgroup reported to transport aminophospholipids between the leaflets of the plasma membrane. Here, we evaluated the potential role of Drs2p in phospholipid transport. When examined by fluorescence microscopy, a drs2 null mutant showed no defect in the uptake or distribution of fluorescent-labeled 1-palmitoyl-2[6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD))aminocaproyl]phosphatidylserine) or 1-myristoyl-2[6-NBD-aminocaproyl]phosphatidylethanolamine. Quantification of the amount of cell-associated NBD fluorescence using flow cytometry indicated a significant decrease in the absence of Drs2p, but this decrease was not restricted to the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) and was dependent on culture conditions. Furthermore, the absence of Drs2p had no effect on the amount of endogenous PE exposed to the outer leaflet of the plasma membrane as detected by labeling with trinitrobenzene sulfonic acid. The steady state pool of Drs2p, which was shown to reside predominantly in the plasma membrane, increased upon shift to low temperature or exposure to various divalent cations (Mn2+, Co2+, Ni2+, and Zn2+ but not Ca2+ or Mg2+), conditions that also inhibited the growth of a drs2 null mutant. The data presented here call into question the identification of Drs2p as the exclusive or major aminophospholipid translocase in yeast plasma membranes (Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497).

Phospholipid transfer protein (PLTP) regulates phospholipid transport in the circulation and is highly expressed within the lung epithelium, where it is secreted into the alveolar space. Since PLTP expression is increased in chronic obstructive pulmonary disease (COPD), this study aimed to determine how PLTP affects lung signaling and inflammation. Despite its increased expression, PLTP activity decreased by 80% in COPD bronchoalveolar lavage fluid (BALF) due to serine protease cleavage, primarily by cathepsin G. Likewise, PLTP BALF activity levels decreased by 20 and 40% in smoke-exposed mice and in the media of smoke-treated small airway epithelial (SAE) cells, respectively. To assess how PLTP affected inflammatory responses in a lung injury model, PLTP siRNA or recombinant protein was administered to the lungs of mice prior to LPS challenge. Silencing PLTP at baseline caused a 68% increase in inflammatory cell infiltration, a 120 and 340% increase in ERK and NF-κB activation, and increased MMP-9, IL1β, and IFN-γ levels after LPS treatment by 39, 140, and 190%, respectively. Conversely, PLTP protein administration countered these effects in this model. Thus, these findings establish a novel anti-inflammatory function of PLTP in the lung and suggest that proteolytic cleavage of PLTP by cathepsin G may enhance the injurious inflammatory responses that occur in COPD.—Brehm, A., Geraghty, P., Campos, M., Garcia-Arcos, I., Dabo, A. J., Gaffney, A., Eden, E., Jiang, X.-C., D'Armiento, J., Foronjy, R. Cathepsin G degradation of phospholipid transfer protein (PLTP) augments pulmonary inflammation.

The influence of the suspension pH (pHo ) on the transmembrane mobility of spin-labeled phospholipid analogues in the human red blood cell was investigated. The passive transverse diffusion of spin-labeled phospholipid analogues was independent of pHo in the investigated range (5.8 to 8.5). However, upon acidification to pHo 5.8, a significant decrease of the rapid adenosine triphosphate (ATP)-dependent inward movement of aminophospholipids was found at physiologic ionic concentration, whereas a change of pH from 7.4 to 8.5 did not affect this transport. Evidence is given that the intracellular pH affects the active transport of aminophospholipids but not the extracellular pH. Suppression of the ATP-dependent outside-inside redistribution of aminophospholipid analogues by low pH was reversible because original transport activity was re-established upon reneutralization. pH dependence of the active phospholipid transport was not caused by the spin-labeled reporter group or by depletion of intracellular ATP. Because the same influence of pH on aminophospholipid movement could be observed for resealed ghosts, constituents of the red blood cell cytoplasm do not mediate the influence of pH on the ATP-dependent inward movement of aminophospholipids.

Multidrug resistance (MDR) describes the phenomenon of simultaneous resistance to unrelated drugs. It has been a decade since the P-glycoprotein (Pgp) gene, which is associated with a form of MDR caused by reduced drug accumulation, was cloned. Thus, this would seem to be an appropriate time to evaluate our understanding of this form of MDR. The two MDR genes identified in humans to date (the MDR-associated protein [MRP] and Pgp genes) are structurally similar and both are members of the ATP-binding cassette (ABC) transporter family. Although the physiological role of MRP is not yet understood, one Pgp gene (mdr1) plays an important role in the blood-tissue barrier and the other (mdr2/3) is involved in phospholipid transport in the liver. A variety of compounds (chemosensitizing agents) can interfere with Pgp and MRP function; such agents may improve the efficacy of conventional therapy when used in combination with such regimens. Determining the roles cellular MDR mechanisms play in patients' response to chemotherapy is a major challenge. Using Pgp and MRP as molecular markers to detect MDR tumor cells is technically demanding, and solid tumors in particular contain heterogeneous cell populations. Since MDR requires Pgp or MRP gene expression, clinically relevant gene expression thresholds need to be established; sequential samples from individual patients are valuable for correlating MDR gene expression with the clinical course of disease. Studies in leukemias, myelomas, and some childhood cancers show that Pgp expression correlates with poor response to chemotherapy. However, in some cases, inclusion of a reversing or chemosensitizing agent such as verapamil or cyclosporin A has improved clinical efficacy. Such agents may inactivate Pgp in tumor cells or affect Pgp function in normal cells, resulting in altered pharmacokinetics. It would be interesting to determine whether patients who fail treatment in the presence of chemosensitizing agents acquire other MDR mechanisms. The ABC transporter superfamily in prokaryotes and eukaryotes is involved in the transport of substrates ranging from ions to large proteins. Of the 15 or more ABC transporter genes characterized in human cells, two (Pgp and MRP) cause MDR. Therefore, it would be relevant to determine the number of such genes present in the human genome; however, extrapolating from the number of ABC transporter genes in bacteria, the human gene probably contains a minimum of 200 ABC transporter superfamily members. Thus, tumor cells can potentially use many ABC transporters to mount resistance to known and future therapeutic agents. The challenge will be to determine which ABC transporters are clinically relevant. Despite the potential of tumor cells to protect themselves, a variety of malignancies can be successfully treated with chemotherapy. This may provide unique insights.

Pulmonary surfactant is required for lung function at birth and throughout postnatal life. Defects in the surfactant system are associated with common pulmonary disorders including neonatal respiratory distress syndrome and acute respiratory distress syndrome in children and adults. Lipogenesis is essential for the synthesis of pulmonary surfactant by type II epithelial cells lining the alveoli. This study sought to identify the role of pulmonary epithelial SREBP, a transcriptional regulator of cellular lipid homeostasis, during a critical time period of perinatal lung maturation in the mouse. Genome wide mRNA expression profiling of lung tissue from transgenic mice with epithelial-specific deletions of Scap (Scap(Δ/Δ), resulting in inactivation of SREBP signaling) or Insig1 and Insig2 (Insig1/2(Δ/Δ), resulting in activation of SREBP signaling) was assessed. Differentially expressed genes responding to SREBP perturbations were identified and subjected to functional enrichment analysis, pathway mapping and literature mining to predict upstream regulators and transcriptional networks regulating surfactant lipid homeostasis. Through comprehensive data analysis and integration, time dependent effects of epithelial SCAP/INSIG/SREBP deletion and defined SCAP/INSIG/SREBP-associated genes, bioprocesses and downstream pathways were identified. SREBP signaling influences epithelial development, cell death and cell proliferation at E17.5, while primarily influencing surfactant physiology, lipid/sterol synthesis, and phospholipid transport after birth. SREBP signaling integrated with the Wnt/β-catenin and glucocorticoid receptor signaling pathways during perinatal lung maturation. SREBP regulates perinatal lung lipogenesis and maturation through multiple mechanisms by interactions with distinct sets of regulatory partners.

ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.

Most P-type ATPases pump specific cations or heavy metals across a membrane to form ion gradients. However, the type IV P-type ATPases evolved the ability to transport specific phospholipid substrates rather than cations and function to establish plasma membrane asymmetry in eukaryotic cells. The mechanism for how a P-type ATPase, or any other transporter, can recognize and flip a phospholipid substrate is unclear. Here, through a combination of genetic screening and directed mutagenesis with the type IV P-type ATPases Dnf1 and Drs2 from budding yeast, we identify more than a dozen residues that determine headgroup specificity for phospholipid transport. These residues cluster at two interfacial regions flanking transmembrane segments 1–4 and lie outside of the canonical substrate binding site operating in cation pumps. Our data imply the presence of two substrate-selecting gates acting sequentially on opposite sides of the membrane: an entry gate, where phospholipid is initially selected from the extracellular leaflet, and an exit gate at the cytosolic leaflet. The entry and exit gates act cooperatively but imperfectly, with neither being able to restrict phosphatidylserine selection completely when the opposing gate is tuned to permit it. This work describes a unique transport mechanism for a P-type ATPase and provides insight into how integral membrane proteins can recognize and transport phospholipid substrate across a lipid bilayer.

The mode of transport of biliary lipids within the hepatocyte and the role of the bile canalicular membrane (BCM) in biliary lipid secretion are not well understood. We hypothesized that biliary cholesterol and phospholipid are co-transported across the hepatocyte in vesicular form from the endoplasmic reticulum to the bile across the BCM. We obtained wedge liver biopsies and fasting gallbladder bile from 15 cholesterol gallstone patients and 10 control subjects. BCM, basolateral membrane (BLM), and many microsomal vesicular fractions were isolated by centrifugation. One of the vesicular fractions (V3) was enriched in both the microsomal and the BCM marker enzymes and had a high phosphatidylcholine proportion in its phospholipid with a fatty acid pattern similar to biliary phosphatidylcholine. Moreover, its cholesterol content was increased in the obese cholesterol gallstone subjects, who had an increase in cholesterol synthesis, as indicated by the increased activity of the HMG-CoA reductase. The cholesterol content correlated with HMG-CoA reductase activity. A direct correlation was found between cholesterol/phospholipid ratio in V3, BCM, and in bile but not in the BLM. These data are in agreement with the assumption that this vesicular fraction is involved in the transport of cholesterol and phospholipid from the endoplasmic reticulum to the site of secretion in the BCM, and thence to bile, and that this transport is enhanced in obese gallstone patients.

The molecular cloning of mouse plasma phospholipid transfer protein (PLTP) and the eukaryotic cell expression of complementary DNA for mouse and human PLTP are described. Mouse PLTP was found to share 83% amino acid sequence identity with human PLTP. PLTP was produced in baby hamster kidney cells. Conditioned medium from BHK cells expressing PLTP possessed both phospholipid transfer activity and high density lipoprotein (HDL) conversion activity. PLTP mRNA was detected in all 16 human tissues examined by Northern blot analysis with the ovary, thymus, and placenta having the highest levels. PLTP mRNA was also examined in eight mouse tissues with the highest PLTP mRNA levels found in the lung, brain, and heart. The effect of purified human plasma-derived PLTP and human recombinant PLTP (rPLTP) on the two human plasma HDL subspecies Lp(A-I) and Lp(A-I/A-II) was evaluated. Plasma PLTP or rPLTP converted the two distinct size subspecies of Lp(A-I) into a larger species, an intermediate species, and a smaller species. Lp(A-I/A-II) particles containing multiple size subspecies were significantly altered by incubation with either plasma or rPLTP with the largest but less prominent subspecies becoming the predominant one, and the smallest subspecies increasing in concentration. Thus, PLTP promoted the conversion of both Lp(A-I) and Lp(A-I/A-II) to populations of larger and smaller particles. Also, both human PLTP and mouse rPLTP were able to convert human or mouse HDL into larger and smaller particles. These observations suggest that PLTP may play a key role in extracellular phospholipid transport and modulation of HDL particles.

Experimental data on the small angle synchrotron X-ray scattering (SAXS) are analyzed on a basis of Separated form factors method (SFF) for a study of the drug delivery Phospholipid Transport Nano System (PTNS). Basic parameters of polydispersed population of PTNS nanoparticles (average radius of PTNS-particles, polydispersity of radius, thickness of membrane) have been determined. The results are discussed in comparison with the SFF results for the "classical" vesicular system of dimyristoylphosphocholine (DMPC).

The P4 subfamily of P-type ATPases includes phospholipid transporters. Moving such bulky amphipathic substrate molecules across the membrane poses unique mechanistic problems. Recently, three papers from three different laboratories have offered insights into some of these problems. One effect of these experiments will be to ignite a healthy debate about the path through the enzyme taken by the substrate. A second effect is to suggest a counterintuitive model for the critical substrate-binding site. By putting concrete hypotheses into play, these papers finally provide a foundation for investigations of mechanism for these proteins.

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.

Protozoans of the Leishmania genus are the etiological agents of a wide spectrum of diseases commonly known as leishmaniases. Lipid organization of the plasma membrane of the parasite may mimic the lipid organization of mammalian apoptotic cells and play a role in phagocytosis and parasite survival in the mammal host. Here, we analyzed the phospholipid dynamics in the plasma membrane of both the L. (Leishmania) and the L. (Viannia) subgenera. We found that the activity and substrate specificity of the inward translocation machinery varied between Leishmania species. The differences in activity of inward phospholipid transport correlated with the different sensitivities of the various species towards the alkyl-phospholipid analogue miltefosine. Furthermore, all species exhibited a phospholipid scramblase activity in their plasma membranes upon stimulation with calcium ionophores. However, binding of annexin V to the parasite surface was only detected for a subpopulation of parasites during the stationary growth phase and only marginally enhanced by scramblase activation.

Erythrocytes infected with mature-stage malaria parasites accumulate phospholipids from exogenous sources. We show that the transport of N-(7-nitrobenzy-2-oxa-1,3-diazol-4-yl)-1,2- dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-NBD-DPPE), from the erythrocyte membrane to the intracellular malaria parasite, is dependent upon metabolic energy. A photoreactive phospholipid analogue, N-[125I]iodo-4-azidosalicylamidyl-1, 2-dilauryl-sn-glycero-3-phosphatidylethanolamine (N-125I-ASA-DLPE), has been synthesised and used in an attempt to identify proteins involved in phospholipid trafficking in malaria-infected erythrocytes. This photoreactive probe was found to preferentially label a protein with an apparent molecular weight of 22 kDa. Photolabelling of the 22-kDa protein was enhanced upon ATP depletion of malaria-infected erythrocytes.

Summary A recent study now shows that opsin catalyzes rapid movement of phospholipids from one leaflet of a membrane bilayer to the other. This capability illuminates a mechanism for this physiologically important process.

While accumulating evidence indicates that P4-ATPases catalyze phospholipid transport across cellular bilayers, their kinship to cation-pumping ATPases has raised fundamental questions concerning the underlying flippase mechanism. Loss of P4-ATPase function perturbs vesicle formation in late secretory and endocytic compartments. An intriguing concept is that P4-ATPases help drive vesicle budding by generating imbalances in transbilayer lipid numbers. Moreover, activation of P4-ATPases by phosphoinositides and other effectors of coat recruitment provide a potential mechanism to confine flippase activity to sites of vesicle biogenesis. These developments have raised considerable interest in understanding the mechanism, regulation and biological implications of P4-ATPase-catalyzed phospholipid transport.

Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.

Background & Aims Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4 −/− mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. Methods To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. Results Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1–CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. Conclusions ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1–CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.

Members of the P4 subfamily of P-type ATPases catalyze phospholipid transport and create membrane lipid asymmetry in late secretory and endocytic compartments. P-type ATPases usually pump small cations and the transport mechanism involved appears conserved throughout the family. How this mechanism is adapted to flip phospholipids remains to be established. P 4-ATPases form heteromeric complexes with CDC50 proteins. Dissociation of the yeast P4-ATPase Drs2p from its binding partner Cdc50p disrupts catalytic activity (Lenoir, G., Williamson, P., Puts, C. F., and Holthuis, J. C. (2009) J. Biol. Chem. 284, 17956-17967), suggesting that CDC50 subunits play an intimate role in the mechanism of transport by P 4-ATPases. The human genome encodes 14 P4-ATPases while only three human CDC50 homologues have been identified. This implies that each human CDC50 protein interacts with multiple P4-ATPases or, alternatively, that some human P4-ATPases function without a CDC50 binding partner. Here we show that human CDC50 proteins each bind multiple class-1 P4-ATPases, and that in all cases examined, association with a CDC50 subunit is required for P4-ATPase export from the ER. Moreover, we find that phosphorylation of the catalytically important Asp residue in human P 4-ATPases ATP8B1 and ATP8B2 is critically dependent on their CDC50 subunit. These results indicate that CDC50 proteins are integral part of the P4-ATPase flippase machinery. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc

The effects of exogenous ethanolamine concentrations on ethanolamine uptake and its subsequent incorporation into phosphatidylethanolamine were examined. Hamster hearts were perfused with 0.04-1000 microM labelled ethanolamine. Analysis of radioactivity distribution in ethanolamine-containing metabolites revealed an accumulation of labelled ethanolamine when the heart was perfused with greater than or equal to 0.4 microM labelled ethanolamine. The changes in radioactivity distribution indicated that the phosphorylation of ethanolamine had become rate-limiting in the CDP-ethanolamine pathway when the heart was perfused with greater than or equal to 0.4 microM ethanolamine. Perfusion with different concentrations of ethanolamine did not significantly change the intracellular ethanolamine pool. The accumulation of labelled ethanolamine without a corresponding change in the ethanolamine pool suggests that the newly imported ethanolamine did not equilibrate with the endogenous ethanolamine pool. We postulate that the newly imported ethanolamine was preferentially utilized for phosphatidylethanolamine biosynthesis.

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans . Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans , we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA 3 ) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA 3 ) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA 3 ). Location profiling using whole-genome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA 3 or Cap1p-CSE-HA 3 (the binding ratio was at least twofold; P ≤ 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3′ ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets ( P ≤ 0.02) included 11 genes involved in the OSR ( CAP1 , GLR1 , TRX1 , SOD1 , CAT1 , and others), 13 genes involved in response to drugs ( PDR16 , MDR1 , FLU1 , YCF1 , FCR1 , and others), 4 genes involved in phospholipid transport ( PDR16 , GIT1 , RTA2 , and orf19.932), and 3 genes involved in the regulation of nitrogen utilization ( GST3 , orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5′-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.

The outer membranes (OMs) of Gram-negative bacteria have an asymmetric lipid distribution with lipopolysaccharides at the outer leaflet and phospholipids (PLs) at the inner leaflet. This lipid arrangement is essential for the barrier function of the OM and for the viability of most Gram-negative bacteria. Cells with OM assembly defects or cells exposed to harsh chemical treatments accumulate PLs in the outer leaflet of the OM and this disrupts lipopolysaccharide organization and increases sensitivity to small toxic molecules. We have identified an ABC transport system in Escherichia coli with predicted import function that serves to prevent PL accumulation in the outer leaflet of the OM. This highly conserved pathway, which we have termed the Mla pathway for its role in preserving OM lipid asymmetry, is composed of at least 6 proteins and contains at least 1 component in each cellular compartment. We propose that the Mla pathway constitutes a bacterial intermembrane PL trafficking system.

Members of the P(4) subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport mechanism that appears to be conserved throughout the family. A challenging problem is to understand how this mechanism is adapted in P(4) ATPases to flip phospholipids. P(4) ATPases form oligomeric complexes with members of the CDC50 protein family. While formation of these complexes is required for P(4) ATPase export from the endoplasmic reticulum, little is known about the functional role of the CDC50 subunits. The Na(+)/K(+)-ATPase and closely-related H(+)/K(+)-ATPase are the only other P-type pumps that are oligomeric, comprising mandatory beta-subunits that are strikingly reminiscent of CDC50 proteins. Besides serving a role in the functional maturation of the catalytic alpha-subunit, the beta-subunit also contributes specifically to intrinsic transport properties of the Na(+)/K(+) pump. As beta-subunits and CDC50 proteins likely adopted similar structures to accomplish analogous tasks, current knowledge of the Na(+)/K(+)-ATPase provides a useful guide for understanding the inner workings of the P(4) ATPase class of lipid pumps.

The association of hypercholesterolemia and obesity with airway hyperresponsiveness has drawn increasing attention to the potential role of cholesterol and lipid homeostasis in lung physiology and in chronic pulmonary diseases such as asthma. We have recently shown that activation of the nuclear hormone receptor liver X receptor (LXR) stimulates cholesterol efflux in human airway smooth muscle (hASM) cells and induces expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, members of a family of proteins that mediate reverse cholesterol and phospholipid transport. We show here that ABCA1 is responsible for all LXR-mediated cholesterol and phospholipid efflux to both apolipoprotein AI and high-density lipoprotein acceptors. In contrast, ABCG1 does not appear to be required for this process. Moreover, we show that hASM cells respond to increased levels of cholesterol by inducing expression of ABCA1 and ABCG1 transporters, a process that is dependent on LXR expression. These findings establish a critical role for ABCA1 in reverse cholesterol and phospholipid transport in airway smooth muscle cells and suggest that dysregulation of cholesterol homeostasis in these cells may be important in the pathogenesis of diseases such as asthma.

Transfer of phosphatidic acid from the outer to the inner membrane within intact rat liver mitochondria was assessed by measuring the ratio of lipid 32P to the marker enzyme of the outer membrane, rotenone-insensitive NADH-cytochrome c reductase, in the outer and inner membrane fractions obtained after incubation of mitochondria under conditions for net synthesis of [32P]phosphatidic acid. This transfer was found to proceed with time, to occur only under high ionic strength of the external medium and to be insensitive to N-ethylmaleimide and factors reducing the number of contact sites between the two mitochondrial membranes. These results are interpreted as supporting the idea that phosphatidic acid transport within the mitochondrion occurs as free diffusion through the aqueous phase and not being mediated by phospholipid transfer protein(s).

Cryptococcus neoformans is a basidiomycetous fungus and an opportunistic human pathogen that causes infections in both immunocompromised and immunocompetent hosts. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this microorganism. Representational difference analysis (RDA) was used to profile gene expression in C. neoformans grown at 37 degrees C or 25 degrees C. Contig assembly of 300 high-quality sequenced cDNAs and comparison analysis to the GenBank database led to the identification of transcripts that may be critical for both pathogen-host interactions and responses to either low or high temperature growth. Gene products involved in cell wall integrity, stress response, filamentation, oxidative metabolism, protein targeting and fatty acids metabolism were induced at 37 degrees C. In addition, genes related to chromatin silencing and phospholipid transport were upregulated at 25 degrees C. Therefore, our RDA analysis, comparing saprophytic and host temperature conditions, revealed new genes with potential involvement in C. neoformans virulence.

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.

Inter- and intramembrane phospholipid transport processes are central features of membrane biogenesis and homeostasis. Relatively recent successes in the molecular genetic analysis of aminoglycerophospholipid transport processes in both yeast and mammalian cells are now providing important new information defining specific protein and lipid components that participate in these reactions. Studies focused on phosphatidylserine (PtdSer) transport to the mitochondria reveal that the process is regulated by ubiquitination. In addition, a specific mutation disrupts PtdSer transport between mitochondrial membranes. Analysis of PtdSer transport from the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 demonstrates the requirement for a phosphatidylinositol-4-kinase, a phosphatidylinositol-binding protein, and the C2 domain of the decarboxylase. Examination of NBD-phosphatidylcholine transport demonstrates the involvement of the prevacuolar compartment and a requirement for multiple genes involved in regulating vacuolar protein sorting for transport of the lipid to the vacuole. In intramembrane transport, multiple genes are now identified including those encoding multidrug resistant protein family members, DNF family members, ATP binding cassette transporters, and pleiotropic drug resistance family members. The scramblase family constitutes a collection of putative transmembrane transporters that function in an ATP-independent manner. The genetic analysis of lipid traffic is uncovering new molecules involved in all aspects of the regulation and execution of the transport steps and also providing essential tools to critically test the involvement of numerous candidate molecules.Key words: lipid transport, lipid sorting, membrane biogenesis, organelles, flippase.