Biomedical subjects

Thomas Pomorski

Publications and source records attributed to Thomas Pomorski.

12 recordsLinked to original sources

PubMed2006

Antigen recognition induces phosphatidylserine exposure on the cell surface of human CD8+ T cells.

In eukaryotic cells the phospholipid phosphatidylserine (PS) is restricted to the inner plasma-membrane leaflet. This lipid asymmetry, which is maintained by the concerted action of phospholipid transport proteins, is mainly lost during apoptosis. Here, we demonstrate that primary human CD8+ cytotoxic T lymphocytes (CTLs) expose PS on T-cell receptor (TCR)-mediated antigen (Ag) recognition. In contrast to PS externalization on apoptotic cells, activation-induced PS exposure is less pronounced and reversible. Fluorescence microscopic analysis revealed that PS is distributed nonhomogenously over the plasma membrane and concentrated in membrane lipid raft domains at the immunologic synapse. By studying the activity of PS transport proteins using a fluorescence-labeled PS analogue, we found that activation of CTLs inhibited the flippase-mediated inward-directed PS transport without affecting the outward transport. Shielding of exposed PS by annexin V protein during Ag recognition diminished cytokine secretion, activation, and cell-to-cell clustering of Ag-specific CTLs. In summary, our data demonstrate for the first time that externalized PS on Ag-stimulated CTLs is linked to T-cell activation and probably involved in cell-to-cell contact formation at the immunologic synapse.

Annexin A5↗

PubMed2006

Loss of P4 ATPases Drs2p and Dnf3p disrupts aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles.

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.

Biological Transport↗

PubMed2005

StarD10, a START domain protein overexpressed in breast cancer, functions as a phospholipid transfer protein.

We originally identified StarD10 as a protein overexpressed in breast cancer that cooperates with the ErbB family of receptor tyrosine kinases in cellular transformation. StarD10 contains a steroidogenic acute regulatory protein (StAR/StarD1)-related lipid transfer (START) domain that is thought to mediate binding of lipids. We now provide evidence that StarD10 interacts with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by electron spin resonance measurement. Interaction with these phospholipids was verified in a fluorescence resonance energy transfer-based assay with 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled lipids. Binding was not restricted to lipid analogs since StarD10 selectively extracted PC and PE from small unilamellar vesicles prepared with endogenous radiolabeled lipids from Vero monkey kidney cells. Mass spectrometry revealed that StarD10 preferentially selects lipid species containing a palmitoyl or stearoyl chain on the sn-1 and an unsaturated fatty acyl chain (18:1 or 18:2) on the sn-2 position. StarD10 was further shown to bind lipids in vivo by cross-linking of protein expressed in transfected HEK-293T cells with photoactivable phosphatidylcholine. In addition to a lipid binding function, StarD10 transferred PC and PE between membranes. Interestingly, these lipid binding and transfer specificities distinguish StarD10 from the related START domain proteins Pctp and CERT, suggesting a distinct biological function.

Animals↗

PubMed2005

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells.

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.

ATP Binding Cassette Transporter 1↗

PubMed2005

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

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

4-Chloro-7-nitrobenzofurazan↗

PubMed2004

The phosphoprotein StarD10 is overexpressed in breast cancer and cooperates with ErbB receptors in cellular transformation.

We have identified that StarD10, a member of the START protein family, is overexpressed in both mouse and human breast tumors. StarD10 was initially discovered on the basis of its cross-reactivity with a phosphoserine-specific antibody in mammary tumors from Neu/ErbB2 transgenic mice and subsequently isolated from SKBR3 human breast carcinoma cells using a multistep biochemical purification strategy. We have shown that StarD10 is capable of binding lipids. StarD10 was found to be overexpressed in 35% of primary breast carcinomas and 64% of human breast cancer cell lines, correlating with their ErbB2/Her2 status. Coexpression of StarD10 with ErbB1/epidermal growth factor receptor in murine fibroblasts enhanced anchorage-independent growth in soft agar, providing evidence for functional cooperation between StarD10 and ErbB receptor signaling. Taken together, these data suggest that overexpression of this lipid-binding protein contributes to breast oncogenesis.

Amino Acid Sequence↗

PubMed2004

Tracking down lipid flippases and their biological functions.

The various organellar membranes of eukaryotic cells display striking differences in the composition, leaflet distribution and transbilayer movement of their lipids. In membranes such as the endoplasmic reticulum, phospholipids can move readily across the bilayer, aided by membrane proteins that facilitate a passive equilibration of lipids between both membrane halves. In the plasma membrane, and probably also in the late Golgi and endosomal compartments, flip-flop of phospholipids is constrained and subject to a dynamic, ATP-dependent regulation that involves members of distinct protein families. Recent studies in yeast, parasites such as Leishmania, and mammalian cells have identified several candidates for lipid flippases, and whereas some of these serve a fundamental role in the release of lipids from cells, others appear to have unexpected and important functions in vesicular traffic: their activities are required to support vesicle formation in the secretory and endocytic pathways.

Adenosine Triphosphate↗

PubMed2003

Protein sorting in the late Golgi of Saccharomyces cerevisiae does not require mannosylated sphingolipids.

Glycosphingolipids are widely viewed as integral components of the Golgi-based machinery by which membrane proteins are targeted to compartments of the endosomal/lysosomal system and to the surface domains of polarized cells. The yeast Saccharomyces cerevisiae creates glycosphingolipids by transferring mannose to the head group of inositol phosphorylceramide (IPC), yielding mannosyl-IPC (MIPC). Addition of an extra phosphoinositol group onto MIPC generates mannosyldi-IPC (M(IP)2C), the final and most abundant sphingolipid in yeast. Mannosylation of IPC is partially dependent on CSG1, a gene encoding a putative sphingolipidmannosyltransferase. Here we show that open reading frame YBR161w, renamed CSH1, is functionally homologous to CSG1 and that deletion of both genes abolishes MIPC and M(IP)2C synthesis without affecting protein mannosylation. Csg1p and Csh1p are closely related polytopic membrane proteins that co-localize with IPC synthase in the medial-Golgi. Loss of Csg1p and Csh1p has no effect on clathrin- or AP-3 adaptor-mediated protein transport from the Golgi to the vacuole. Moreover, segregation of the periplasmic enzyme invertase, the plasma membrane ATPase Pma1p and the glycosylphosphatidylinositol-anchored protein Gas1p into distinct classes of secretory vesicles occurs independently of Csg1p and Csh1p. Our results indicate that protein sorting in the late Golgi of yeast does not require production of mannosylated sphingolipids.

Adenosine Triphosphatases↗

PubMed2003

Rapid transport of phospholipids across the plasma membrane of Leishmania infantum.

The internalization of fluorescent phospholipid analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM) in Leishmania infantum promastigotes was studied. We observed a rapid inward redistribution of NBD-PC, -PE, and -PS across the plasma membrane at 28 and 4 degrees C. This internalization was shown to be independent of the endocytic activity of parasites. Rapid inward movement was coupled to an energy-dependent transporter because it was almost inhibited by depletion of cellular ATP and was blocked after pretreatment with N-ethylmaleimide (NEM). In contrast, NBD-SM traverses the plasma membrane by passive flip-flop. By comparing this pattern of phospholipid transbilayer movement with those known from other eukaryotic cells, candidate lipid transporters are discussed.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters.

Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids. Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP. Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP. A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells. Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells. This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis. The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed.

ATP-Binding Cassette Transporters↗

PubMed2003

Drs2p-related P-type ATPases Dnf1p and Dnf2p are required for phospholipid translocation across the yeast plasma membrane and serve a role in endocytosis.

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 ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of Deltadnf1Deltadnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by Deltadnf1Deltadnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.

ATP-Binding Cassette Transporters↗

PubMed2002

Transport of phosphatidylserine via MDR1 (multidrug resistance 1)P-glycoprotein in a human gastric carcinoma cell line.

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗