The ins(ide) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum.

The precursor oligosaccharide donor for protein N-glycosylation in eukaryotes, Glc3Man9GlcNAc(2)-P-P-dolichol, is synthesized in two stages on both leaflets of the rough endoplasmic reticulum (ER). There is good evidence that the level of dolichyl monophosphate (Dol-P) is one rate-controlling factor in the first stage of the assembly process. In the current topological model it is proposed that ER proteins (flippases) then mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol, and Man5GlcNAc(2)-P-P-Dol from the cytoplasmic leaflet to the lumenal leaflet. The rate of flipping of the three intermediates could plausibly influence the conversion of Man5GlcNAc(2)-P-P-Dol to Glc3Man(9)GlcNAc(2)-P-P-Dol in the second stage on the lumenal side of the rough ER. This article reviews the current understanding of the enzymes involved in the de novo biosynthesis of Dol-P and other polyisoprenoid glycosyl carrier lipids and speculates about the role of membrane proteins and enzymes that could be involved in the transbilayer movement of the lipid intermediates and the recycling of Dol-P and Dol-P-P discharged during glycosylphosphatidylinositol anchor biosynthesis, N-glycosylation, and O- and C-mannosylation reactions on the lumenal surface of the rough ER.     

Opsin is a phospholipid flippase

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<10 s) flipping of phospholipid probes across the vesicle membrane. This is the first molecular identification of an ATP-independent phospholipid flippase in any system. It reveals an unexpected activity for opsin and, in conjunction with recently available structural information on this G protein-coupled receptor [6, 7], significantly advances our understanding of the mechanism of ATP-independent lipid flip-flop.     

Role of Flippases,Scramblases and Transfer Proteins in Phosphatidylserine Subcellular Distribution

It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then distributed to different cellular locations in order to create the distinct membrane compositions observed in eukaryotes. However, the mechanisms by which specific lipid species are trafficked to and maintained in specific areas of the cell are poorly understood and constitute an active area of research. Of particular interest is the distribution of phosphatidylserine (PS), an anionic lipid that is enriched in the cytosolic leaflet of the plasma membrane. PS transport occurs by both vesicular and non‐vesicular routes, with members of the oxysterol‐binding protein family (Osh6 and Osh7) recently implicated in the latter route. In addition, the flippase activity of P4‐ATPases helps build PS membrane asymmetry by preferentially translocating PS to the cytosolic leaflet. This asymmetric PS distribution can be used as a signaling device by the regulated activation of scramblases, which rapidly expose PS on the extracellular leaflet and play important roles in blood clotting and apoptosis. This review will discuss recent advances made in the study of phospholipid flippases, scramblases and PS‐specific lipid transfer proteins, as well as how these proteins contribute to subcellular PS distribution.      

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton, and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity, and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.     

Emerging new paradigms for ABCG transporters

Every cell is separated from its external environment by a lipid membrane. Survival depends on the regulated and selective transport of nutrients, waste products and regulatory molecules across these membranes, a process that is often mediated by integral membrane proteins. The largest and most diverse of these membrane transport systems is the ATP binding cassette (ABC) family of membrane transport proteins. The ABC family is a large evolutionary conserved family of transmembrane proteins (> 250 members) present in all phyla, from bacteria to Homo sapiens, which require energy in the form of ATP hydrolysis to transport substrates against concentration gradients. In prokaryotes the majority of ABC transporters are involved in the transport of nutrients and other macromolecules into the cell. In eukaryotes, with the exception of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), ABC transporters mobilize substrates from the cytoplasm out of the cell or into specific intracellular organelles. This review focuses on the members of the ABCG subfamily of transporters, which are conserved through evolution in multiple taxa. As discussed below, these proteins participate in multiple cellular homeostatic processes, and functional mutations in some of them have clinical relevance in humans.     

BAR domain competition during directional cellular migration

While directed cellular migration facilitates the coordinated movement of cells during development and tissue repair, the precise mechanisms regulating the interplay between the extracellular environment, the actin cytoskeleton and the overlying plasma membrane remain inadequately understood. The BAR domain family of lipid binding, actin cytoskeletal regulators are gaining greater appreciation for their role in these critical processes. BAR domain proteins are involved as both positive and negative regulators of endocytosis, membrane plasticity and directional cell migration. This review focuses on the functional relationship between different classes of BAR domain proteins and their role in guiding cell migration through regulation of the endocytic machinery. Competition for key signaling substrates by positive and negative BAR domain endocytic regulators appears to mediate control of directional cell migration, and may have wider applicability to other trafficking functions associated with development and carcinogenesis.Key words: BAR domain, MIM, directed cell migration, competition, membrane dynamics, actin cytoskeleton, endocytosis     

Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum

The process of N-linked glycosylation of secretory proteins is characterized by enzymatic reactions occurring on both sides of the endoplasmic reticulum (ER) membrane. On either side multiple glycosyltransferases participate in the stepwise addition of monosaccharides to core oligosaccharide unit that is attached to the lipid carrier dolichyl pyrophosphate. Cytoplasm-oriented glycosyltransferases use nucleotide-activated sugars as substrates, whereas lumen-oriented transferases that act later in the pathway make use of dolichyl phosphate-linked monosaccharides. The completely assembled core oligosaccharide is transferred to proteins on the lumenal side of the ER. The topological organization of this biosynthetic pathway requires the translocation of lipid-linked mono- and oligo-saccharides across the ER membrane. The transfer of the substrates and intermediates depend on specific translocators, i.e. so called flippases.     

Regulation of transbilayer plasma membrane phospholipid asymmetry

Lipids in biological membranes are asymmetrically distributed across the bilayer; the amine-containing phospholipids are enriched on the cytoplasmic surface of the plasma membrane, while the choline-containing and sphingolipids are enriched on the outer surface. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathologic conditions. Lipid asymmetry is generated primarily by selective synthesis of lipids on one side of the membrane. Because passive lipid transbilayer diffusion is slow, a number of proteins have evolved to either dissipate or maintain this lipid gradient. These proteins fall into three classes: 1) cytofacially-directed, ATP-dependent transporters ("flippases"); 2) exofacially-directed, ATP-dependent transporters ("floppases"); and 3) bidirectional, ATP-independent transporters ("scramblases"). The flippase is highly selective for phosphatidylserine and functions to keep this lipid sequestered from the cell surface. Floppase activity has been associated with the ABC class of transmembrane transporters. Although they are primarily nonspecific, at least two members of this class display selectivity for their substrate lipid. Scramblases are inherently nonspecific and function to randomize the distribution of newly synthesized lipids in the endoplasmic reticulum or plasma membrane lipids in activated cells. It is the combined action of these proteins and the physical properties of the membrane bilayer that generate and maintain transbilayer lipid asymmetry.     

Cargo trafficking from the trans‐Golgi network towards the endosome

The trans‐Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low‐level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching‐off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot–Marie–Tooth disease, I‐cell disease and Wiskott–Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.     

Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis

Cell membranes contain various transporter proteins, some of which are responsible for transferring amino acids across membrane. In this study, we report another class of carrier proteins, termed Serinc1-5, that incorporates a polar amino acid serine into membranes and facilitates the synthesis of two serine-derived lipids, phosphatidylserine and sphingolipids. Serinc is a unique protein family that shows no amino acid homology to other proteins but is highly conserved among eukaryotes. The members contain 11 transmembrane domains, and rat Serinc1 protein co-localizes with lipid biosynthetic enzymes in endoplasmic reticulum membranes. A Serinc protein forms an intracellular complex with key enzymes involved in serine and sphingolipid biosyntheses, and both functions, serine synthesis and membrane incorporation, are linked to each other. In the rat brain, expression of Serinc1 and Serinc2 mRNA was rapidly up-regulated by kainate-induced seizures in neuronal cell layers of the hippocampus. In contrast, myelin throughout the brain is enriched with Serinc5, which was down-regulated in the hippocampus by seizures. These results indicate a novel mechanism linking neural activity and lipid biosynthesis.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

SMP domain proteins in membrane lipid dynamics

Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved family of proteins in eukaryotes that localize between the endoplasmic reticulum (ER) and either the plasma membrane (PM) or other organelles. They are involved in tethering of these membrane contact sites through interaction with other proteins and membrane lipids. Recent structural and biochemical studies have demonstrated that SMP domain proteins transport a wide variety of lipid species by the ability of the SMP domain to harbor lipids through its unique hydrophobic cavity. Growing evidence suggests that SMP domain proteins play critical roles in cell physiology by their actions at membrane contact sites. In this review, we summarize the functions of SMP domain proteins and their direct roles in lipid transport across different membrane compartments. We also discuss their physiological functions in organisms as well as “bypass” pathways that act in parallel with SMP domain proteins at membrane contact sites.     

Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.     

The Role of the Membrane in Transporter Folding and Activity

The synthesis, folding, and function of membrane transport proteins are critical factors for defining cellular physiology. Since the stability of these proteins evolved amidst the lipid bilayer, it is no surprise that we are finding that many of these membrane proteins demonstrate coupling of their structure or activity in some way to the membrane. More and more transporter structures are being determined with some information about the surrounding membrane, and computational modeling is providing further molecular details about these solvation structures. Thus, the field is moving towards identifying which molecular mechanisms - lipid interactions, membrane perturbations, differential solvation, and bulk membrane effects - are involved in linking membrane energetics to transporter stability and function. In this review, we present an overview of these mechanisms and the growing evidence that the lipid bilayer is a major determinant of the fold, form, and function of membrane transport proteins in membranes.     

How lipid flippases can modulate membrane structure

Phospholipid flippases, are proteins able to translocate phospholipids from one side of a membrane to the other even against a gradient of concentration and thereby able to establish, or annihilate, a transmembrane asymmetrical lipid distribution. This lipid shuttling forms new membrane structures, in particular vesicles, which are associated with diverse physiological functions in eukaryotic cells such as lipid and protein traffic via vesicles between organelles or towards the plasma membrane, and the stimulation of fluid phase endocytosis. The transfer of lipids is also responsible for the triggering of membrane associated events such as blood coagulation, the recognition and elimination of apoptotic or aged cells, and the regulation of phosphatidylserine dependent enzymes. Exposure of new lipid-head groups on a membrane leaflet by rapid flip-flop can serve as a specific signal and, upon recognition, can be the cause of physiological modifications. Membrane bending is one of the mechanisms by which such activities can be triggered. We show that the lateral membrane tension is an important physical factor for the regulation of the size of the membrane invaginations. Finally, we suggest in this review that this diversity of functions benefits from the diversity of the lipids existing in a cell and the ability of proteins to recognize specific messenger molecules.     

Evidence that the wzxE gene of Escherichia coli K-12 encodes a protein involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen

The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.     

Decoding P4-ATPase substrate interactions

Cellular membranes display a diversity of functions that are conferred by the unique composition and organization of their proteins and lipids. One important aspect of lipid organization is the asymmetric distribution of phospholipids (PLs) across the plasma membrane. The unequal distribution of key PLs between the cytofacial and exofacial leaflets of the bilayer creates physical surface tension that can be used to bend the membrane; and like Ca²⁺, a chemical gradient that can be used to transduce biochemical signals. PL flippases in the type IV P-type ATPase (P4-ATPase) family are the principle transporters used to set and repair this PL gradient and the asymmetric organization of these membranes are encoded by the substrate specificity of these enzymes. Thus, understanding the mechanisms of P4-ATPase substrate specificity will help reveal their role in membrane organization and cell biology. Further, decoding the structural determinants of substrate specificity provides investigators the opportunity to mutationally tune this specificity to explore the role of particular PL substrates in P4-ATPase cellular functions. This work reviews the role of P4-ATPases in membrane biology, presents our current understanding of P4-ATPase substrate specificity, and discusses how these fundamental aspects of P4-ATPase enzymology may be used to enhance our knowledge of cellular membrane biology.     

Seeking the determinants of the elusive functions of Sco proteins

Sco proteins are present in all types of organisms, including the vast majority of eukaryotes and many prokaryotes. It is well established that Sco proteins in eukaryotes are involved in the assembly of the Cu(A) cofactor of mitochondrial cytochrome c oxidase; however their precise role in this process has not yet been elucidated at the molecular level. In particular, some but not all eukaryotes including humans possess two Sco proteins whose individual functions remain unclear. There is evidence that eukaryotic Sco proteins are also implicated in other cellular processes such as redox signalling and regulation of copper homeostasis. The range of physiological functions of Sco proteins appears to be even wider in prokaryotes, where Sco-encoding genes have been duplicated many times during evolution. While some prokaryotic Sco proteins are required for the biosynthesis of cytochrome c oxidase, others are most likely to take part in different processes such as copper delivery to other enzymes and protection against oxidative stress. The detailed understanding of the multiplicity of roles ascribed to Sco proteins requires the identification of the subtle determinants that modulate the two properties central to their known and potential functions, i.e. copper binding and redox properties. In this review, we provide a comprehensive summary of the current knowledge on Sco proteins gained by genetic, structural and functional studies on both eukaryotic and prokaryotic homologues, and propose some hints to unveil the elusive molecular mechanisms underlying their functions.     

Gamma-secretase is a functional component of phagosomes

Gamma-secretase is a high molecular mass protein complex that catalyzes the intramembrane cleavage of its protein substrates. Two proteins involved in phagocytosis, CD44 and the low density lipoprotein receptor-related protein, are gamma-secretase substrates, suggesting that this complex might regulate some aspects of phagocytosis. Our results indicate that the four components of gamma-secretase, viz. presenilin, nicastrin, APH-1, and PEN-2, are present and enriched on phagosome membranes from both murine macrophages and Drosophila S2 phagocytes. The gamma-secretase components form high molecular mass complexes in lipid microdomains of the phagosome membrane with the topology expected for the functional enzyme. In contrast to the majority of the phagosome proteins studied so far, which appear to associate transiently with this organelle, gamma-secretase resides on newly formed phagosomes and remains associated throughout their maturation into phagolysosomes. Finally, our results indicate that interferon-gamma stimulates gamma-secretase-dependent cleavages on phagosomes and that gamma-secretase activity may be involved in the phagocytic response of macrophages to inflammatory cytokines.     

HRD gene dependence of endoplasmic reticulum-associated degradation

Work from several laboratories has indicated that many different proteins are subject to endoplasmic reticulum (ER) degradation by a common ER-associated machinery. This machinery includes ER membrane proteins Hrd1p/Der3p and Hrd3p and the ER-associated ubiquitin-conjugating enzymes Ubc7p and Ubc6p. The wide variety of substrates for this degradation pathway has led to the reasonable hypothesis that the HRD (Hmg CoA reductase degradation) gene-encoded proteins are generally involved in ER protein degradation in eukaryotes. We have tested this model by directly comparing the HRD dependency of the ER-associated degradation for various ER membrane proteins. Our data indicated that the role of HRD genes in protein degradation, even in this highly defined subset of proteins, can vary from absolute dependence to complete independence. Thus, ER-associated degradation can occur by mechanisms that do not involve Hrd1p or Hrd3p, despite their apparently broad envelope of substrates. These data favor models in which the HRD gene-encoded proteins function as specificity factors, such as ubiquitin ligases, rather than as factors involved in common aspects of ER degradation.