Multiple Members of a Third Subfamily of P-Type ATPases Identified by Genomic Sequences and ESTs

The Saccharomyces cerevisiae genome contains five P-type ATPases divergent from both of the well-known subfamilies of these membrane ion transporters. This newly recognized third subfamily can be further divided into four classes of genes with nearly equal relatedness to each other. Genes of this new subfamily are also present and expressed in multicellular organisms such as Caenorhabditis elegans and mammals; some, but not all, can be assigned to the classes identified in yeast. Different classes of genes and different genes within a class are expressed differentially in tissues of the mouse. The recently cloned gene for the mammalian aminophospholipid translocase belongs to this new subfamily, suggesting that other subfamily members may transport other lipids or lipid-like molecules from one leaflet of the membrane bilayer to the other

Aminophospholipid Translocase TAT-1 Promotes Phosphatidylserine Exposure during C. elegans Apoptosis

SummaryPhospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an “eat-me” signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals

Transbilayer Phospholipid Movements in ABCA1-Deficient Cells

Tangier disease is an inherited disorder that results in a deficiency in circulating levels of HDL. Although the disease is known to be caused by mutations in the ABCA1 gene, the mechanism by which lesions in the ABCA1 ATPase effect this outcome is not known. The inability of ABCA1 knockout mice (ABCA1−/−) to load cholesterol and phospholipids onto apoA1 led to a proposal that ABCA1 mediates the transbilayer externalization of phospholipids, an activity integral not only to the formation of HDL particles but also to another, distinct process: the recognition and clearance of apoptotic cells by macrophages. Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient engulfment of the apoptotic cells, and it has been proposed that ABCA1 is required for transbilayer externalization of PS to the surface of both cell types. To determine whether ABCA1 is responsible for any of the catalytic activities known to control transbilayer phospholipid movements, these activities were measured in cells from ABCA1−/− mice and from Tangier individuals as well as ABCA1-expressing HeLa cells. Phospholipid movements in either normal or apoptotic lymphocytes or in macrophages were not inhibited when cells from knockout and wildtype mice or immortalized cells from Tangier individuals vs normal individuals were compared. Exposure of PS on the surface of normal thymocytes, apoptotic thymocytes and elicited peritoneal macrophages from wildtype and knockout mice or B lymphocytes from normal and Tangier individuals, as measured by annexin V binding, was also unchanged. No evidence was found of ABCA1-stimulated active PS export, and spontaneous PS movement to the outer leaflet in the presence or absence of apoA1 was unaffected by the presence or absence of ABCA1. Normal or Tangier B lymphocytes and macrophages were also identical in their ability to serve as targets or phagocytes, respectively, in apoptotic cell clearance assays. No evidence was found to support the suggestion that ABCA1 is involved in transport to the macrophage cell surface of annexins I and II, known to enhance phagocytosis of apoptotic cells. These results show that mutations in ABCA1 do not measurably reduce the rate of transbilayer movements of phospholipids in either the engulfing macrophage or the apoptotic target, thus discounting catalysis of transbilayer movements of phospholipids as the mechanism by which ABCA1 facilitates loading of phospholipids and cholesterol onto apoA1

Endogenous PS externalization in apoptotic EBV-transformed human B lymphocytes.

<p>EBV-transformed normal (A–D) or Tangier (E–H) B lymphocytes were untreated (A&B, E&F) or treated with camptothecin (C&D, G&H) to induce apoptosis, stained with fluorescent annexin V, and examined by flow cytometry. Left panel, forward (FS) vs side (SS) light scatter plots, with cells of normal size and shape in the R2 gate and shrunken cells in the R1 gate. Right panel, fluorescence profile of cells in the R2 gate.</p

Internalization (translocation) of NBD-labeled phospholipids by peritoneal macrophages from wildtype and <i>ABCA1^−/−</i> mice.

<p>The outer leaflet of the plasma membrane of macrophages from wildtype (diamonds) or <i>ABCA1^−/−</i> (squares) mice was labeled with NBD-PC (open symbols) or NBD-PS (closed symbols). At various times samples were removed into dithionate to reduce outer leaflet probe and after 5 min remaining inner leaflet probe was measured by flow cytometry at room temperature and expressed as percent transported. Cells from wildtype (triangles) and <i>ABCA1^−/−</i> (circles) mice in the S gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone-0000729-g006" target="_blank">Figure 6</a> are shown for comparison.</p

Basal (or Ca²⁺-activated) NBD-PS externalization in normal and ABCA1-deficient cells.

<p>(A) Normal human or Tangier fibroblasts, (B) EBV-transformed normal or Tangier B lymphocytes, (C) Control or ABCA1-GFP-transfected HeLa cells, (D) thymocytes from wildtype or <i>ABCA1^−/−</i> mice, (E) normal human fibroblasts or (F) Tangier fibroblasts were allowed to internalize NBD-PS, dithionite added to reduce and render non-fluorescent externalized NBD-PS, and cellular fluorescence (unexternalized NBD-PS) measured continuously over time at room temperature. (A–D) normal/wildtype, filled symbols; ABCA1-deficient or replete (HeLa), open symbols. (D) untreated, circles; treated with Ca²⁺ and Ca²⁺ ionophore, squares. (E and F) presence (open triangles) or absence (closed triangles) of apoA1.</p

Summary of Assays

<p>ND = Not Determined T = as targets P = as phagocytes</p

Basal NBD-PS externalization in peritoneal macrophages from wildtype or <i>ABCA1^−/−</i> mice.

<p>Macrophages (squares) or cells in the S gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone-0000729-g006" target="_blank">Figure 6</a> (circles) from wildtype (filled symbols) or <i>ABCA1^−/−</i> (open symbols) mice were allowed to internalize NBD-PS, dithionite was added to reduce and render non-fluorescent externalized NBD-PS, and cellular fluorescence (unexternalized NBD-PS) was measured continuously over time (A). Rate constants derived by simple exponential fit to the data for macrophages (black) and gated S cells (grey) are compared in (B).</p

Phagocytosis of apoptotic targets by wildtype or <i>ABCA1^−/−</i> macrophages.

<p>A&B, phagocytosis of non-apoptotic or apoptotic thymocytes from wildtype (black) or <i>ABCA1^−/−</i> (gray) mice by peritoneal macrophages from wildtype (A) or <i>ABCA1^−/−</i> (B) mice. C, phagocytosis of camptothecin-induced apoptotic EBV-transformed normal (black) or Tangier (gray) human B lymphocytes, either untreated or pre-treated with 10 µM Annexin V, then washed prior to presentation to mouse J774 macrophages. Error bars represent the standard error of 3 replicates.</p