Crystal structure of a human plasma membrane phospholipid flippase

AbstractATP11C, a member of P4-ATPase flippase, exclusively translocates phosphatidylserine from the outer to the inner leaflets of the plasma membrane, and maintains the asymmetric distribution of phosphatidylserine in the living cell. However, the mechanisms by which ATP11C translocates phosphatidylserine remain elusive. Here we show the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in an outward-open E2P conformation. Two phosphatidylserine molecules are in a conduit that continues from the cell surface to the occlusion site in the middle of the membrane. Mutations in either of the phosphotidylserine binding sites or along the pathway between significantly impairs specific ATPase and transport activities. We propose a model for phosphatidylserine translocation from the outer to the inner leaflet of the plasma membrane.

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Crystal structure of a human plasma membrane phospholipid flippase

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014144 ◽

2020 ◽

Vol 295 (30) ◽

pp. 10180-10194 ◽

Cited By ~ 8

Author(s):

Hanayo Nakanishi ◽

Katsumasa Irie ◽

Katsumori Segawa ◽

Kazuya Hasegawa ◽

Yoshinori Fujiyoshi ◽

...

Keyword(s):

Plasma Membrane ◽

Human Plasma ◽

Binding Sites ◽

The Other ◽

Asymmetric Distribution ◽

Transmembrane Helices ◽

Outer Leaflet ◽

Extracellular Side ◽

Phospholipid Flippase ◽

Plasma Membrane Phospholipid

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C–CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p–Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1–CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

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Faculty Opinions recommendation of Crystal structure of a human plasma membrane phospholipid flippase.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738080812.793576971 ◽

2020 ◽

Author(s):

Tom Rapoport ◽

Navdar Sever

Keyword(s):

Crystal Structure ◽

Plasma Membrane ◽

Human Plasma ◽

Membrane Phospholipid ◽

Phospholipid Flippase ◽

Plasma Membrane Phospholipid

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Calpain cleaves phospholipid flippase ATP8A1 during apoptosis in platelets

Blood Advances ◽

10.1182/bloodadvances.2018023473 ◽

2019 ◽

Vol 3 (3) ◽

pp. 219-229 ◽

Cited By ~ 5

Author(s):

Weidong Jing ◽

Mehmet Yabas ◽

Angelika Bröer ◽

Lucy Coupland ◽

Elizabeth E. Gardiner ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cysteine Protease ◽

Calcium Influx ◽

Human Platelets ◽

Asymmetric Distribution ◽

Resting Cells ◽

Activated Platelets ◽

Caspase Inhibition ◽

Phospholipid Flippase

Abstract The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

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Participation of concanavalin A binding sites in the interaction between Trypanosoma cruzi and macrophages

Journal of Cell Science ◽

10.1242/jcs.62.1.287 ◽

1983 ◽

Vol 62 (1) ◽

pp. 287-299

Author(s):

M.N. Meirelles ◽

A. Martinez-Palomo ◽

T. Souto-Padron ◽

W. De Souza

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Trypanosoma Cruzi ◽

Uniform Distribution ◽

Concanavalin A ◽

Binding Sites ◽

Peritoneal Macrophages ◽

Untreated Mouse ◽

Mouse Peritoneal Macrophages

Untreated mouse peritoneal macrophages as well as macrophages treated with concanavalin A (ConA) were incubated in the presence of untreated or ConA-treated epimastigotes and trypomastigotes of Trypanosoma cruzi. Treatment of epimastigotes or trypomastigotes with ConA increased or decreased their uptake by macrophages, respectively. Treatment of their macrophages with ConA reduced by 70% and increased by five times the ingestion of epimastigotes and trypomastigotes, respectively. These results are discussed in relation to previous studies on the mobility of ConA receptors in the membrane of the parasite. Using fluorescein- or ferritin-labelled ConA we observed that ConA binding sites located on the plasma membrane of macrophages are internalized during endocytosis of T. cruzi, and observed in association with the membrane of the endocytic vacuole. Vacuoles without parasites showed a uniform distribution of ConA binding sites, while these sites were distributed in patches in vacuoles containing parasites. These results, in association with others previously reported, suggest the involvement of glycoproteins and/or glycolipids localized on the cell surface of T. cruzi and macrophages during the T. cruzi-macrophage interaction.

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Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites

Natural Structural Biology ◽

10.1038/1869 ◽

1998 ◽

Vol 5 (9) ◽

pp. 827-835 ◽

Cited By ~ 918

Author(s):

Stephen Curry ◽

Hendrik Mandelkow ◽

Peter Brick ◽

Nick Franks

Keyword(s):

Crystal Structure ◽

Fatty Acid ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Binding Sites ◽

Asymmetric Distribution

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Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate.

The Journal of Cell Biology ◽

10.1083/jcb.64.3.538 ◽

1975 ◽

Vol 64 (3) ◽

pp. 538-550 ◽

Cited By ~ 60

Author(s):

P P Silva ◽

A Martínez-Palomo ◽

A Gonzalez-Robles

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Binding Sites ◽

Membrane Structure ◽

Ruthenium Red ◽

Iron Binding ◽

Con A ◽

Colloidal Iron ◽

Acidic Sites ◽

Membrane Components

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.

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Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts.

Journal of Histochemistry & Cytochemistry ◽

10.1177/36.1.2447153 ◽

1988 ◽

Vol 36 (1) ◽

pp. 95-101 ◽

Cited By ~ 12

Author(s):

M Takagi ◽

H Yagasaki ◽

T Baba ◽

H Baba

Keyword(s):

Plasma Membrane ◽

Cell Membrane ◽

Cell Surface ◽

Binding Sites ◽

Peanut Agglutinin ◽

Coated Pits ◽

Cytoplasmic Surface ◽

Coated Membranes ◽

Entire Cell ◽

Ruffled Border

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.

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Phospholipid flipping facilitates annexin translocation across membranes

10.1101/241976 ◽

2018 ◽

Cited By ~ 1

Author(s):

Sarah E Stewart ◽

Avraham Ashkenazi ◽

Athena Williamson ◽

David C Rubinsztein ◽

Kevin Moreau

Keyword(s):

Plasma Membrane ◽

Cell Migration ◽

Cell Surface ◽

Binding Proteins ◽

Annexin A2 ◽

Plasminogen Activation ◽

Anoctamin 6 ◽

Outer Leaflet ◽

Membrane Cell ◽

Plasma Membrane Phospholipid

AbstractAnnexins are phospholipid binding proteins that somehow translocate from the inner leaflet of the plasma membrane to the outer leaflet. For example, Annexin A2 is known to localise to the outer leaflet of the plasma membrane (cell surface) where it is involved in plasminogen activation leading to fibrinolysis and cell migration, among other functions. Despite having well described extracellular functions, the mechanism of annexin transport from the cytoplasmic inner leaflet to the extracellular outer leaflet of the plasma membrane remains unclear. Here, we show that phospholipid flipping activity is crucial for the transport of annexins A2 and A5 across membranes in cells and in liposomes. We identified TMEM16F (anoctamin-6) as a lipid scramblase required for transport of these annexins to the outer leaflet of the plasma membrane. This work reveals a mechanism for annexin translocation across membranes which depends on plasma membrane phospholipid flipping.

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