Phosphatidylserine transport in cell life and death

Phosphatidylserine (PS) is a negatively charged glycerophospholipid found mainly in the plasma membrane (PM) and in the late secretory/endocytic compartments, where it regulates cellular activity and can mediate apoptosis. Export of PS from the endoplasmic reticulum, its site of synthesis, to other compartments, and its transbilayer asymmetry must therefore be precisely regulated. We review recent findings on nonvesicular transport of PS by lipid transfer proteins (LTPs) at membrane contact sites, on PS flip-flop between membrane leaflets by flippases and scramblases, and on PS nanoclustering at the PM. We also discuss emerging data on cooperation between scramblases and LTPs, how perturbation of PS distribution can lead to disease, and the specific role of PS in viral infection.     

Phosphatidylserine exposure in living cells

Abstract

P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.     

Mechanisms of apoptotic phosphatidylserine exposure

It has been a long-standing enigma which scramblase causes phosphatidylserine residues to be exposed on the surface of apoptotic cells, thereby facilitating the phagocytic recognition, engulfment and destruction of apoptotic corpses. In a recent paper in Science, Nagata and coworkers reveal that the scramblases Xkr8 and its C. elegans ortholog, CED-8, are activated by caspase cleavage in apoptotic cells.All cells are separated from the extracellular environment by the plasma membrane, a phospholipid bilayer that prevents diffusion of proteins, ions and other essential molecules into the extracellular space and constitutes the structure in which membrane proteins are embedded. In animal cells, the lipid composition of the outer and inner leaflets of the plasma membrane is not symmetrical. Phosphatidylcholine (PC) and sphingomyelin (SM) are mainly present in the outer leaflet of the plasma membrane, whereas phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) are restricted to the inner leaflet. This lipid asymmetry is maintained by the combined action of ATP-dependent enzymes called flippases and floppases, which specifically translocate phospholipids and other molecules from the outer to the inner membrane leaflet and from the inner to the outer membrane leaflet, respectively¹. Lipid composition asymmetry not only defines the curvature and electrochemical properties of the plasma membrane, but is also essential for the correct function of determined lipids, as for instance, PI, which only functions as a second messenger if present in the inner leaflet². Nonetheless, several physiologically relevant processes as diverse as platelet activation, neurotransmitter release, sperm capacitation or apoptosis, require dissipation of plasma membrane lipid asymmetry, a process known as scrambling. The enzymes responsible for this activity are called scramblases, and function to randomize the distribution of phospholipids between both membrane leaflets in an ATP-independent manner^2,3,4.Although plasma membrane asymmetry and the existence of flippases, floppases and scramblases have been known for decades, the identity of the specific enzymes involved in these activities has only begun to be revealed during the last few years. Very recently, the group of Shigekazu Nagata identified TMEM16F as the long sought-after calcium-dependent phospholipid scramblase³. However, to date, the identity of the scramblase(s) involved in apoptosis-related (and calcium-independent) PS exposure had remained elusive. Cell surface PS exposure is a classic feature of apoptotic cells and acts as an “eat me” signal allowing phagocytosis of post-apoptotic bodies. In a recent paper in Science, Nagata''s group identified Xk-Related Protein 8 (Xkr8) as the enzyme responsible for this activity and demonstrated an evolutionarily conserved role of this protein in apoptosis-induced lipid scrambling⁵.To identify enzymes involved in membrane lipid scrambling, Nagata''s group took advantage of their previously generated mouse Ba/F3 pro-B cell line³, which presented a high basal level of PS exposure. They then generated a cDNA library from Ba/F3 cells and overexpressed it in the parental cell line. Through sequential enrichment of cells with increased PS exposure, they were able to isolate a cDNA encoding the Xkr8 protein, which enhanced PS scrambling when overexpressed. Xkr8 overexpression (but not that of TMEM16F) was able to increase apoptosis-associated PS exposure. The authors then noticed that both impaired apoptosis-induced PS exposure and deficient post-apoptotic body clearance were correlated with low Xkr8 expression in leukemia and lymphoma cell lines, which was linked to hypermethylation of its promoter. Interestingly, these two alterations were reverted either by overexpressing Xkr8 or by restitution of endogenous Xkr8 expression after treatment with the demethylating agent 5-aza-2′-deoxycytidine (DAC), suggesting that methylation of the Xrk8 promoter may be a mechanism by which tumor cells evade their phagocytosis after apoptotic death, which may result in increased local inflammation, thus favoring tumor progression. Far from being restricted only to PS exposure, Xrk8 overexpression was able to promote scrambling of multiple lipid species during apoptosis, which was demonstrated by incorporation of fluorescent PC and SM analogues. This scrambling activity was restricted to apoptotic events, as Xkr8 overexpression had no effect on Ca²⁺-induced PS exposure. This specificity may be explained by the presence of an evolutionarily conserved caspase recognition site near Xkr8 C-terminal region, whose mutation prevented both Xkr8 cleavage by caspase-3 or -7 and PS exposure during the course of apoptosis (Figure 1). These results from human cell lines were confirmed in Xkr8^−/− mouse embryonic fibroblasts and fetal thymocytes, which were unable to expose PS upon induction of apoptosis, underscoring the broad physiological relevance of Xkr8 in the apoptotic process. Finally, the authors moved to the nematode Caenorhabditis elegans to analyze whether the role of Xpr8 as lipid scramblase is evolutionarily conserved. C. elegans harbors only one ortholog of Xk proteins, CED-8, known to participate in the phagocytic removal of apoptotic corpses⁶. To determine the role of CED-8 in PS exposure, the authors took advantage of the “floater” assay, which is based on the appearance of floating cells (“floaters”) that have detached from developing C. elegans embryos defective for apoptotic cell phagocytosis⁷. Nagata''s group discovered that ced-8 deficiency leads to the accumulation of floaters. Moreover, ced-8 deficiency synergistically enhanced the number of floaters found in other engulfment mutants, which suggests that CED-8 function is not redundant to that developed by previously known engulfment mutants. This enhancing effect of ced-8 deletion was dependent on CED-3, the C. elegans ortholog of caspase-3, confirming the aforementioned results in mammalian cells. The authors then characterized that floaters resulting from ced-8 deletion show a largely deficient PS exposure after developmental apoptosis, confirming the evolutionarily conserved role of Xk-related proteins in apoptosis-induced lipid scrambling. However, they observed that ced-8 deletion does not lead to a total impairment in apoptotic PS presentation, suggesting that additional proteins must be involved in this process. Indeed, apoptosis-inducing factor can induce PS exposure in mammalian cells in a caspase-independent fashion⁸, and the C. elegans AIF ortholog, WAF-1, physically interacts with and activates another scramblase, SCRM-1⁴.Open in a separate windowFigure 1Xrp8 acts as apoptosis-induced lipid scramblase. Under normal conditions, the combined action of multiple mechanisms, including the activity of flippases and floppases, maintains lipid asymmetry between the outer and inner leaflets of the plasma membrane. Once apoptotic program is activated, caspases-3 and -7 are able to cleave and activate Xrp8 protein, which acts as a lipid scramblase and leads to the loss of lipid asymmetry, resulting in PS exposure to the extracellular space. This acts as the “eat-me” signal that will allow phagocytosis of post-apoptotic cell corpses. PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PS, phosphatidylserine.In summary, through a series of elegant manipulations, Nagata''s group has found the long-sought caspase-activated lipid scramblase that mediates the exposure of “eat-me” signals in post-apoptotic cell corpses. Further studies involving Xkr8 protein, including the mechanisms participating in its epigenetic repression may open new roads for the study of autoimmune diseases, such as lupus erythematosus, which is associated with failure in the post-apoptotic corpse clearance system.     

Ist2 recruits the lipid transporters Osh6/7 to ER–PM contacts to maintain phospholipid metabolism

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER–PM contacts through a binding site located in Ist2’s disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.     

Endosomal lipid flippases and their related diseases

 In eukaryotic cell membranes, phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer. For example, the extracellular leaflet of the plasma membrane (PM) is enriched with phosphatidylcholine and sphingomyelin, while the cytosolic leaflet of the PM is enriched with phosphatidylserine (PS) and phosphatidylethanolamine. The asymmetric distribution of PS in the PM is crucial for cell life, since PS in the extracellular leaflet of the PM is recognized as an “eat-me” signal by phagocytes. Inside the cells, a high PS concentration in the cytosolic leaflet of the PM is essential to facilitate various cellular events through the recruitment of signaling molecules such as protein kinase C and Akt.The asymmetric distribution of phospholipids is believed to be generated in part by phospholipid translocases, or “flippases.” The proteins responsible for flippase activity are type IV P-type ATPase (P₄-ATPases). P-type ATPases are multispan transmembrane pumps that use ATP hydrolysis as an energy source. P-type ATPases undergo autophosphorylation of a conserved aspartate residue during the catalytic cycle, hence the designation of “P”-type. P₄-ATPases are unique in that they are phospholipid transporters whereas other types of P-type ATPases are ion transporters.The human genome contains 14 P₄-ATPases, and mutations in some P₄-ATPases cause inherited genetic diseases. For example, mutations in ATP8B1 are associated with intrahepatic cholestasis and also cause hearing loss. Mutations in ATP8A2 are associated with a severe neurological disorder characterized by cerebellar ataxia, mental retardation, and dysequilibrium syndrome (CAMRQ).¹ Despite the accumulating evidence highlighting the physiological importance of P₄-ATPases, how dysfunction of P₄-ATPases causes diseases is poorly understood.In a recent study, we revealed the cellular function of the P₄-ATPase, ATP8A1.² ATP8A1 localizes at recycling endosomes (REs), an organelle that functions in recycling transport of internalized molecules back to the PM, thus defining the amount of proteins at the PM. PS is most concentrated in REs among intracellular organelles and we roughly estimated that 70 and 30% of PS are localized in the cytosolic and the luminal leaflets of RE membranes, respectively.² ATP8A1 generates the asymmetric transbilayer distribution of PS at REs. The knockdown of ATP8A1 halted recycling traffic from REs to the PM. At the mechanistic level, we found that EHD1, a dynamin-like membrane fission protein, lost its RE localization upon ATP8A1 knockdown and EHD1 knockdown also blocked recycling traffic. EHD1 bound PS in vitro and lost its membrane localization in cells that are defective in PS synthesis. Thus, we propose that PS flipping by ATP8A1 recruits EHD1 to RE membranes, thereby regulating the recycling traffic from REs to the PM (Fig. 1). Open in a separate windowFigure 1.Model of flippase-related diseases. Under normal conditions, flippases (e.g., ATP8A1 and ATP8A2) translocate PS to the cytosolic leaflet of RE membranes. PS recruits EHD1 to REs, and then EHD1 participates in the fission of RE membranes to generate transport vesicles that contain cell surface receptors. In flippase-dysfunctional situations, PS levels in the cytosolic leaflet of REs would be low. This impairs the PS/EHD1/membrane traffic axis, leading to a lower abundance of cell surface receptors that are critical for responses to extracellular ligands.ATP8A2 is a tissue-specific ATP8A1 paralogue. We found that a CAMRQ-causative mutation of ATP8A2 (I376M) lost its ATPase and flippase activity toward PS. ATP8A2 is not endogenously expressed in COS-1 cells. Interestingly, the phenotype that was caused by the loss of ATP8A1 in COS-1 cells, was restored by the exogenous expression of wild-type ATP8A2, but not I376M mutant ATP8A2. Moreover, cortical neurons prepared from ATP8A2 knockout mice showed lower abundance of transferrin receptors at the PM. Together, these results indicate that ATP8A2 functions in the recycling traffic in neurons, and that CAMRQ may result from the defect in recycling of important neurological receptor proteins from REs to the PM. One possible candidate protein is very low-density lipoprotein receptor (VLDLR). VLDLR is a receptor for reelin, an extracellular protein that guides neuronal migration in the cerebral cortex and cerebellum. VLDLR circulates between the PM and endosomes (possibly REs) by recycling traffic.³ Significantly, mutations in VLDLR gene are also linked to CAMRQ.^4,5 Therefore, impaired recycling traffic of VLDLR to the PM in neurons with dysfunctional ATP8A2 (I376M) may cause lower expression of VLDLR at the PM, leading to reduced reelin signaling, abnormal neuronal development, and neurological disorder.dATP8B, a P₄-ATPase in Drosophila melanogaster was recently reported to cause an impaired response to cVA pheromone (a sex-specific social cue) and mislocalization of the pheromone receptor in cVA-sensing neurons.⁶ The impaired response to the pheromone in dATP8B mutant was rescued by expressing bovine ATP8A2. Therefore, from insects to mammals, phospholipid flippases may define the localization of neuronal receptors to the PM.Lastly, our findings may explain the phenotype of ATP8A1 knockout mice.⁷ ATP8A1 knockout mice are vital but show deficiencies in hippocampus-dependent learning. Hippocampus-dependent learning involves modification of synaptic strength, and one cellular mechanism for tuning synaptic strength is long-term potentiation (LTP). During LTP, REs supply glutamate receptors to the post-synaptic membrane.⁸ Therefore, we speculate that impaired glutamate receptor traffic from REs to the post-synaptic membranes during LTP may underlie the deficiency in learning in ATP8A1 knockout mice. In agreement with this hypothesis, the dominant-negative form of EHD1 inhibits glutamate receptor traffic during LTP.⁸Many P₄-ATPases are expressed in the Golgi/endosomes and the PM. We expect that they contribute redundantly to the phospholipid asymmetry and membrane traffic through organelles. Simultaneous ablations of P₄-ATPases may dissect their roles and will give more insight into flippase-mediated cellular processes and -related diseases.     

Dynamic Transbilayer Lipid Asymmetry

Cells have thousands of different lipids. In the plasma membrane, and in membranes of the late secretory and endocytotic pathways, these lipids are not evenly distributed over the two leaflets of the lipid bilayer. The basis for this transmembrane lipid asymmetry lies in the fact that glycerolipids are primarily synthesized on the cytosolic and sphingolipids on the noncytosolic surface of cellular membranes, that cholesterol has a higher affinity for sphingolipids than for glycerolipids. In addition, P4-ATPases, “flippases,” actively translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine to the cytosolic surface. ABC transporters translocate lipids in the opposite direction but they generally act as exporters rather than “floppases.” The steady state asymmetry of the lipids can be disrupted within seconds by the activation of phospholipases and scramblases. The asymmetric lipid distribution has multiple implications for physiological events at the membrane surface. Moreover, the active translocation also contributes to the generation of curvature in the budding of transport vesicles.A lipid bilayer consisting of phosphatidylcholine (PC) with one saturated and one unsaturated acyl chain is stable, flexible, and semipermeable. It is the simplest model of a biomembrane. In such membranes, PC with a spin label on its choline headgroup diffused rapidly in the plane of the membrane with a diffusion coefficient of 1.8 µm²/sec (Devaux and McConnell 1972). In contrast, PC movement between leaflets, “flip-flop,” was slow with a half-time of >6 h at 30°C (Kornberg and McConnell 1971). Similar half-times for PC flip-flop were measured in erythrocyte membranes, a mammalian plasma membrane with a complex lipid composition (Rousselet et al. 1976; Renooij and Van Golde 1977; van Meer et al. 1980). Interestingly, the erythrocyte membrane maintains an asymmetric lipid distribution across the lipid bilayer with all of its phosphatidylserine (PS) and most of its phosphatidylethanolamine (PE) in the cytosolic leaflet (Bretscher 1972; Verkleij et al. 1973). A critical discussion of these early data and the techniques used can be found in (Op den Kamp 1979).It was then observed that the enrichment of aminophospholipids in the cytosolic leaflet is maintained by an ATP-consuming translocator that flips these lipids from the outer leaflet across the lipid bilayer (Seigneuret and Devaux 1984). The flippase was later identified as a P4-ATPase (Tang et al. 1996; Soupene and Kuypers 2006). Around the same time it was found that an ABC transporter, ABCB4, was involved in transporting PC into the bile (Smit et al. 1993), and studies on the closely related ABCB1 proved that these transporters can translocate lipids across the plasma membrane onto acceptors in the extracellular space (van Helvoort et al. 1996). Finally, evidence was provided for passive, bidirectional movement of lipids across the ER membrane and under some conditions across the plasma membrane, in which cases the responsible proteins have not yet been unequivocally identified (Sanyal and Menon 2009; Bevers and Williamson 2010). Thus, we now have a general picture of how lipid asymmetry is generated, maintained, and disrupted. However, there are still important gaps in our knowledge. For example, the transbilayer orientation of the sterols that make up one-third of the lipids in eukaryotic plasma membranes has still not been resolved satisfactorily. Moreover, we do not understand mechanistically how translocators and exporters work and how their activity is regulated.     

Lipid microdomains,lipid translocation and the organization of intracellular membrane transport (Review)

Eukaryotic cells contain hundreds of different lipid species that are not uniformly distributed among their membranes. For example, sphingolipids and sterols form gradients along the secretory pathway with the highest levels in the plasma membrane and the lowest in the endoplasmic reticulum. Moreover, lipids in late secretory organelles display asymmetric transbilayer arrangements with the aminophospholipids concentrated in the cytoplasmic leaflet. This lipid heterogeneity can be viewed as a manifestation of the fact that cells exploit the structural diversity of lipids in organizing intracellular membrane transport. Lipid immiscibility and the generation of phase-separated lipid domains provide a molecular basis for sorting membrane proteins into specific vesicular pathways. At the same time, energy-driven aminophospholipid transporters participate in membrane deformation during vesicle biogenesis. This review will focus on how selective membrane transport relies on a dynamic interplay between membrane lipids and proteins.     

Enhanced translocation of amphiphilic peptides across membranes by transmembrane proteins

Cell membranes are phospholipid bilayers with a large number of embedded transmembrane proteins. Some of these proteins, such as scramblases, have properties that facilitate lipid flip-flop from one membrane leaflet to another. Scramblases and similar transmembrane proteins could also affect the translocation of other amphiphilic molecules, including cell-penetrating or antimicrobial peptides. We studied the effect of transmembrane proteins on the translocation of amphiphilic peptides through the membrane. Using two very different models, we consistently demonstrate that transmembrane proteins with a hydrophilic patch enhance the translocation of amphiphilic peptides by stabilizing the peptide in the membrane. Moreover, there is an optimum amphiphilicity because the peptide could become overstabilized in the transmembrane state, in which the peptide-protein dissociation is hampered, limiting the peptide translocation. The presence of scramblases and other proteins with similar properties could be exploited for more efficient transport into cells. The described principles could also be utilized in the design of a drug-delivery system by the addition of a translocation-enhancing peptide that would integrate into the membrane.     

Book reviews

Eukaryotic cells contain hundreds of different lipid species that are not uniformly distributed among their membranes. For example, sphingolipids and sterols form gradients along the secretory pathway with the highest levels in the plasma membrane and the lowest in the endoplasmic reticulum. Moreover, lipids in late secretory organelles display asymmetric transbilayer arrangements with the aminophospholipids concentrated in the cytoplasmic leaflet. This lipid heterogeneity can be viewed as a manifestation of the fact that cells exploit the structural diversity of lipids in organizing intracellular membrane transport. Lipid immiscibility and the generation of phase-separated lipid domains provide a molecular basis for sorting membrane proteins into specific vesicular pathways. At the same time, energy-driven aminophospholipid transporters participate in membrane deformation during vesicle biogenesis. This review will focus on how selective membrane transport relies on a dynamic interplay between membrane lipids and proteins.     

Involvement of VAT‐1 in Phosphatidylserine Transfer from the Endoplasmic Reticulum to Mitochondria

Mitochondria receive phosphatidylserine (PS) from the endoplasmic reticulum (ER), but how PS is moved from the ER to mitochondria is unclear. Current models postulate a physical link between the organelles, but no involvement of cytosolic proteins. Here, we have reconstituted PS transport from the ER to mitochondria in vitro using Xenopus egg components. Transport is independent of ER proteins, but is dependent on a cytosolic factor that has a preferential affinity for PS. Crosslinking with a photoactivatable PS analog identified VAT‐1 as a candidate for a cytosolic PS transport protein. Recombinant, purified VAT‐1 stimulated PS transport into mitochondria and depletion of VAT‐1 from Xenopus cytosol with specific antibodies led to a reduction of transport. Our results suggest that cytosolic factors have a role in PS transport from the ER to mitochondria, implicate VAT‐1 in the transport process, and indicate that physical contact between the organelles is not essential.      

Suicidal Membrane Repair Regulates Phosphatidylserine Externalization during Apoptosis

One of the hallmarks of apoptosis is the redistribution of phosphatidylserine (PS) from the inner-to-outer plasma membrane (PM) leaflet, where it functions as a ligand for phagocyte recognition and the suppression of inflammatory responses. The mechanism by which apoptotic cells externalize PS has been assumed to involve “scramblases” that randomize phospholipids across the PM bilayer. These putative activities, however, have not been unequivocally proven to be responsible for the redistribution of lipids. Because elevated cytosolic Ca²⁺ is critical to this process and is also required for activation of lysosome-PM fusion during membrane repair, we hypothesized that apoptosis could activate a “pseudo”-membrane repair response that results in the fusion of lysosomes with the PM. Using a membrane-specific probe that labels endosomes and lysosomes and fluorescein-labeled annexin 5 that labels PS, we show that the appearance of PS at the cell surface during apoptosis is dependent on the fusion of lysosomes with the PM, a process that is inhibited with the lysosomotrophe, chloroquine. We demonstrate that apoptotic cells evoke a persistent pseudo-membrane repair response that likely redistributes lysosomal-derived PS to the PM outer leaflet that leads to membrane expansion and the formation of apoptotic blebs. Our data suggest that inhibition of lysosome-PM fusion-dependent redistribution of PS that occurs as a result of chemotherapy- and radiotherapy-induced apoptosis will prevent PS-dependent anti-inflammatory responses that preclude the development of tumor- and patient-specific immune responses.There is increasing evidence that damaged plasma membranes (PM)² trigger an emergency Ca²⁺-dependent exocytotic repair response that patches the affected area by adding lysosome-derived membranes at the cell surface disruption site (1–5). Because high cytosolic Ca²⁺ concentrations trigger lysosome-PM fusion, the elevated cytosolic Ca²⁺ levels characteristic to apoptotic cells may also evoke a pseudo-repair mechanism that promotes lysosome-PM fusion. Indeed, similar to normal emergency repair responses, apoptosis is characterized by the appearance of organelle proteins and lipids at the PM surface (6–8). One critical distinction between the apoptotic and physiologic repair processes is the preservation of membrane lipid asymmetry. In normal cells, any perturbation in PS sidedness is corrected by restoration of basal cytosolic [Ca²⁺], reactivation of the Ca²⁺-inhibited aminophospholipid translocase (9, 10), and subsequent facilitated transport of PS back to the inner membrane leaflet of the cell. In apoptotic cells, however, persistent high cytosolic [Ca²⁺] precludes reactivation of the aminophospholipid translocase, and the redistributed PS remains in the outer membrane leaflet (11). The apparent similarities in these processes combined with observations that apoptotic cells express PS at the cell surface prompted us to investigate whether lysosome to PM fusion plays a role in the redistribution of PS during apoptosis.     

A Highlights from MBoC Selection: Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles

Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane–missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN.     

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.     

A Highlights from MBoC Selection: Osh4p is needed to reduce the level of phosphatidylinositol-4-phosphate on secretory vesicles as they mature

Phosphatidylinositol-4-phosphate (PI4P) is produced on both the Golgi and the plasma membrane. Despite extensive vesicular traffic between these compartments, genetic analysis suggests that the two pools of PI4P do not efficiently mix with one another. Several lines of evidence indicate that the PI4P produced on the Golgi is normally incorporated into secretory vesicles, but the fate of that pool has been unclear. We show here that in yeast the oxysterol-binding proteins Osh1–Osh7 are collectively needed to maintain the normal distribution of PI4P and that Osh4p is critical in this function. Osh4p associates with secretory vesicles at least in part through its interaction with PI4P and is needed, together with lipid phosphatases, to reduce the level of PI4P as vesicles approach sites of exocytosis. This reduction in PI4P is necessary for a switch in the regulation of the Sec4p exchange protein, Sec2p, from an interaction with the upstream Rab, Ypt31/32, to an interaction with a downstream Sec4p effector, Sec15p. Spatial regulation of PI4P levels thereby plays an important role in vesicle maturation.     

The role of S‐acylation in protein trafficking

Protein S‐acylation, also known as palmitoylation, consists of the addition of a lipid molecule to one or more cysteine residues through a thioester bond. This modification, which is widespread in eukaryotes, is thought to affect over 12% of the human proteome. S‐acylation allows the reversible association of peripheral proteins with membranes or, in the case of integral membrane proteins, modulates their behavior within the plane of the membrane. This review focuses on the consequences of protein S‐acylation on intracellular trafficking and membrane association. We summarize relevant information that illustrates how lipid modification of proteins plays an important role in dictating precise intracellular movements within cells by regulating membrane‐cytosol exchange, through membrane microdomain segregation, or by modifying the flux of the proteins by means of vesicular or diffusional transport systems. Finally, we highlight some of the key open questions and major challenges in the field.      

Yeast oxysterol-binding proteins: sterol transporters or regulators of cell polarization?

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) are a conserved family of soluble cytoplasmic proteins that can bind sterols, translocate between membrane compartments, and affect sterol trafficking. These properties make ORPs attractive candidates for lipid transfer proteins (LTPs) that directly mediate nonvesicular sterol transfer to the plasma membrane. To test whether yeast ORPs (the Osh proteins) are sterol LTPs, we studied endoplasmic reticulum (ER)-to-plasma membrane (PM) sterol transport in OSH deletion mutants lacking one, several, or all Osh proteins. In conditional OSH mutants, ER-PM ergosterol transport slowed ~20-fold compared with cells expressing a full complement of Osh proteins. Although this initial finding suggested that Osh proteins act as sterol LTPs, the situation is far more complex. Osh proteins have established roles in Rho small GTPase signaling. Osh proteins reinforce cell polarization and they specifically affect the localization of proteins involved in polarized cell growth such as septins, and the GTPases Cdc42p, Rho1p, and Sec4p. In addition, Osh proteins are required for a specific pathway of polarized secretion to sites of membrane growth, suggesting that this is how Osh proteins affect Cdc42p- and Rho1p-dependent polarization. Our findings suggest that Osh proteins integrate sterol trafficking and sterol-dependent cell signaling with the control of cell polarization.     

A detour for yeast oxysterol binding proteins

Oxysterol binding protein-related proteins, including the yeast proteins encoded by the OSH gene family (OSH1-OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh proteins are sterol transfer proteins and present new models consistent with known Osh protein functions. These models focus on the role of Osh proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer proteins, we propose that Osh proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites.     

Detection of intracellular phosphatidylserine in living cells

To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.     

Regulation of hepatic HMG-CoA reductase in vivo by reversible phosphorylation

The transbilayer distribution of aminophospholipids in trout intestinal brush-border membrane has been investigated using trinitrobenzene sulfonic acid (TNBS). In the middle intestine, phosphatidylethanolamine (PE) is symmetrically distributed between the two leaflets while 68% of the phosphatidylserine (PS) are located in the inner membrane leaflet. In the posterior intestine, 64% of the PE and 69% of the PS are located in the inner membrane leaflet. When asymmetrically distributed, the inner species of PE and PS have a higher content of 22:6(n-3) than the outer ones. This asymmetric distribution of docosahexaenoic acid in trout intestinal brush-border membrane might be related to the rod-like shape of the microvillus membrane and to its metabolism to hydroxylated derivatives.     

Investigation of the phosphatidylserine binding properties of the lipid biosensor,Lactadherin C2 (LactC2), in different membrane environments

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner.