Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides. cholera toxin | endocytosis

P4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2−/− mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.

Two new electron-paramagnetic-resonance (EPR) spectra, tentatively labeled NIRIM-1 and NIRIM-2, have been studied using synthetic diamond crystals grown from the Ni solvent to which various amounts of nitrogen getters (Ti, Zr) and/or boron were added. The NIRIM-1 spectrum (g =2.0112) having the effective spin S =~, which has been determined from the microwave pulsewidth dependence of the two-pulse echo intensity, is assigned to be isolated interstitial Ni+ with electronic configuration 3d'. The anisotropic spectra of powderlike line shape at 4 K might be ascribed to an intermediate Jahn-Teller effect coupled predominantly with trigonal distortions. The NIRIM-2 spectrum having an axially symmetric g tensor (S= -, ', g~~= 2.3285, g, =0) arises from a 3d' ion in a crystal field of trigonally distorted octahedron. It is proposed that the center is interstitial Ni+ associated with a vacancy or with local charge compensation.

α-Tocopherol (vitamin E) transfer protein (α-TTP) regulates the secretion of α-tocopherol from liver cells. Missense mutations of some arginine residues at the surface of α-TTP cause severe vitamin E deficiency in humans, but the role of these residues is unclear. Here, we found that wild-type α-TTP bound phosphatidylinositol phosphates (PIPs), whereas the arginine mutants did not. In addition, PIPs in the target membrane promoted the intermembrane transfer of α-tocopherol by α-TTP. The crystal structure of the α-TTP-PIPs complex revealed that the disease-related arginine residues interacted with phosphate groups of the PIPs and that the PIPs binding caused the lid of the α-tocopherol-binding pocket to open. Thus, PIPs have a role in promoting the release of a ligand from a lipid-transfer protein.

Phosphatidylinositol (PI) is a relatively minor component of membrane phospholipids but plays important roles in signal transduction through distinct phosphorylated derivatives of the inositol head group ( 1, 2 ). Threefourths or more of membrane PI obtained from mammalian tissues consist of the 1-stearoyl-2-arachidonoyl (18:0/20:4) species ( 3, 4 ), which is thought to be formed by a fatty acid remodeling reaction after the de novo synthesis of PI ( 5-10 ). The remodeling reaction involves the hydrolysis of a fatty acyl ester bond at the sn -1 or sn -2 position of the newly synthesized PI and subsequent incorporation of the appropriate fatty acid into the position. In an RNA interference (RNAi)-based genetic screen using Caenorhabditis elegans , we identifi ed mboa-7/ LPIAT1 as an acyltransferase that selectively incorporates arachidonic acid into the sn-2 position of PI ( 11 ). More recently, we demonstrated that C. elegans acl-8 , acl-9 , and acl-10 , which show signifi cant sequence homology to each other, encode acyltransferases that incorporate stearic acid (18:0) into the sn-1 position of PI ( 12 ). Stearic acid attached at the sn -1 position of PI Abstract Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identifi ed three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10 ) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identifi ed as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-defi cient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCATdefi cient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn -1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals. Abbreviations: AGPAT, 1-acylglycerol-3-phosphate O -acyltransferase; A-P axis, anterior-posterio axis; CL, cardiolipin; ER, endoplasmic reticulum; LPCAT, lysophosphatidylcholine acyltransferase; LPEAT, lysophosphatidylethanolamine acyltransferase; LPGAT, lysophosphatidylglycerol acyltransferase; LPIAT, lysophosphatidylinositol acyltransferase; LPSAT, lysophosphatidylserine acyltransferase; LYCAT, lysocardiolipin acyltransferase; MEF, mouse embryonic fi broblast; PC, phosphatidylcholi...

Photoemission studies of (La~-"Sr")2Cu04and YBa2Cu3067 have revealed that their electronic structure is essentially that of a Mott insulator, i.e. , Cu 3d electrons are localized, and the density of states at the Fermi level is very low. This result combined with the magnetic properties suggest a fluctuation of the local moment due to intersite exchange, which may be consistent with a bipolaron state or a resonating valence-bond state.Recently discovered high-T, superconductivity in the La-Ba-Cu-0 system' and related Cu oxides has stimulated extensive research in these materials. So far, most theoretical studies have been based on the twodimensional character of the electronic energy bands giving a high density of states (DOS) near the Fermi level (EF).Density-functional band-structure calculations have yielded a strongly hybridized Cu3d-02p antibonding band at EF, which leads to a strong electron-phonon coupling for a breathing-type displacement of 0 atoms.On the other hand, Anderson has proposed the resonating-valence-bond or quantum-spin-liquid state, which may occur in frustrated Heisenberg spin systems, and also discussed their unusual magnetic and transport properties in the normal state. ' In the light of the above two extreme points of view, the knowledge of the electronic structure is a key step toward understanding not only the superconducting but also the normal-state properties. This Rapid Communication reports a photoemission study on (La~-"Sr") 2Cu04-s and YBa2CusOs 7. The results demonstrate that the Cu 3d electrons are essentially localized due to strong electron correlation at the Cu sites. We have studied sintered samples of (La~"Sr")2Cu04-~and YBa2Cu30s7 which were prepared as described in Refs. 2 and 4 and whose properties are listed in Table I. Room-temperature x-ray and ultraviolet photoemission (XPS and UPS) experiments were performed using a spectrometer fitted with a Mg Ka x-ray source (hv=1253. 6 eV) and a helium resonance lamp (h v =21.2 and 40.8 eV). The energy resolution was -0. 1 eV for UPS and -0.8 eV for XPS. The base pressure in the spectrometer was -1 x 10 ' Torr. The spectra have been corrected for the Mg Ea3 4 satellite and the analyzer transmission efficiency. Clean surfaces were obtained by in situ scraping with a diamond file. Scraping was repeated frequently so that no change in the spectra was observed during the measurements. Figure 1 shows XPS spectra in the Cu 2p core-level region, where one can see that each of the j = 2 and components exhibits a two-peak structure as in previous reports. " The higher and lower binding-energy (satellite and main) peaks correspond, respectively, to 2p3d9 and 2p3d' final-state configurations (2p denotes a Cu 2p core hole), so that the separation between the two peaks is approximately the intra-atomic Coulomb energy between the core hole and the 3d electron U,d. The satellite structure arises not only from the coexistence of the d (Cu +) and d ' (Cu '+ ) configurations in the ground state but also from a ligand-to-d charge transfer in the...

Electron-paramagnetic-resonance and electron-nuclear-double-resonance (ENDOR) methods are used to identify the negatively charged state of the isolated vacancy in electron-irradiated synthetic diamond crystals. The Td symmetry is confirmed by determining the arrangement of both nearest neighbors and next-nearest neighbors. ENDOR measurements reveal that the effective spin is -', .

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