A facile method for monitoring sphingomyelin synthase activity in HeLa cells using liquid chromatography/mass spectrometry.

Sphingomyelin synthase (SMS) is a sphingolipid-metabolizing enzyme involved in the de novo synthesis of sphingomyelin (SM) from ceramide (Cer). Recent studies have indicated that SMS is a key therapeutic target for metabolic diseases such as fatty liver, type 2 diabetes, atherosclerosis, and colorectal cancer. However, very few SMS inhibitors have been identified because of the limited sensitivity and selectivity of the current fluorescence-based screening assay. In this study, we developed a simple cell-based assay coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to screen for SMS inhibitors. HeLa cells stably expressing SMS1 or SMS2 were used for the screening. A non-fluorescent unnatural C6-Cer was used as a substrate for SMS to produce C6-SM. C6-Cer and C6-SM levels in the cells were monitored and quantified using LC-MS/MS. The activity of ginkgolic acid C15:1 (GA), a known SMS inhibitor, was measured. GA had half-maximal inhibitory concentrations of 5.5 µM and 3.6 µM for SMS1 and SMS2, respectively. To validate these findings, hSMS1 and hSMS2 proteins were optimized for molecular docking studies. In silico analyses were conducted to assess the interaction of GA with SMS1 and SMS2, and its binding affinity. This study offers an analytical approach for screening novel SMS inhibitors and provides in silico support for the experimental findings.

Glucosylceramide flippases contribute to cellular glucosylceramide homeostasis.

Lipid transport is an essential cellular process with importance to human health, disease development, and therapeutic strategies. Type IV P-type ATPases (P4-ATPases) have been identified as membrane lipid flippases by utilizing nitrobenzoxadiazole (NBD)-labeled lipids as substrates. Among the 14 human type IV P-type ATPases, ATP10D was shown to flip NBD-glucosylceramide (GlcCer) across the plasma membrane. Here, we found that conversion of incorporated GlcCer (d18:1/12:0) to other sphingolipids is accelerated in cells exogenously expressing ATP10D but not its ATPase-deficient mutant. These findings suggest that 1) ATP10D flips unmodified GlcCer as well as NBD-GlcCer at the plasma membrane and 2) ATP10D can translocate extracellular GlcCer, which is subsequently converted to other metabolites. Notably, exogenous expression of ATP10D led to the reduction in cellular hexosylceramide levels. Moreover, the expression of GlcCer flippases, including ATP10D, also reduced cellular hexosylceramide levels in fibroblasts derived from patients with Gaucher disease, which is a lysosomal storage disorder with excess GlcCer accumulation. Our study highlights the contribution of ATP10D to the regulation of cellular GlcCer levels and maintaining lipid homeostasis.

Lysosomal membrane integrity in fibroblasts derived from patients with Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.

Phosphatidylserine exposure modulates adhesion GPCR BAI1 (ADGRB1) signaling activity.

Brain-specific angiogenesis inhibitor 1 (BAI1; also called ADGRB1 or B1) is an adhesion G protein-coupled receptor known from studies on macrophages to bind to phosphatidylserine (PS) on apoptotic cells via its N-terminal thrombospondin repeats. A separate body of work has shown that B1 regulates postsynaptic function and dendritic spine morphology via signaling pathways involving Rac and Rho. However, it is unknown if PS binding by B1 has any effect on the receptor's signaling activity. To shed light on this subject, we studied G protein-dependent signaling by B1 in the absence and presence of coexpression with the PS flippase ATP11A in human embryonic kidney 293T cells. ATP11A expression reduced the amount of PS exposed extracellularly and also strikingly reduced the signaling activity of coexpressed full-length B1 but not a truncated version of the receptor lacking the thrombospondin repeats. Further experiments with an inactive mutant of ATP11A showed that the PS flippase function of ATP11A was required for modulation of B1 signaling. In coimmunoprecipitation experiments, we made the surprising finding that ATP11A not only modulates B1 signaling but also forms complexes with B1. Parallel studies in which PS in the outer leaflet was reduced by an independent method, deletion of the gene encoding the endogenous lipid scramblase anoctamin 6 (ANO6), revealed that this manipulation also markedly reduced B1 signaling. These findings demonstrate that B1 signaling is modulated by PS exposure and suggest a model in which B1 serves as a PS sensor at synapses and in other cellular contexts.

Regulatory Roles of N- and C-Terminal Cytoplasmic Regions of P4-ATPases.

P4-ATPases, which are subfamily members of P-type ATPase superfamily, translocate membrane lipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet, thus regulating trans-bilayer lipid asymmetry. Mammalian P4-ATPases localize to the specific subcellular organelles or the plasma membrane where they translocate the specific lipids. Although recent advances in the structural analysis of P4-ATPases have improved our understanding of lipid transporting machinery, the mechanism of substrate specificity and the regulatory mechanism of the enzymes remain largely unknown. Recent studies have uncovered several specific localization and regulatory mechanisms of P4-ATPases. Here, we review the current understanding of the regulatory mechanism of P4-ATPase activity and localization in mammalian cells.

The interaction of ATP11C-b with ezrin contributes to its polarized localization.

ATP11C, a member of the P4-ATPase family, translocates phosphatidylserine and phosphatidylethanolamine at the plasma membrane. We previously revealed that its C-terminal splice variant ATP11C-b exhibits polarized localization in motile cell lines, such as MDA-MB-231 and Ba/F3. In the present study, we found that the C-terminal cytoplasmic region of ATP11C-b interacts specifically with ezrin. Notably, the LLxY motif in the ATP11C-b C-terminal region is crucial for its interaction with ezrin as well as its polarized localization on the plasma membrane. A constitutively active, C-terminal phosphomimetic mutant of ezrin was colocalized with ATP11C-b in polarized motile cells. ATP11C-b was partially mislocalized in cells depleted of ezrin alone, and exhibited greater mislocalization in cells simultaneously depleted of the family members ezrin, radixin and moesin (ERM), suggesting that ERM proteins, particularly ezrin, contribute to the polarized localization of ATP11C-b. Furthermore, Atp11c knockout resulted in C-terminally phosphorylated ERM protein mislocalization, which was restored by exogenous expression of ATP11C-b but not ATP11C-a. These observations together indicate that the polarized localizations of ATP11C-b and the active form of ezrin to the plasma membrane are interdependently stabilized.

The N- or C-terminal cytoplasmic regions of P4-ATPases determine their cellular localization.

Mammalian P4-ATPases specifically localize to the plasma membrane and the membranes of intracellular compartments. P4-ATPases contain 10 transmembrane domains, and their N- and C-terminal (NT and CT) regions face the cytoplasm. Among the ATP10 and ATP11 proteins of P4-ATPases, ATP10A, ATP10D, ATP11A, and ATP11C localize to the plasma membrane, while ATP10B and ATP11B localize to late endosomes and early/recycling endosomes, respectively. We previously showed that the NT region of ATP9B is critical for its localization to the Golgi apparatus, while the CT regions of ATP11C isoforms are critical for Ca2+-dependent endocytosis or polarized localization at the plasma membrane. Here, we conducted a comprehensive analysis of chimeric proteins and found that the NT region of ATP10 proteins and the CT region of ATP11 proteins are responsible for their specific subcellular localization. Importantly, the ATP10B NT and the ATP11B CT regions were found to harbor a trafficking and/or targeting signal that allows these P4-ATPases to localize to late endosomes and early/recycling endosomes, respectively. Moreover, dileucine residues in the NT region of ATP10B were required for its trafficking to endosomal compartments. These results suggest that the NT and CT sequences of P4-ATPases play a key role in their intracellular trafficking.

Phosphatidylserine exposure in living cells.

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.

ATPase reaction cycle of P4-ATPases affects their transport from the endoplasmic reticulum.

P4-ATPases belonging to the P-type ATPase superfamily mediate active transport of phospholipids across cellular membranes. Most P4-ATPases, except ATP9A and ATP9B proteins, form heteromeric complexes with CDC50 proteins, which are required for transport of P4-ATPases from the endoplasmic reticulum (ER) to their final destinations. P-type ATPases form autophosphorylated intermediates during the ATPase reaction cycle. However, the association of the catalytic cycle of P4-ATPases with their transport from the ER and their cellular localization has not been studied. Here, we show that transport of ATP9 and ATP11 proteins as well as that of ATP10A from the ER depends on the ATPase catalytic cycle, suggesting that conformational changes in P4-ATPases during the catalytic cycle are crucial for their transport from the ER.

The cytoplasmic C-terminal region of the ATP11C variant determines its localization at the polarized plasma membrane.

ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs.

Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.

Substrates of P4-ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine).

P4-ATPases, a subfamily of P-type ATPases, were initially identified as aminophospholipid translocases in eukaryotic membranes. These proteins generate and maintain membrane lipid asymmetry by translocating aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the exoplasmic/lumenal leaflet to the cytoplasmic leaflet. The human genome encodes 14 P4-ATPases, and the cellular localizations, substrate specificities, and cellular roles of these proteins were recently revealed. Numerous P4-ATPases, including ATP8A1, ATP8A2, ATP11A, ATP11B, and ATP11C, transport phosphatidylserine. By contrast, ATP8B1, ATP8B2, and ATP10A transport phosphatidylcholine but not aminophospholipids, although there is a discrepancy regarding the substrate of ATP8B1 in the literature. Some yeast and plant P4-ATPases can also translocate phosphatidylcholine. At least 2 P4-ATPases (ATP8A2 and ATP8B1) are associated with severe human diseases, and other P4-ATPases are implicated in various pathophysiologic conditions in mouse models. Here, we discuss the cellular functions of phosphatidylcholine flippases and suggest a model for the phenotype of progressive familial intrahepatic cholestasis 1 caused by a defect in ATP8B1.-Shin, H.-W., Takatsu, H. Substrates of P4-ATPases: beyond aminophospholipids (phosphatidylserine and phosphatidylethanolamine).

Phospholipid-flipping activity of P4-ATPase drives membrane curvature.

P4-ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM). Enhanced phosphatidylcholine-flippase activity at the PM due to expression of ATP10A, a member of the P4-ATPase family, promoted membrane tubulation upon recruitment of BAR domains to the PM This is the important evidence that changes in the transbilayer lipid composition induced by P4-ATPases can deform biological membranes.

Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation.

We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-flippase activity is regulated in non-apoptotic cells. Here we report that the PS-flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.

Uptake of glucose-conjugated MGMT inhibitors in cancer cells: role of flippases and type IV P-type ATPases.

The DNA repair protein O 6-methylguanine-DNA-methyltransferase (MGMT) is a key determinant of cancer resistance. The MGMT inhibitors O 6-benzylguanine (O6BG) and O 6-(4-bromothenyl)guanine (O6BTG) failed to enhance the therapeutic response due to toxic side effects when applied in combination with alkylating chemotherapeutics, indicating a need of inhibitor targeting. We assessed MGMT targeting that relies on conjugating the inhibitors O6BG and O6BTG to ß-D-glucose, resulting in O6BG-Glu and O6BTG-Glu, respectively. This targeting strategy was selected by taking advantage of high demand of glucose in cancers. Contrary to our expectation, the uptake of O6BG-Glu and O6BTG-Glu was not dependent on glucose transporters. Instead, it seems that after membrane binding the conjugates are taken up via flippases, which normally transport phospholipids. This membrane binding is the consequence of the amphiphilic character of the conjugates, which at higher concentrations lead to the formation of micelle-like particles in aqueous solution. The unusual uptake mechanism of the conjugates highlights the importance of proper linker selection for a successful ligand-based drug delivery strategy. We also demonstrate that proteins of the P4-Type ATPase family are involved in the transport of the glucose conjugates. The findings are not only important for MGMT inhibitor targeting, but also for other amphiphilic drugs.

The phospholipid flippase ATP9A is required for the recycling pathway from the endosomes to the plasma membrane.

Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that translocate phospholipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of lipid bilayers. In Saccharomyces cerevisiae, P4-ATPases are localized to specific subcellular compartments and play roles in compartment-mediated membrane trafficking; however, roles of mammalian P4-ATPases in membrane trafficking are poorly understood. We previously reported that ATP9A, one of 14 human P4-ATPases, is localized to endosomal compartments and the Golgi complex. In this study, we found that ATP9A is localized to phosphatidylserine (PS)-positive early and recycling endosomes, but not late endosomes, in HeLa cells. Depletion of ATP9A delayed the recycling of transferrin from endosomes to the plasma membrane, although it did not affect the morphology of endosomal structures. Moreover, depletion of ATP9A caused accumulation of glucose transporter 1 in endosomes, probably by inhibiting their recycling. By contrast, depletion of ATP9A affected neither the early/late endosomal transport and degradation of epidermal growth factor (EGF) nor the transport of Shiga toxin B fragment from early/recycling endosomes to the Golgi complex. Therefore ATP9A plays a crucial role in recycling from endosomes to the plasma membrane.

Alteration of transbilayer phospholipid compositions is involved in cell adhesion, cell spreading, and focal adhesion formation.

We previously showed that P4-ATPases, ATP10A/ATP8B1, and ATP11A/ATP11C have flippase activities toward phosphatidylcholine (PC), and aminophospholipids [phosphatidylserine (PS) and phosphatidylethanolamine], respectively. Here, we investigate the effect of PC-specific flippases versus aminophospholipid-specific flippases in cell spreading on the extracellular matrix. Expression of PC-flippases, but not PS-flippases, delayed cell adhesion, cell spreading and inhibited formation of focal adhesions. In addition, overexpression of a PS-binding probe that sequesters PS in the cytoplasmic leaflet delayed cell spreading and inhibited formation of focal adhesions. These results suggest that elevation of PC at the cytoplasmic leaflet of the plasma membrane by expression of PC-flippases may reduce the local concentration of PS or phosphoinositides, required for efficient cell adhesion, focal adhesion formation, and cell spreading.

ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells.

Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells.

Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics.

We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543-33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology.