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Anant K Menon - One of the best experts on this subject based on the ideXlab platform.
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The Photoreceptor Rhodopsin is a Constitutively Active Lipid Flippase
Biophysical Journal, 2013Co-Authors: Michael A. Goren, Oliver P. Ernst, Anant K MenonAbstract:The transbilayer transport of lipids is essential in both eukaryotes and prokaryotes, yet the spontaneous rate of flipping is too slow to support cellular life. Physiologically relevant rates of flipping are achieved through the activity of two classes of lipid transporters. The first couples ATP hydrolysis to unidirectional lipid flipping in order to maintain asymmetric membranes, such as the eukaryotic plasma membrane. The second class of transporters facilitates bi-directional, ATP-independent movement of lipids across biogenic and specialized membranes. In a recent report (Menon et al. (2011) Curr. Biol. 21, 149-153) we identified opsin as the first ATP-independent Flippase. We now report that opsin's Flippase activity is constitutive, and not linked to its established function as a light sensor. We expressed thermostable variants of opsin in COS-7 cells and assayed activity of the purified proteins in a reconstituted system. We tested three structurally discrete signaling states of opsin: dark-adapted rhodopsin with the endogenous inverse agonist 9-cis retinal; the metarhodopsin II intermediate containing the agonist all-trans retinal in the constitutively active M257Y background; and the ligand-free light-adapted opsin. All constructs demonstrated rapid (t ½ < 10 s), ATP-independent flip-flop of zwitterionic phospholipid probes in both dark- and light-adapted conditions. These results suggest that the Flippase activity of opsin, and likely other Type-A GPCRs such as the β1-adrenergic receptor which we also previously showed to have Flippase activity, is localized to the relatively immobile transmembrane helices 1 - 4, or the amphipathic helix 8. These data, as well as the results of ongoing experiments on the Flippase activity of dynamically constrained rhodopsin constructs, will be presented.Supported by NIH grant GM71041
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reconstitution of glucosylceramide flip flop across endoplasmic reticulum implications for mechanism of glycosphingolipid biosynthesis
Journal of Biological Chemistry, 2012Co-Authors: Madhavan Chalat, Indu Menon, Zeynep Turan, Anant K MenonAbstract:Abstract Most glycosphingolipids are synthesized by the sequential addition of monosaccharides to glucosylceramide (GlcCer) in the lumen of the Golgi apparatus. Because GlcCer is synthesized on the cytoplasmic face of Golgi membranes, it must be flipped to the non-cytoplasmic face by a lipid Flippase in order to nucleate glycosphingolipid synthesis. Halter et al. (Halter, D., Neumann, S., van Dijk, S. M., Wolthoorn, J., de Maziere, A. M., Vieira, O. V., Mattjus, P., Klumperman, J., van Meer, G., and Sprong, H. (2007) Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. J. Cell Biol. 179, 101–115) proposed that this essential flipping step is accomplished via a complex trafficking itinerary; GlcCer is moved from the cytoplasmic face of the Golgi to the endoplasmic reticulum (ER) by FAPP2, a cytoplasmic lipid transfer protein, flipped across the ER membrane, then delivered to the lumen of the Golgi complex by vesicular transport. We now report biochemical reconstitution studies to analyze GlcCer flipping at the ER. Using proteoliposomes reconstituted from Triton X-100-solubilized rat liver ER membrane proteins, we demonstrate rapid (t½ < 20 s), ATP-independent flip-flop of N-(6-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)hexanoyl)-d-glucosyl-β1–1′-sphingosine, a fluorescent GlcCer analog. Further studies involving protein modification, biochemical fractionation, and analyses of flip-flop in proteoliposomes reconstituted with ER membrane proteins from yeast indicate that GlcCer translocation is facilitated by well characterized ER phospholipid Flippases that remain to be identified at the molecular level. By reason of their abundance and membrane bending activity, we considered that the ER reticulons and the related Yop1 protein could function as phospholipid-GlcCer Flippases. Direct tests showed that these proteins have no Flippase activity.
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opsin is a phospholipid Flippase
Current Biology, 2011Co-Authors: Indu Menon, Thomas Huber, Sumana Sanyal, Sourabh Banerjee, Patrick Barre, Sam Canis, David J Warren, John Hwa, Thomas P Sakmar, Anant K MenonAbstract:Summary Polar lipids must flip-flop rapidly across biological membranes to sustain cellular life [1, 2], but flipping is energetically costly [3] and its intrinsic rate is low. To overcome this problem, cells have membrane proteins that function as lipid transporters (Flippases) to accelerate flipping to a physiologically relevant rate. Flippases that operate at the plasma membrane of eukaryotes, coupling ATP hydrolysis to unidirectional lipid flipping, have been defined at a molecular level [2]. On the other hand, ATP-independent bidirectional Flippases that translocate lipids in biogenic compartments, e.g., the endoplasmic reticulum, and specialized membranes, e.g., photoreceptor discs [4, 5], have not been identified even though their activity has been recognized for more than 30 years [1]. Here, we demonstrate that opsin is the ATP-independent phospholipid Flippase of photoreceptor discs. We show that reconstitution of opsin into large unilamellar vesicles promotes rapid (τ
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stereoselective transbilayer translocation of mannosyl phosphoryl dolichol by an endoplasmic reticulum Flippase
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Sumana Sanyal, Anant K MenonAbstract:Mannose-phosphate-dolichol (MPD) is a multifunctional glycolipid that is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER) and used on the opposite side of the membrane in the ER lumen as a mannose donor for protein N-glycosylation, glycosylphosphatidylinositol-anchoring, and C- and O-mannosylation. For this, it must be translocated, i.e., flipped, across the ER membrane. The molecular identity of the MPD translocator (MPD Flippase) is not known. Here we show that MPD-Flippase activity can be reconstituted in large unilamellar proteoliposomes prepared from phosphatidylcholine and Triton X-100-solubilized rat liver ER-membrane proteins. Using carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl NO+ as a topological probe to selectively oxidize MPD molecules in the outer leaflet of the reconstituted vesicles, we demonstrate rapid, protein-dependent, ATP-independent transbilayer translocation of MPD from the inner to the outer leaflet. MPD flipping is highly specific. A stereoisomer of MPD was weakly translocated (> 10-fold lower rate) compared with natural MPD. Competition experiments with water-soluble isoprenyl monophosphates showed that MPD Flippase recognizes the dolichol chain of MPD, preferring a saturated α-isoprene to unsaturated trans- or cis- α-isoprene units. Chromatography of the detergent-solubilized ER protein mixture prior to reconstitution indicated that MPD Flippase (i) is not a Con A-binding glycoprotein and (ii) can be resolved from the oligosaccharide-diphosphate dolichol Flippase that translocates Man5GlcNAc2-PP-dolichol, a lipid intermediate of N-glycosylation. These data provide a mechanistic framework for understanding MPD flipping, as well as a biochemical basis for identifying MPD Flippase.
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Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum Flippase
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Sumana Sanyal, Anant K MenonAbstract:The oligosaccharide donor for protein N-glycosylation, Glc3Man9GlcNAc2-PP-dolichol, is synthesized via a multistep pathway that starts on the cytoplasmic face of the endoplasmic reticulum (ER) and ends in the lumen where the glycosylation reaction occurs. This necessitates transbilayer translocation or flipping of the lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) across the ER membrane. The mechanism by which M5-DLO—or any other lipid—is flipped across the ER is unknown, except that specific transport proteins or Flippases are required. We recently demonstrated M5-DLO flipping activity in proteoliposomes reconstituted from detergent-solubilized ER membrane proteins and showed that it was ATP-independent and required a trypsin-sensitive protein that sedimented at approximately 4S. By using an activity-enriched fraction devoid of glycerophospholipid Flippase activity, we now report that M5-DLO is rapidly flipped in the reconstituted system with a time constant τ 200 min. DLOs larger than M5-DLO are also poorly translocated, with τ ranging from approximately 10 min to >200 min. We conclude that (i) the number and arrangement of mannoses in the DLO glycan has a profound effect on the ability of the DLO to be translocated by the Flippase, (ii) glycan size per se does not dictate whether a DLO will be flipped, and (iii) the Flippase is highly specific for M5-DLO. Our results suggest a simple structural model for the interaction between the DLO head group and the Flippase.
Hye Won Shin - One of the best experts on this subject based on the ideXlab platform.
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Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs
The Journal of biological chemistry, 2018Co-Authors: Bartholomew P. Roland, Hiroyuki Takatsu, Hye Won Shin, Tomoki Naito, Jordan T Best, Cayetana Arnaiz-yépez, Todd R. GrahamAbstract:Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid Flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in Flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these Flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.
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Identification and characterization of yeast and human glycosphingolipid Flippases
2018Co-Authors: Bartholomew P. Roland, Hiroyuki Takatsu, Hye Won Shin, Tomoki Naito, Jordan T Best, Cayetana Arnaiz-yépez, Todd R. GrahamAbstract:Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid Flippases (P4-ATPases) transport lipids across the membrane bilayer, and are involved in signal transduction, cell division, and vesicular transport. Mutations in Flippase genes cause or contribute to a host of diseases such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disease. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis; and ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids are strong contributors to metabolic disease, little is known about how GlcCer is transported across cell membranes. We have identified a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer. Further, we establish the structural determinants necessary for the recognition of this sphingolipid substrate. Our molecular observations clarify the relationship between these Flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.
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Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature
The EMBO journal, 2018Co-Authors: Naoto Takada, Hiroyuki Takatsu, Kazuhisa Nakayama, Tomoki Naito, Takanari Inoue, Hye Won ShinAbstract:P4-ATPases are phospholipid Flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by Flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by Flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-Flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.
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Phospholipid Flippase ATP11C is endocytosed and downregulated following Ca 2+ -mediated protein kinase C activation
Nature communications, 2017Co-Authors: Hiroyuki Takatsu, Kazuhisa Nakayama, Tomoki Naito, Naoto Takada, Masahiro Takayama, Kazuya Tsumagari, Yasushi Ishihama, Hye Won ShinAbstract:We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-Flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-Flippase activity is regulated in non-apoptotic cells. Here we report that the PS-Flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.
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Alteration of transbilayer phospholipid compositions is involved in cell adhesion, cell spreading, and focal adhesion formation.
FEBS letters, 2016Co-Authors: Rie Miyano, Hiroyuki Takatsu, Kazuhisa Nakayama, Takashi Matsumoto, Hye Won ShinAbstract:We previously showed that P4-ATPases, ATP10A/ATP8B1, and ATP11A/ATP11C have Flippase activities toward phosphatidylcholine (PC), and aminophospholipids [phosphatidylserine (PS) and phosphatidylethanolamine], respectively. Here, we investigate the effect of PC-specific Flippases versus aminophospholipid-specific Flippases in cell spreading on the extracellular matrix. Expression of PC-Flippases, but not PS-Flippases, delayed cell adhesion, cell spreading and inhibited formation of focal adhesions. In addition, overexpression of a PS-binding probe that sequesters PS in the cytoplasmic leaflet delayed cell spreading and inhibited formation of focal adhesions. These results suggest that elevation of PC at the cytoplasmic leaflet of the plasma membrane by expression of PC-Flippases may reduce the local concentration of PS or phosphoinositides, required for efficient cell adhesion, focal adhesion formation, and cell spreading.
Shigekazu Nagata - One of the best experts on this subject based on the ideXlab platform.
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Transport Cycle of Plasma Membrane Flippase ATP11C by Cryo-EM.
Cell reports, 2020Co-Authors: Hanayo Nakanishi, Katsumori Segawa, Shigekazu Nagata, Tomohiro Nishizawa, Osamu Nureki, Yoshinori Fujiyoshi, Kazuhiro AbeAbstract:Summary ATP11C, a plasma membrane phospholipid Flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential for an apoptotic “eat me” signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. We show six cryo-EM structures of ATP11C at 3.0–4.0 A resolution in five different states of the transport cycle. A structural comparison reveals phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site, and thus they detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C, and a common basic mechanism for Flippases is emerging.
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phosphorylation mediated activation of mouse xkr8 scramblase for phosphatidylserine exposure
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Takaharu Sakuragi, Hidetaka Kosako, Shigekazu NagataAbstract:The exposure of phosphatidylserine (PtdSer) to the cell surface is regulated by the down-regulation of Flippases and the activation of scramblases. Xkr8 has been identified as a scramblase that is activated during apoptosis, but its exogenous expression in the mouse Ba/F3 pro B cell line induces constitutive PtdSer exposure. Here we found that this Xkr8-mediated PtdSer exposure occurred at 4 °C, but not at 20 °C, although its scramblase activity was observed at 20 °C. The Xkr8-mediated PtdSer exposure was inhibited by a kinase inhibitor and enhanced by phosphatase inhibitors. Phosphorylated Xkr8 was detected by Phos-tag PAGE, and a mass spectrometric and mutational analysis identified three phosphorylation sites. Their phosphomimic mutation rendered Xkr8 resistant to the kinase inhibitor for PtdSer exposure at 4 °C, but unlike phosphatase inhibitors, it did not induce constitutive PtdSer exposure at 20 °C. On the other hand, when the Flippase genes were deleted, the Xkr8 induced constitutive PtdSer exposure at high temperature, indicating that the Flippase activity normally counteracted Xkr8’s ability to expose PtdSer. These results indicate that PtdSer exposure can be increased by the phosphorylation-mediated activation of Xkr8 scramblase and Flippase down-regulation.
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The CDC50A extracellular domain is required for forming a functional complex with and chaperoning phospholipid Flippases to the plasma membrane
The Journal of biological chemistry, 2017Co-Authors: Katsumori Segawa, Sachiko Kurata, Shigekazu NagataAbstract:Flippases are enzymes that translocate phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) from the outer to the inner leaflet in the lipid bilayer of the plasma membrane, leading to the asymmetric distribution of aminophospholipids in the membrane. One mammalian phospholipid Flippase at the plasma membrane is ATP11C, a type IV P-type ATPase (P4-ATPase) that forms a heterocomplex with the transmembrane protein CDC50A. However, the structural features in CDC50A that support the function of ATP11C and other P4-ATPases have not been characterized. Here, using error-prone PCR-mediated mutagenesis of human CDC50A cDNA followed by functional screening and deep sequencing, we identified 14 amino acid residues that affect ATP11C's Flippase activity. These residues were all located in CDC50A's extracellular domain and were evolutionarily well-conserved. Most of the mutations decreased CDC50A's ability to chaperone ATP11C and other P4-ATPases to their destinations. The CDC50A mutants failed to form a stable complex with ATP11C and could not induce ATP11C's PtdSer-dependent ATPase activity. Notably, one mutant variant could form a stable complex with ATP11C and transfer ATP11C to the plasma membrane, yet the ATP11C complexed with this CDC50A variant had very weak or little PtdSer- or PtdEtn-dependent ATPase activity. These results indicated that the extracellular domain of CDC50A has important roles both in CDC50A's ability to chaperone ATP11C to the plasma membrane and in inducing ATP11C's ATP hydrolysis-coupled Flippase activity.
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Flippases and Scramblases at Plasma Membranes that Regulate Phosphatidylserine Exposure
Blood, 2015Co-Authors: Shigekazu NagataAbstract:One of the hallmarks of apoptosis is the caspase-dependent exposure of phosphatidylserine (PtdSer) on cell surface, which is recognized by macrophages for engulfment of dead cells (1). How PtdSer is exposed to the cell surface had been elusive for a long time. We recently identified two membrane proteins (TMEM16F and Xkr8) that are involved in scrambling of phospholipids in plasma membrane (2, 3). TMEM16F carries 8 transmembrane regions, and requires Ca2+ to mediate phospholipid scrambling. It plays a role in the PtdSer-exposure in activated platelets for blood clotting, and patients of Scott Syndrome who suffer bleeding disorder carry a mutation in TMEM16F gene. Xkr8 is a protein carrying 6 transmembrane regions. Caspase 3 and 7 cleave off the C-terminal tail of Xkr8, and the cleaved Xkr8 promotes the PtdSer-exposure. In addition to the activation of scramblase, the Flippase that translocates PtdSer from outer to inner leaflets was thought to be inactivated during apoptosis. In fact, we recently found that a pair of molecules, ATP11C of a P4-type ATPase and its chaperon CDC50A work as a Flippase at plasma membrane (4, 5). ATP11C carries three caspase recognition sites in the middle of the molecule, and is cleaved during apoptosis. When ATP11C gene is mutated, the cells lose most of the Flippase activity, but the asymmetrical distribution of PtdSer was still maintained at plasma membrane. Whereas, the cells lacking CDC50A completely lost the Flippase activity and constitutively exposed PtdSer. The PtdSer-exposing living CDC50A-null cells were engulfed by thioglycollate-elicited macrophages, indicating that PtdSer exposed on the cell surface is necessary and sufficient to be recognized by macrophages for engulfment. Several molecules such as MFG-E8, Tim-4, Gas6, and Protein S specifically bind to PtdSer with high affinity, and promote the engulfment of PtdSer-exposing cells. However, how they work for the engulfment of apoptotic cells in certain macrophages has not been clear. We recently found that that resident peritoneal macrophages require both Tim4 and Protein S for engulfment, and Tim4, PtdSer-receptor, was involved in tethering of apoptotic cells, while Protein S promoted the engulfment of apoptotic cells by binding to MerTK, a tyrosine kinase receptor (6, 7). Here, I discuss how PdtSer is exposed during apoptotic cell death, and how dead cells are engulfed by macrophages. 1. Nagata S, Hanayama R, Kawane K. Autoimmunity and the clearance of dead cells. Cell. 2010;140:619-630. 2. Suzuki J, Umeda M, Sims PJ, Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 2010;468:834-838. 3. Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science. 2013;341:403-406. 4. Segawa K, Suzuki J, Nagata S. Flippases and scramblases in the plasma membrane. Cell Cycle. 2014;13:2990-2991. 5. Segawa K, Kurata S, Yanagihashi Y, Brummelkamp T, Matsuda F, Nagata S. Caspase-mediated cleavage of phospholipid Flippase for apoptotic phosphatidylserine exposure. Science. 2014;344:1164-1168. 6. Nishi C, Toda S, Segawa K, Nagata S. Tim4- and MerTK-mediated engulfment of apoptotic cells by mouse resident peritoneal macrophages. Mol Cell Biol. 2014;34:1512-1520. 7. Toda S, Segawa K, Nagata S. MerTK-mediated engulfment of pyrenocytes by central macrophages in erythroblastic islands. Blood. 2014;123:3963-3971. Disclosures No relevant conflicts of interest to declare.
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Human Type IV P-type ATPases That Work as Plasma Membrane Phospholipid Flippases and Their Regulation by Caspase and Calcium
The Journal of biological chemistry, 2015Co-Authors: Katsumori Segawa, Sachiko Kurata, Shigekazu NagataAbstract:In plasma membranes, Flippases translocate aminophospholipids such as phosphatidylserine and phosphatidylethanolamine from the extracellular to the cytoplasmic leaflet. Mammalian ATP11C, a type IV P-type ATPase, acts as a Flippase at the plasma membrane. Here, by expressing 12 human type IV P-type ATPases in ATP11C-deficient cells, we determined that ATP8A2 and ATP11A can also act as plasma membrane Flippases. As with ATP11C, ATP8A2 and ATP11A localized to the plasma membrane in a CDC50A-dependent manner. ATP11A was cleaved by caspases during apoptosis, and a caspase-resistant ATP11A blocked apoptotic PtdSer exposure. In contrast, ATP8A2 was not cleaved by caspase, and cells expressing ATP8A2 did not expose PtdSer during apoptosis. Similarly, high Ca(2+) concentrations inhibited the ATP11A and ATP11C PtdSer Flippase activity, but ATP8A2 Flippase activity was relatively resistant to Ca(2+). ATP11A and ATP11C were ubiquitously expressed in human and mouse adult tissues. In contrast, ATP8A2 was expressed in specific tissues, such as the brain and testis. Thus, ATP8A2 may play a specific role in translocating PtdSer in these tissues.
Kazuma Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Cfs1p, a Novel Membrane Protein in the PQ-Loop Family, Is Involved in Phospholipid Flippase Functions in Yeast
G3 (Bethesda Md.), 2017Co-Authors: Takaharu Yamamoto, Konomi Fujimura-kamada, Eno Shioji, Risa Suzuki, Kazuma TanakaAbstract:Type 4 P-type ATPases (P4-ATPases) function as phospholipid Flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer, to generate and maintain asymmetric distribution of phospholipids at the plasma membrane and endosomal/Golgi membranes. The budding yeast Saccharomyces cerevisiae has four heteromeric Flippases ([Drs2p][1], [Dnf1p][2], [Dnf2p][3], and [Dnf3p][4]), associated with the [Cdc50p][5] family noncatalytic subunit, and one monomeric Flippase, [Neo1p][6]. They have been suggested to function in vesicle formation in membrane trafficking pathways, but details of their mechanisms remain to be clarified. Here, to search for novel factors that functionally interact with Flippases, we screened transposon insertional mutants for strains that suppressed the cold-sensitive growth defect in the [cdc50][5] Δ mutant. We identified a mutation of [YMR010W][7] encoding a novel conserved membrane protein that belongs to the PQ-loop family including the cystine transporter cystinosin and the SWEET sugar transporters. We named this gene [CFS1][7] ( cdc fifty suppressor 1). GFP-tagged [Cfs1p][7] was partially colocalized with [Drs2p][1] and [Neo1p][6] to endosomal/late Golgi membranes. Interestingly, the [cfs1][7] Δ mutation suppressed growth defects in all Flippase mutants. Accordingly, defects in membrane trafficking in the Flippase mutants were also suppressed. These results suggest that [Cfs1p][7] and Flippases function antagonistically in membrane trafficking pathways. A growth assay to assess sensitivity to duramycin, a phosphatidylethanolamine (PE)-binding peptide, suggested that the [cfs1][7] Δ mutation changed PE asymmetry in the plasma membrane. [Cfs1p][7] may thus be a novel regulator of phospholipid asymmetry. [1]: http://www.yeastgenome.org/locus/S000000024/overview [2]: http://www.yeastgenome.org/locus/S000000968/overview [3]: http://www.yeastgenome.org/locus/S000002500/overview [4]: http://www.yeastgenome.org/locus/S000004772/overview [5]: http://www.yeastgenome.org/locus/S000000690/overview [6]: http://www.yeastgenome.org/locus/S000001310/overview [7]: http://www.yeastgenome.org/locus/S000004612/overview
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Inositol depletion restores vesicle transport in yeast phospholipid Flippase mutants.
PloS one, 2015Co-Authors: Kanako Yamagami, Takaharu Yamamoto, Shota Sakai, Tetsuo Mioka, Takamitsu Sano, Yasuyuki Igarashi, Kazuma TanakaAbstract:In eukaryotic cells, type 4 P-type ATPases function as phospholipid Flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of Flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that Flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five Flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a Flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a Flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of Flippases.
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Asymmetric distribution of phosphatidylserine is generated in the absence of phospholipid Flippases in Saccharomyces cerevisiae.
MicrobiologyOpen, 2014Co-Authors: Tetsuo Mioka, Konomi Fujimura-kamada, Kazuma TanakaAbstract:In eukaryotic cells, phosphatidylserine (PS) is predominantly located in the cytosolic leaflet of the plasma membrane; this asymmetry is generated by an unknown mechanism. In this study, we used the PS-specific probe mRFP-Lact-C2 to investigate the possible involvement of type 4 P-type ATPases, also called phospholipid Flippases, in the generation of this asymmetry in Saccharomyces cerevisiae. PS was not found in the trans-Golgi Network in wild-type cells, but it became exposed when vesicle formation was compromised in the sec7 mutant, and it was also exposed on secretory vesicles (SVs), as reported previously. However, Flippase mutations did not reduce the exposure of PS in either case, even at low levels that would only be detectable by quantitative analysis of mRFP-Lact-C2 fluorescence in isolated SVs. Furthermore, no reduction in the PS level was observed in a mutant with multiple Flippase mutations. Because PS was not exposed in a mutant that accumulates ER or cis/medial-Golgi membranes, Golgi maturation seems to be a prerequisite for PS translocation. Our results suggest that an unknown mechanism, possibly a protein with Flippase-like activity, acts in conjunction with known Flippases to regulate PS translocation.
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Role of Phosphatidylserine in Phospholipid Flippase-Mediated Vesicle Transport in Saccharomyces cerevisiae
Eukaryotic cell, 2014Co-Authors: Miyoko Takeda, Kanako Yamagami, Kazuma TanakaAbstract:Phospholipid Flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric Flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric Flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in Flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for Flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all Flippases, suggesting that the suppression was dependent on Flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that Flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.
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interaction of the phospholipid Flippase drs2p with the f box protein rcy1p plays an important role in early endosome to trans golgi network vesicle transport in yeast
Journal of Biochemistry, 2014Co-Authors: Hisatoshi Hanamatsu, Takaharu Yamamoto, Konomi Fujimurakamada, Nobumichi Furuta, Kazuma TanakaAbstract:Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid Flippases that generate such membrane phospholipid asymmetry. Drs2p, a Flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.
Hiroyuki Takatsu - One of the best experts on this subject based on the ideXlab platform.
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Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs
The Journal of biological chemistry, 2018Co-Authors: Bartholomew P. Roland, Hiroyuki Takatsu, Hye Won Shin, Tomoki Naito, Jordan T Best, Cayetana Arnaiz-yépez, Todd R. GrahamAbstract:Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid Flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in Flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these Flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.
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Identification and characterization of yeast and human glycosphingolipid Flippases
2018Co-Authors: Bartholomew P. Roland, Hiroyuki Takatsu, Hye Won Shin, Tomoki Naito, Jordan T Best, Cayetana Arnaiz-yépez, Todd R. GrahamAbstract:Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid Flippases (P4-ATPases) transport lipids across the membrane bilayer, and are involved in signal transduction, cell division, and vesicular transport. Mutations in Flippase genes cause or contribute to a host of diseases such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disease. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis; and ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids are strong contributors to metabolic disease, little is known about how GlcCer is transported across cell membranes. We have identified a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer. Further, we establish the structural determinants necessary for the recognition of this sphingolipid substrate. Our molecular observations clarify the relationship between these Flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.
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Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature
The EMBO journal, 2018Co-Authors: Naoto Takada, Hiroyuki Takatsu, Kazuhisa Nakayama, Tomoki Naito, Takanari Inoue, Hye Won ShinAbstract:P4-ATPases are phospholipid Flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by Flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by Flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-Flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.
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Phospholipid Flippase ATP11C is endocytosed and downregulated following Ca 2+ -mediated protein kinase C activation
Nature communications, 2017Co-Authors: Hiroyuki Takatsu, Kazuhisa Nakayama, Tomoki Naito, Naoto Takada, Masahiro Takayama, Kazuya Tsumagari, Yasushi Ishihama, Hye Won ShinAbstract:We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-Flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-Flippase activity is regulated in non-apoptotic cells. Here we report that the PS-Flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.
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Alteration of transbilayer phospholipid compositions is involved in cell adhesion, cell spreading, and focal adhesion formation.
FEBS letters, 2016Co-Authors: Rie Miyano, Hiroyuki Takatsu, Kazuhisa Nakayama, Takashi Matsumoto, Hye Won ShinAbstract:We previously showed that P4-ATPases, ATP10A/ATP8B1, and ATP11A/ATP11C have Flippase activities toward phosphatidylcholine (PC), and aminophospholipids [phosphatidylserine (PS) and phosphatidylethanolamine], respectively. Here, we investigate the effect of PC-specific Flippases versus aminophospholipid-specific Flippases in cell spreading on the extracellular matrix. Expression of PC-Flippases, but not PS-Flippases, delayed cell adhesion, cell spreading and inhibited formation of focal adhesions. In addition, overexpression of a PS-binding probe that sequesters PS in the cytoplasmic leaflet delayed cell spreading and inhibited formation of focal adhesions. These results suggest that elevation of PC at the cytoplasmic leaflet of the plasma membrane by expression of PC-Flippases may reduce the local concentration of PS or phosphoinositides, required for efficient cell adhesion, focal adhesion formation, and cell spreading.