Anant K. Menon

Thematic review series: Lipid Posttranslational Modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids

Glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins is the most complex and metabolically expensive of the lipid posttranslational modifications described to date. The GPI anchor is synthesized via a membrane-bound multistep pathway in the endoplasmic reticulum (ER) requiring >20 gene products. The pathway is initiated on the cytoplasmic side of the ER and completed in the ER lumen, necessitating flipping of a glycolipid intermediate across the membrane. The completed GPI anchor is attached to proteins that have been translocated across the ER membrane and that display a GPI signal anchor sequence at the C terminus. GPI proteins transit the secretory pathway to the cell surface; in yeast, many become covalently attached to the cell wall. Genes encoding proteins involved in all but one of the predicted steps in the assembly of the GPI precursor glycolipid and its transfer to protein in mammals and yeast have now been identified. Most of these genes encode polytopic membrane proteins, some of which are organized in complexes. The steps in GPI assembly, and the enzymes that carry them out, are highly conserved. GPI biosynthesis is essential for viability in yeast and for embryonic development in mammals. In this review, we describe the biosynthesis of mammalian and yeast GPIs, their transfer to protein, and their subsequent processing.

Lipid landscapes and pipelines in membrane homeostasis.

The lipid composition of cellular organelles is tailored to suit their specialized tasks. A fundamental transition in the lipid landscape divides the secretory pathway in early and late membrane territories, allowing an adaptation from biogenic to barrier functions. Defending the contrasting features of these territories against erosion by vesicular traffic poses a major logistical problem. To this end, cells evolved a network of lipid composition sensors and pipelines along which lipids are moved by non-vesicular mechanisms. We review recent insights into the molecular basis of this regulatory network and consider examples in which malfunction of its components leads to system failure and disease.

Cytoplasmic and Nuclear Delivery of a TAT-derived Peptide and a β-Peptide after Endocytic Uptake into HeLa Cells

Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural β-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47–57), as well as a similarly labeled 12-residue β-peptide, β-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47–57)) or nucleolus (β-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47–57) and β-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.

Lipid flippases and their biological functions.

Abstract.The typically distinct phospholipid composition of the two leaflets of a membrane bilayer is generated and maintained by bi-directional transport (flip-flop) of lipids between the leaflets. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. Energy-independent flippases allow common phospholipids to equilibrate rapidly between the two monolayers and also play a role in the biosynthesis of a variety of glycoconjugates such as glycosphingolipids, N-glycoproteins, and glycosylphosphatidylinositol (GPI)-anchored proteins. ATP-dependent flippases, including members of a conserved subfamily of P-type ATPases and ATP-binding cassette transporters, mediate the net transfer of specific phospholipids to one leaflet of a membrane and are involved in the creation and maintenance of transbilayer lipid asymmetry of membranes such as the plasma membrane of eukaryotes. Energy-dependent flippases also play a role in the biosynthesis of glycoconjugates such as bacterial lipopolysaccharide. This review summarizes recent progress on the identification and characterization of the various flippases and the demonstration of their biological functions.

Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosoma brucei : involvement of mannosyl-phosphoryldolichol as the mannose donor

Trypanosome variant surface glycoproteins (VSGs) exemplify a class of eukaryotic cell‐surface glycoproteins that rely on a covalently attached lipid, glycosyl‐phosphatidylinositol, for membrane attachment. The glycolipid anchor is acquired soon after translation of the polypeptide, apparently by replacement of a short sequence of carboxyl‐terminal amino acids with a precursor glycolipid. A candidate glycolipid precursor (P2) and a related glycolipid (P3) have been identified in polar lipid extracts from trypanosomes. Both lipids are glycosylphosphatidylinositol species containing a Man3GlcN core glycan indistinguishable from the backbone sequence of the VSG glycolipid anchor. We and others have recently described the cell‐free synthesis of P2, P3, and a spectrum of putative biosynthetic lipid intermediates using crude preparations of trypanosome membranes. In this paper we use these preparations to show that all three mannose residues in the glycosyl‐phosphatidylinositol glycan are derived from dolichol‐P‐mannose.

Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (Review)

Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of nonprotein-linked GPIs can approach 107 molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.

Ca2+-dependent phospholipid scrambling by a reconstituted TMEM16 ion channel

Phospholipid scramblases disrupt the lipid asymmetry of the plasma membrane, externalizing phosphatidylserine to trigger blood coagulation and mark apoptotic cells. Recently, members of the TMEM16 family of Ca2+-gated channels have been shown to be involved in Ca2+-dependent scrambling. It is however controversial whether they are scramblases or channels regulating scrambling. Here we show that purified afTMEM16, from Aspergillus fumigatus, is a dual-function protein: it is a Ca2+-gated channel, with characteristics of other TMEM16 homologues, and a Ca2+-dependent scramblase, with the expected properties of mammalian phospholipid scramblases. Remarkably, we find that a single Ca2+ site regulates separate transmembrane pathways for ions and lipids. Two other purified TMEM16-channel homologues do not mediate scrambling, suggesting that the family diverged into channels and channel/scramblases. We propose that the spatial separation of the ion and lipid pathways underlies the evolutionary divergence of the TMEM16 family, and that other homologues, such as TMEM16F, might also be dual-function channel/scramblases.

A new family of StART domain proteins at membrane contact sites has a role in ER-PM sterol transport

Sterol traffic between the endoplasmic reticulum (ER) and plasma membrane (PM) is a fundamental cellular process that occurs by a poorly understood non-vesicular mechanism. We identified a novel, evolutionarily diverse family of ER membrane proteins with StART-like lipid transfer domains and studied them in yeast. StART-like domains from Ysp2p and its paralog Lam4p specifically bind sterols, and Ysp2p, Lam4p and their homologs Ysp1p and Sip3p target punctate ER-PM contact sites distinct from those occupied by known ER-PM tethers. The activity of Ysp2p, reflected in amphotericin-sensitivity assays, requires its second StART-like domain to be positioned so that it can reach across ER-PM contacts. Absence of Ysp2p, Ysp1p or Sip3p reduces the rate at which exogenously supplied sterols traffic from the PM to the ER. Our data suggest that these StART-like proteins act in trans to mediate a step in sterol exchange between the PM and ER. DOI: http://dx.doi.org/10.7554/eLife.07253.001

Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum

BACKGROUND A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop. RESULTS To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins. CONCLUSIONS Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP‐dependent, non‐vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol‐binding protein (Osh1‐7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM‐derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔosh4‐1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse‐chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM‐enriched fractions as well as by extracting sterols from intact cells with methyl‐β‐cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh‐deficient cells, the rate at which it moves within the PM to equilibrate with the methyl‐β‐cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non‐vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.