An unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the C. elegans putative aminophospholipid translocases.

BACKGROUND: P-type ATPases in subfamily IV are exclusively eukaryotic transmembrane proteins that have been proposed to directly translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine from the exofacial to the cytofacial monolayer of the plasma membrane. Eukaryotic genomes contain many genes encoding members of this subfamily. At present it is unclear why there are so many genes of this kind per organism or what individual roles these genes perform in organism development. RESULTS: We have systematically investigated expression and developmental function of the six, tat-1 through 6, subfamily IV P-type ATPase genes encoded in the Caenorhabditis elegans genome. tat-5 is the only ubiquitously-expressed essential gene in the group. tat-6 is a poorly-transcribed recent duplicate of tat-5. tat-2 through 4 exhibit tissue-specific developmentally-regulated expression patterns. Strong expression of both tat-2 and tat-4 occurs in the intestine and certain other cells of the alimentary system. The two are also expressed in the uterus, during spermatogenesis and in the fully-formed spermatheca. tat-2 alone is expressed in the pharyngeal gland cells, the excretory system and a few cells of the developing vulva. The expression pattern of tat-3 is almost completely different from those of tat-2 and tat-4. tat-3 expression is detectable in the steroidogenic tissues: the hypodermis and the XXX cells, as well as in most cells of the pharynx (except gland), various tissues of the reproductive system (except uterus and spermatheca) and seam cells. Deletion of tat-1 through 4 individually interferes little or not at all with the regular progression of organism growth and development under normal conditions. However, tat-2 through 4 become essential for reproductive growth during sterol starvation. CONCLUSION: tat-5 likely encodes a housekeeping protein that performs the proposed aminophospholipid translocase function routinely. Although individually dispensable, tat-1 through 4 seem to be at most only partly redundant. Expression patterns and the sterol deprivation hypersensitivity deletion phenotype of tat-2 through 4 suggest that these genes carry out subtle metabolic functions, such as fine-tuning sterol metabolism in digestive or steroidogenic tissues. These findings uncover an unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the genes encoding the putative aminophospholipid translocases.

Cognate putative nuclear localization signal effects strong nuclear localization of a GFP reporter and facilitates gene expression studies in Caenorhabditis elegans.

Targeting a gene expression reporter, usually the green fluorescent protein (GFP), to the nucleus via a translationally fused nuclear localization signal (NLS) greatly facilitates recognition and identification of the reporter-expressing cells in Caenorhabditis elegans. Presently circulating nematode transcriptional gene expression vectors use the viral NLS from simian virus 40 (SV40) large T antigen. This NLS, however fails to ensure sufficient localization of the GFP peptide to the nucleus. We modified the common transcriptional reporter SV40 NLS-GFP by adding to its C terminus a cognate putative NLS from the transcription factor egl-13. The EGL-13 NLS effected clear contrast in fluorescence intensity between the nucleus and the cytoplasm in cells with strong reporter signal and efficiently highlighted the nucleus in tissues with weak reporter expression in a wide range of tested tissues. The SV40 NLS-GFP-EGL-13 NLS vector should become a valuable tool for gene expression studies in C. elegans.

P.S. to PS (phosphatidylserine)--pertinent proteins in apoptotic cell clearance.

The psr protein has been proposed as the critical receptor that detects phosphatidylserine (PS) on the surface of apoptotic cells. However, for some time there has been evidence that this protein is not at the cell surface but in the nucleus. Now, the phenotype of a knockout of the Drosophila psr protein (dPSR) has discredited the identification altogether, lending impetus both to uncovering the real function of the protein and to identifying the real PS receptor. Interpretations of studies of two other genes supposedly involved in PS transport may be built on similarly shaky foundations.

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.

Aminophospholipid translocase TAT-1 promotes phosphatidylserine exposure during C. elegans apoptosis.

Phospholipids 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.

Signal transduction induced by apoptotic cells inhibits HIV transcription in monocytes/macrophages.

The primary targets of HIV are CD4(+) T cells and macrophages. HIV infection is associated with an increase in apoptosis of infected and uninfected CD4(+) T cells, and these infected cells undergo apoptosis and produce HIV virions with phosphatidylserine (PS) on their surface. During phagocytosis of apoptotic cells, macrophages, using an array of receptors, are able to perceive various surface changes on apoptotic cells. The engagement of phagocytic receptors by ligands on the apoptotic cell surface results in the activation of signaling cascades, which facilitate engulfment. In this study, we examined how PS associated with virions and apoptotic cells influences HIV replication. We demonstrate that virus-associated PS is required for HIV infection of macrophages at a step prior to integration but following strong-stop, indicating that PS-initiated signals alter the establishment of HIV provirus. Conversely, apoptotic cells inhibited HIV transcription in infected macrophages, although this ability to suppress transcription was independent of PS. Furthermore, we show that ELMO, a key signaling molecule that participates in the phagocytosis of apoptotic cells, inhibited HIV transcription; however, knocking down endogenous ELMO expression in infected U937 cells rescued HIV transcription when these cells were coincubated with apoptotic targets. Taken together, these data show that apoptotic cells and the signals, which they initiate upon recognition by macrophages, influence the successful establishment of HIV infection and provirus transcription.

Lipid specific activation of the murine P4-ATPase Atp8a1 (ATPase II).

The asymmetric transbilayer distribution of phosphatidylserine (PS) in the mammalian plasma membrane and secretory vesicles is maintained, in part, by an ATP-dependent transporter. This aminophospholipid "flippase" selectively transports PS to the cytosolic leaflet of the bilayer and is sensitive to vanadate, Ca(2+), and modification by sulfhydryl reagents. Although the flippase has not been positively identified, a subfamily of P-type ATPases has been proposed to function as transporters of amphipaths, including PS and other phospholipids. A candidate PS flippase ATP8A1 (ATPase II), originally isolated from bovine secretory vesicles, is a member of this subfamily based on sequence homology to the founding member of the subfamily, the yeast protein Drs2, which has been linked to ribosomal assembly, the formation of Golgi-coated vesicles, and the maintenance of PS asymmetry. To determine if ATP8A1 has biochemical characteristics consistent with a PS flippase, a murine homologue of this enzyme was expressed in insect cells and purified. The purified Atp8a1 is inactive in detergent micelles or in micelles containing phosphatidylcholine, phosphatidic acid, or phosphatidylinositol, is minimally activated by phosphatidylglycerol or phosphatidylethanolamine (PE), and is maximally activated by PS. The selectivity for PS is dependent upon multiple elements of the lipid structure. Similar to the plasma membrane PS transporter, Atp8a1 is activated only by the naturally occurring sn-1,2-glycerol isomer of PS and not the sn-2,3-glycerol stereoisomer. Both flippase and Atp8a1 activities are insensitive to the stereochemistry of the serine headgroup. Most modifications of the PS headgroup structure decrease recognition by the plasma membrane PS flippase. Activation of Atp8a1 is also reduced by these modifications; phosphatidylserine-O-methyl ester, lysophosphatidylserine, glycerophosphoserine, and phosphoserine, which are not transported by the plasma membrane flippase, do not activate Atp8a1. Weakly translocated lipids (PE, phosphatidylhydroxypropionate, and phosphatidylhomoserine) are also weak Atp8a1 activators. However, N-methyl-phosphatidylserine, which is transported by the plasma membrane flippase at a rate equivalent to PS, is incapable of activating Atp8a1 activity. These results indicate that the ATPase activity of the secretory granule Atp8a1 is activated by phospholipids binding to a specific site whose properties (PS selectivity, dependence upon glycerol but not serine, stereochemistry, and vanadate sensitivity) are similar to, but distinct from, the properties of the substrate binding site of the plasma membrane flippase.

Hide and seek: the secret identity of the phosphatidylserine receptor.

Phosphatidylserine on the dying cell surface helps identify apoptotic cells to phagocytes, which then engulf them. A candidate phagocyte receptor for phosphatidylserine was identified using phage display, but the phenotypes of knockout mice lacking this presumptive receptor, as well as the location of the protein within cells, cast doubt on the assignment of this protein as the phosphatidylserine receptor.

Macrophage surface expression of annexins I and II in the phagocytosis of apoptotic lymphocytes.

When cells undergo apoptosis, or programmed cell death, they expose phosphatidylserine (PS) on their surface. Macrophages that efficiently phagocytose apoptotic cells also express PS on their surface, although at a lower level. The PS exposed on both cells is required for phagocytosis, because uptake is inhibited by masking PS on either cell with annexin V, a PS-binding protein. The inhibition is not additive, suggesting that the exposed PS molecules on the two cells participate in a common process. We asked whether this dual requirement reflects bridging of the target cell and macrophage by bivalent, PS-binding annexins. Monoclonal antibodies (mAbs) against annexins I or II stained a variety of live phagocytes. Apoptotic Jurkat T lymphocytes and human peripheral T lymphocytes, but not apoptotic thymocytes, were stained by anti-annexin I but not II. Phagocytosis of apoptotic targets was inhibited by mAbs to annexins I or II, or by pretreatment of macrophages with the same mAbs. Pretreatment of apoptotic thymocytes had no effect, whereas pretreating Jurkat cells with anti-annexin I or removing annexin I with EGTA was inhibitory. Annexin bridging is vectorial, because annexin is bound to PS molecules on targets but not on macrophages, suggesting annexins serve as both ligand and receptor in promoting phagocytosis.

Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells.

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Reanalysis of ATP11B, a type IV P-type ATPase.

P-type ATPases are a venerable family of ATP-dependent ion transporters. Recently, evidence was presented that a rabbit gene in the type IV subfamily of P-type ATPases was missing a transmembrane helix (transmembrane domain 4) thought to be critical for ion transport, a deletion that would place the two major catalytic loops of the enzyme on opposite sides of the membrane. It was proposed that the resulting protein was a RING finger-binding protein that targets transcription factors to specific domains within the nucleus. From analysis of human genomic sequence data, it is shown here that the region containing transmembrane domain 4, corresponding to exon 12, is present in the human homolog of the gene, ATP11B. PCR analysis indicates that the predominant Atp11b transcripts in a rabbit cDNA library and in a mouse cDNA library also contain exon 12. The results suggest that the transcript proposed to encode the RING finger-binding protein is a minor rabbit-specific splice variant. The ATP11B gene thus may not encode a protein with a function radically different from that of other P-type ATPase transporters.

Transbilayer phospholipid movement and the clearance of apoptotic cells.

When lymphocytes (and other cells) die by apoptosis, they orchestrate their own orderly removal by macrophages, and thereby prevent the inflammation that would otherwise attend cell lysis. As part of their demise, apoptotic cells disrupt the normal asymmetric distribution of phospholipids across their plasma membranes, an asymmetry normally maintained by an aminophospholipid translocase. This disruption of asymmetry, mediated by an activity known as the scramblase, generates ligands on the cell surface that trigger phagocytosis of the dying cell before lysis can occur. This crucial alteration of the plasma membrane is not dependent on caspase-mediated proteolysis, but quite unexpectedly, it is required both on the apoptotic target cell and on the phagocyte that engulfs it. At least in the phagocyte, this rearrangement may depend on the activity of an ABC ATPase, termed ABC1 in mammals and ced-7 in C. elegans.