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Robert C. Murphy - One of the best experts on this subject based on the ideXlab platform.
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analysis of polyunsaturated Aminophospholipid molecular species using isotope tagged derivatives and tandem mass spectrometry mass spectrometry mass spectrometry
Analytical Biochemistry, 2006Co-Authors: Karin Zemski A Berry, Robert C. MurphyAbstract:Abstract When Aminophospholipids with only saturated and monounsaturated fatty acids esterified to the glycerol backbone were labeled with isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, it was found that they could be readily detected as N-methylpiperazine-amide-tagged Aminophospholipids using a precursor scan of the stable isotope reporter ion (m/z 114–117) formed by tandem mass spectrometry/mass spectrometry. However, it was found in the current study that these precursor ion scans are not useful in determining the changes of Aminophospholipids with polyunsaturated fatty acids (PUFAs) esterified to the glycerol backbone due to the presence of interfering ions in the reporter ion region. Therefore, a method was developed using tandem mass spectrometry/mass spectrometry/mass spectrometry (MS3) to obtain reporter ion ratios that were not distorted by interfering ions present in the collision-induced dissociation spectra of nontagged Aminophospholipids with PUFAs. This new MS3 method for N-methylpiperazine- amide-tagged Aminophospholipids was used to examine the fate of diacyl, ether, or plasmalogen glycerophosphoethanolamine (GPEtn) species after exposure of human polymorphonuclear leukocytes to A23187 and granulocyte macrophage–colony-stimulating factor/formyl-methionyl-leucyl-phenylalanine stimuli, which can induce eicosanoid biosynthesis, to follow those GPEtn molecular species which were the source of arachidonic acid released. Upon stimulation of the human polymorphonuclear leukocyte, it was found that the abundant arachidonoyl GPEtn plasmalogen molecular species were uniquely reduced in relative content compared to ether or diacyl species and this subclass of GPEtn may be a source of the arachidonic acid converted to leukotrienes by the 5-lipoxygenase pathway activated in this cell.
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Analysis of polyunsaturated Aminophospholipid molecular species using isotope-tagged derivatives and tandem mass spectrometry/mass spectrometry/mass spectrometry
Analytical Biochemistry, 2005Co-Authors: Karin A. Zemski Berry, Robert C. MurphyAbstract:Abstract When Aminophospholipids with only saturated and monounsaturated fatty acids esterified to the glycerol backbone were labeled with isotopically enriched N -methylpiperazine acetic acid N -hydroxysuccinimide ester reagents, it was found that they could be readily detected as N -methylpiperazine-amide-tagged Aminophospholipids using a precursor scan of the stable isotope reporter ion ( m / z 114–117) formed by tandem mass spectrometry/mass spectrometry. However, it was found in the current study that these precursor ion scans are not useful in determining the changes of Aminophospholipids with polyunsaturated fatty acids (PUFAs) esterified to the glycerol backbone due to the presence of interfering ions in the reporter ion region. Therefore, a method was developed using tandem mass spectrometry/mass spectrometry/mass spectrometry (MS 3 ) to obtain reporter ion ratios that were not distorted by interfering ions present in the collision-induced dissociation spectra of nontagged Aminophospholipids with PUFAs. This new MS 3 method for N -methylpiperazine- amide-tagged Aminophospholipids was used to examine the fate of diacyl, ether, or plasmalogen glycerophosphoethanolamine (GPEtn) species after exposure of human polymorphonuclear leukocytes to A23187 and granulocyte macrophage–colony-stimulating factor/formyl-methionyl-leucyl-phenylalanine stimuli, which can induce eicosanoid biosynthesis, to follow those GPEtn molecular species which were the source of arachidonic acid released. Upon stimulation of the human polymorphonuclear leukocyte, it was found that the abundant arachidonoyl GPEtn plasmalogen molecular species were uniquely reduced in relative content compared to ether or diacyl species and this subclass of GPEtn may be a source of the arachidonic acid converted to leukotrienes by the 5-lipoxygenase pathway activated in this cell.
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Analysis of cell membrane Aminophospholipids as isotope-tagged derivatives
Journal of lipid research, 2005Co-Authors: Karin A. Zemski Berry, Robert C. MurphyAbstract:Glycerophosphoethanolamine (GPEtn) and glyc- erophosphoserine (GPSer) lipids were reacted with a multi- plexed set of differentially isotopically enriched N -methylpiper- azine acetic acid N -hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The re- sulting derivatized Aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions ( m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectro- metric response of N -methylpiperazine amide-tagged amino- phospholipids was probed using glycerophosphoethanol- amine and glycerophosphoserine lipid standards. The (MH) � of each tagged Aminophospholipid shifted 144 Da, and dur- ing collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting ami- nophospholipids was useful for an unbiased analysis of plas- malogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the (M-H) � ions. The isotope-tagged re- agents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phos- pholipids extracted from RAW 264.7 cells to Cu 2 � /H 2 O 2 to illustrate the ability of these reagents to aid in the mass spec- trometric identification of Aminophospholipid changes that occur during biological stimuli. —Zemski Berry, K. A., and R. C. Murphy. Analysis of cell membrane Aminophospholipids as isotope-tagged derivatives. J. Lipid Res. 2005. 46: 1038-1046.
Philippe F. Devaux - One of the best experts on this subject based on the ideXlab platform.
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RECONSTITUTION OF FLIPPASE ACTIVITY INTO LIPOSOMES
2014Co-Authors: Philippe F. DevauxAbstract:Phospholipids are asymmetrically distributed in the plasma membrane of eucaryotic cells, the Aminophospholipids being mostly on the cytosolic leaflet, whilst the choline containing phospholipids (phosphatidylcholine and sphingomyelin) are predominantly exposed on the outer leaflet [1]. There is strong evidence in support of the role of specific proteins ( ” flippases„) that are involved in the transmembrane segregation of phospholipids [1-3]. Among these proteins, some are active transporters. The Aminophospholipid translocase is a ubiquitous protein that transports selectively phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE) from the outer to the inner monolayer of the plasma membranes of eucaryotic cells [1]. In the same cells, the P-glycoprotein (P-gp) and several proteins of the ABC family have been reported to transport phospholipids from the inner monolayer to the outer monolayer [2, 4]. Finally a calcium dependent ” scramblase „ plays a key role in the ATP-independent rapid lipid randomization, which takes place in stimulated platelets or during apoptotis, leading in particular to phosphatidylserine exposure on the outer cel
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Aminophospholipids Have No Access to the Luminal Side of the Biliary Canaliculus IMPLICATIONS FOR THE SPECIFIC LIPID COMPOSITION OF THE BILE FLUID
The Journal of biological chemistry, 2003Co-Authors: Astrid Tannert, Philippe F. Devaux, Daniel Wüstner, Josefine Bechstein, Peter Müller, Andreas HerrmannAbstract:About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both Aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both Aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. DiacylAminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an Aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylAminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylAminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of Aminophospholipids to the bile.
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drs2p related p type atpases dnf1p and dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis
Molecular Biology of the Cell, 2003Co-Authors: Thomas Günther Pomorski, Philippe F. Devaux, Ruben Lombardi, Howard Riezman, Gerrit Van Meer, Joost C. M. HolthuisAbstract:Plasma membranes in eukaryotic cells display asymmetric lipid distributions with Aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by...
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Aminophospholipids Have No Access to the Luminal Side of the Biliary Canaliculus
2003Co-Authors: Astrid Tannert, Philippe F. Devaux, Josefine Bechstein, Andreas HerrmannAbstract:About 95% of the bile phospholipids are phosphatidylcholine. Although the fractions of phosphatidylcholine and of both Aminophospholipids phosphatidylserine and phosphatidylethanolamine in the canalicular membrane are in the same order of about 35% of total lipids, both Aminophospholipids are almost absent from the bile. To rationalize this observation, we studied the intracellular uptake of various fluorescent phospholipid analogues and their subsequent enrichment in the bile canaliculus (BC) of HepG2 cells. DiacylAminophospholipid analogues but not phosphatidylcholine analogues became rapidly internalized by an Aminophospholipid translocase (APLT) activity in the plasma membrane of HepG2 cells. We observed only low labeling of BC by diacylAminophospholipids but extensive staining by phosphatidylcholine analogues. In the presence of suramin, known to inhibit APLT, a strong labeling of BC by diacylAminophospholipid analogues was found that declined to a level observed for control cells after removal of suramin. Unlike diacylphosphatidylserine, diether phosphatidylserine analogue, which is not an appropriate substrate of APLT, accumulated in the BC. The correlation between low labeling of BC and an APLT-mediated transbilayer movement suggests the presence of an APLT activity in the canalicular membrane that prevents exposure of Aminophospholipids to the bile.
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Characterization of the Correlation Between ATP‐Dependent Aminophospholipid Translocation and Mg2+‐ATPase Activity in Red Blood Cell Membranes
European journal of biochemistry, 1997Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Peter OttAbstract:Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and Aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, Aminophospholipid translocation from the outer to the inner leaflets of re-sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and Aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of Aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in Aminophospholipid translocation.
Peter Ott - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the Correlation Between ATP‐Dependent Aminophospholipid Translocation and Mg2+‐ATPase Activity in Red Blood Cell Membranes
European journal of biochemistry, 1997Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Peter OttAbstract:Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and Aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, Aminophospholipid translocation from the outer to the inner leaflets of re-sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and Aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of Aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in Aminophospholipid translocation.
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characterization of the correlation between atp dependent Aminophospholipid translocation and mg2 atpase activity in red blood cell membranes
FEBS Journal, 1997Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Peter OttAbstract:Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and Aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, Aminophospholipid translocation from the outer to the inner leaflets of re-sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and Aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of Aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in Aminophospholipid translocation.
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ATP-dependent Aminophospholipid translocation in erythrocyte vesicles: stoichiometry of transport.
Biochemistry, 1993Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Marta Puente Navazo, Peter OttAbstract:Vesicles released from human red blood cells by incubation with a suspension of sonicated dimyristoylphosphatidylcholine were purified by gel filtration. Purified vesicles and intact red cells had a very similar composition with respect to phospholipids and integral membrane proteins, but spectrin, the major component of the membrane skeleton, was not found in vesicles. Comparison of red cell and vesicle ATP levels (expressed as micromolar ATP per millimolar hemoglobin) showed a marked difference with a reduced content of only about 30% in vesicles, whatever the initial concentration in the erythrocytes. Spin-labeled Aminophospholipids (phosphatidylserine and phosphatidylethanolamine) were translocated to the inner vesicle membrane layer at a comparable rate as in intact red cells provided that vesicles contained enough ATP. The maximum fraction of spin-labeled phospholipids translocated to the inner membrane layer was 84% for phosphatidylserine, 65% for phosphatidylethanolamine, 20-40% for phosphatidylcholine, and below 20% for sphingomyelin. The apparent Km of translocation, expressed as percent of total membrane phospholipid, was 0.14% for spin-labeled phosphatidylserine and 1.19% for spin-labeled phosphatidylethanolamine. This compares well to values established earlier for intact red blood cells. The fact that no ATP was synthesized in vesicles allowed determination of ATP consumption by Aminophospholipid transport. The basic ATP hydrolysis rate was increased upon the addition of labeled Aminophospholipids but not of labeled phosphatidylcholine or sphingomyelin. The stoichiometry between lipid translocation and ATP consumption, calculated from the respective initial velocities, was 1.13 +/- 0.2 for phosphatidylserine and 1.11 +/- 0.16 for phosphatidylethanolamine.
Alain Zachowski - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the Correlation Between ATP‐Dependent Aminophospholipid Translocation and Mg2+‐ATPase Activity in Red Blood Cell Membranes
European journal of biochemistry, 1997Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Peter OttAbstract:Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and Aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, Aminophospholipid translocation from the outer to the inner leaflets of re-sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and Aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of Aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in Aminophospholipid translocation.
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characterization of the correlation between atp dependent Aminophospholipid translocation and mg2 atpase activity in red blood cell membranes
FEBS Journal, 1997Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Peter OttAbstract:Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and Aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, Aminophospholipid translocation from the outer to the inner leaflets of re-sealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and Aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of Aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in Aminophospholipid translocation.
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Protein-dependent translocation of Aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.
Biochemistry, 1994Co-Authors: Karin Mueller, Alain Zachowski, Thomas Günther Pomorski, Peter Mueller, Andreas HerrmannAbstract:We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of Aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated Aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an Aminophospholipid translocase and an asymmetric transversal lipid distribution with Aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.
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ATP-dependent Aminophospholipid translocation in erythrocyte vesicles: stoichiometry of transport.
Biochemistry, 1993Co-Authors: Zsuzsa Beleznay, Alain Zachowski, Philippe F. Devaux, Marta Puente Navazo, Peter OttAbstract:Vesicles released from human red blood cells by incubation with a suspension of sonicated dimyristoylphosphatidylcholine were purified by gel filtration. Purified vesicles and intact red cells had a very similar composition with respect to phospholipids and integral membrane proteins, but spectrin, the major component of the membrane skeleton, was not found in vesicles. Comparison of red cell and vesicle ATP levels (expressed as micromolar ATP per millimolar hemoglobin) showed a marked difference with a reduced content of only about 30% in vesicles, whatever the initial concentration in the erythrocytes. Spin-labeled Aminophospholipids (phosphatidylserine and phosphatidylethanolamine) were translocated to the inner vesicle membrane layer at a comparable rate as in intact red cells provided that vesicles contained enough ATP. The maximum fraction of spin-labeled phospholipids translocated to the inner membrane layer was 84% for phosphatidylserine, 65% for phosphatidylethanolamine, 20-40% for phosphatidylcholine, and below 20% for sphingomyelin. The apparent Km of translocation, expressed as percent of total membrane phospholipid, was 0.14% for spin-labeled phosphatidylserine and 1.19% for spin-labeled phosphatidylethanolamine. This compares well to values established earlier for intact red blood cells. The fact that no ATP was synthesized in vesicles allowed determination of ATP consumption by Aminophospholipid transport. The basic ATP hydrolysis rate was increased upon the addition of labeled Aminophospholipids but not of labeled phosphatidylcholine or sphingomyelin. The stoichiometry between lipid translocation and ATP consumption, calculated from the respective initial velocities, was 1.13 +/- 0.2 for phosphatidylserine and 1.11 +/- 0.16 for phosphatidylethanolamine.
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Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry
Blood, 1991Co-Authors: Nadia Blumenfeld, Alain Zachowski, Yves Beuzard, Frederic Galacteros, Pf DevauxAbstract:We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of Aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the Aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower Aminophospholipid transport rate. These data favor the relationship between Aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of Aminophospholipid translocase pathology.
Thomas Günther Pomorski - One of the best experts on this subject based on the ideXlab platform.
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a flippase independent function of atp8b1 the protein affected in familial intrahepatic cholestasis type 1 is required for apical protein expression and microvillus formation in polarized epithelial cells
Hepatology, 2010Co-Authors: Patricia M Verhulst, Lieke M. Van Der Velden, Roderick H.j. Houwen, Ernst E. Van Faassen, Joost C. M. Holthuis, Thomas Günther Pomorski, Viola Oorschot, Judith Klumperman, Leo W. J. KlompAbstract:Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P4 P-type adenosine triphosphatase (ATPase) family of putative Aminophospholipid translocases, and loss of Aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific Aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cell-fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither Aminophospholipid transport nor the asymmetrical distribution of Aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Conclusion: Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed Aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency. (HEPATOLOGY 2010)
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Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory Vesicles
Molecular biology of the cell, 2006Co-Authors: Nele Alder-baerens, Thomas Günther Pomorski, Quirine Lisman, Lambert Luong, Joost C. M. HolthuisAbstract:Eukaryotic plasma membranes generally display asymmetric lipid distributions with the Aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by Aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that Aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.
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drs2p related p type atpases dnf1p and dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis
Molecular Biology of the Cell, 2003Co-Authors: Thomas Günther Pomorski, Philippe F. Devaux, Ruben Lombardi, Howard Riezman, Gerrit Van Meer, Joost C. M. HolthuisAbstract:Plasma membranes in eukaryotic cells display asymmetric lipid distributions with Aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by...
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Protein-dependent translocation of Aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.
Biochemistry, 1994Co-Authors: Karin Mueller, Alain Zachowski, Thomas Günther Pomorski, Peter Mueller, Andreas HerrmannAbstract:We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of Aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated Aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an Aminophospholipid translocase and an asymmetric transversal lipid distribution with Aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.