Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila.

In mammals, lipids are selectively transported to specific sites using multiple classes of lipoproteins. However, in Drosophila, a single class of lipoproteins, lipophorin, carries more than 95% of the lipids in the hemolymph. Although a unique ability of the insect lipoprotein system for cargo transport has been demonstrated, it remains unclear how this single class of lipoproteins selectively transports lipids. In this study, we carried out a comparative analysis of the fatty-acid composition among lipophorin, the CNS, and CNS-derived cell lines and investigated the transport mechanism of fatty acids, particularly focusing on the transport of PUFAs in Drosophila We showed that PUFAs are selectively incorporated into the acyl chains of lipophorin phospholipids and effectively transported to CNS through lipophorin receptor-mediated endocytosis of lipophorin. In addition, we demonstrated that C14 fatty acids are selectively incorporated into the diacylglycerols (DAGs) of lipophorin and that C14 fatty-acid-containing DAGs are spontaneously transferred from lipophorin to the phospholipid bilayer. These results suggest that PUFA-containing phospholipids and C14 fatty-acid-containing DAGs in lipophorin could be transferred to different sites by different mechanisms to selectively transport fatty acids using a single class of lipoproteins.

Role for phospholipid flippase complex of ATP8A1 and CDC50A proteins in cell migration.

Type IV P-type ATPases (P4-ATPases) and CDC50 family proteins form a putative phospholipid flippase complex that mediates the translocation of aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to inner leaflets of the plasma membrane. In Chinese hamster ovary (CHO) cells, at least eight members of P4-ATPases were identified, but only a single CDC50 family protein, CDC50A, was expressed. We demonstrated that CDC50A associated with and recruited P4-ATPase ATP8A1 to the plasma membrane. Overexpression of CDC50A induced extensive cell spreading and greatly enhanced cell migration. Depletion of either CDC50A or ATP8A1 caused a severe defect in the formation of membrane ruffles, thereby inhibiting cell migration. Analyses of phospholipid translocation at the plasma membrane revealed that the depletion of CDC50A inhibited the inward translocation of both PS and PE, whereas the depletion of ATP8A1 inhibited the translocation of PE but not that of PS, suggesting that the inward translocation of cell-surface PE is involved in cell migration. This hypothesis was further examined by using a PE-binding peptide and a mutant cell line with defective PE synthesis; either cell-surface immobilization of PE by the PE-binding peptide or reduction in the cell-surface content of PE inhibited the formation of membrane ruffles, causing a severe defect in cell migration. These results indicate that the phospholipid flippase complex of ATP8A1 and CDC50A plays a major role in cell migration and suggest that the flippase-mediated translocation of PE at the plasma membrane is involved in the formation of membrane ruffles to promote cell migration.

ATP9B, a P4-ATPase (a putative aminophospholipid translocase), localizes to the trans-Golgi network in a CDC50 protein-independent manner.

Type IV P-type ATPases (P4-ATPases) are putative phospholipid flippases that translocate phospholipids from the exoplasmic (lumenal) to the cytoplasmic leaflet of lipid bilayers and are believed to function in complex with CDC50 proteins. In Saccharomyces cerevisiae, five P4-ATPases are localized to specific cellular compartments and are required for vesicle-mediated protein transport from these compartments, suggesting a role for phospholipid translocation in vesicular transport. The human genome encodes 14 P4-ATPases and three CDC50 proteins. However, the subcellular localization of human P4-ATPases and their interactions with CDC50 proteins are poorly understood. Here, we show that class 5 (ATP10A, ATP10B, and ATP10D) and class 6 (ATP11A, ATP11B, and ATP11C) P4-ATPases require CDC50 proteins, primarily CDC50A, for their exit from the endoplasmic reticulum (ER) and final subcellular localization. In contrast, class 2 P4-ATPases (ATP9A and ATP9B) are able to exit the ER in the absence of exogenous CDC50 expression: ATP9B, but not ATP11B, was able to exit the ER despite depletion of CDC50 proteins by RNAi. Although ATP9A and ATP9B show a high overall sequence similarity, ATP9A localizes to endosomes and the trans-Golgi network (TGN), whereas ATP9B localizes exclusively to the TGN. A chimeric ATP9 protein in which the N-terminal cytoplasmic region of ATP9A was replaced with the corresponding region of ATP9B was localized exclusively to the Golgi. These results indicate that ATP9B is able to exit the ER and localize to the TGN independently of CDC50 proteins and that this protein contains a Golgi localization signal in its N-terminal cytoplasmic region.

Transbilayer phospholipid flipping regulates Cdc42p signaling during polarized cell growth via Rga GTPase-activating proteins.

An important problem in polarized morphogenesis is how polarized transport of membrane vesicles is spatiotemporally regulated. Here, we report that a local change in the transbilayer phospholipid distribution of the plasma membrane regulates the axis of polarized growth. Type 4 P-type ATPases Lem3p-Dnf1p and -Dnf2p are putative heteromeric phospholipid flippases in budding yeast that are localized to polarized sites on the plasma membrane. The lem3Delta mutant exhibits prolonged apical growth due to a defect in the switch to isotropic bud growth. In lem3Delta cells, the small GTPase Cdc42p remains polarized at the bud tip where phosphatidylethanolamine remains exposed on the outer leaflet. Intriguingly, phosphatidylethanolamine and phosphatidylserine stimulate GTPase-activating protein (GAP) activity of Rga1p and Rga2p toward Cdc42p, whereas PI(4,5)P(2) inhibits it. We propose that a redistribution of phospholipids to the inner leaflet of the plasma membrane triggers the dispersal of Cdc42p from the apical growth site, through activation of GAPs.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Cdc50p, a protein required for polarized growth, associates with the Drs2p P-type ATPase implicated in phospholipid translocation in Saccharomyces cerevisiae.

Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2Delta mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50Delta mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50Delta mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2Delta cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

The role of NADRIN, a Rho GTPase-activating protein, in the morphological differentiation of astrocytes.

Reorganization of the actin cytoskeleton caused by inactivation of the Rho GTPase RhoA is critical for the morphological differentiation of astrocytes into process-bearing stellate cells. The molecular mechanisms underlying the RhoA inactivation and, in particular, the factors that inactivate RhoA, remain to be elucidated. We show here that the expression of a GTPase-activating protein (GAP) for Rho GTPases, neuron-associated developmentally regulated protein (NADRIN) also known as RICH and ARHGAP17, was significantly increased in stellate astrocytes and induced expression of NADRIN accelerated the morphological differentiation of cultured astrocytes into stellate cells. A GAP activity-negative mutant or truncated forms of NADRIN failed to induce the stellation. Immunoprecipitation analyses revealed that, in response to inductive signals such as dibutyryl cyclic AMP and epidermal growth factor, NADRIN formed a complex with ezrin-radixin-moesin (ERM) protein by interacting with ERM-binding phosphoprotein 50 via its carboxy-terminal PSD95/DlgA/ZO-1-binding motif. We also showed that NADRIN formed a dimer via the interaction between the amino- and carboxy-terminal domains, which was disrupted in response to the inductive signals. These results suggest that the inductive signals cause the structural change of NADRIN, which allows NADRIN to associate with the ERM protein complex, where it inactivates RhoA and leads to the morphological differentiation of astrocytes.

Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.

A novel membrane protein, Ros3p, is required for phospholipid translocation across the plasma membrane in Saccharomyces cerevisiae.

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.