Yasunori Uchida

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes

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.

Impaired α-TTP-PIPs Interaction Underlies Familial Vitamin E Deficiency

Vitamin E Out Familial vitamin E deficiency is caused by mutations in the α-tocopherol transfer protein (α-TTP) gene. Kono et al. (p. 1106, published online 18 April; see the Perspective by Mesmin and Antonny) studied natural mutations in α-TTP. α-TTP bound phosphatidylinositol polyphosphates (PIPs), especially PI(4,5)P2, and a disease-related missense mutation abolished PIP binding but not α-tocopherol binding. The x-ray crystal structure of the α-TTP–PIP complex suggested that PIP binding opens the lid of the α-tocopherol–binding pocket to facilitate the release of α-tocopherol. Thus, PIP binding to α-TTP at the target membrane may facilitate the release of α-tocopherol in the hydrophobic pocket of α-TTP to the lipid bilayer of the target membrane, providing a mechanism for the transfer of lipids from the lipid-transfer protein to the target membrane. Phosphatidylinositol phosphates may play a role in lipid-transfer protein–mediated vitamin E efflux from hepatocytes. [Also see Perspective by Mesmin and Antonny] 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.

Transport through recycling endosomes requires EHD1 recruitment by a phosphatidylserine translocase

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.

Subcellular localization of sphingomyelin revealed by two toxin-based probes in mammalian cells

Sphingomyelin (SM) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS‐1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin‐II (EqtII). Both toxins stained SM in the plasma membrane (PM), and the stains were abolished by sphingomyelin synthase 2 (SMS2) knockdown or sphingomyelinase (SMase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by EqtII, and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes (LEs) among intracellular organelles, whereas EqtII stained recycling endosomes (REs) in addition to LEs. The intracellular SM stains by EqtII were abolished by sphingomyelin synthase 1 (SMS1) knockdown, but not by SMS2 knockdown. These results indicate that lysenin and EqtII label different SM pools and that SMS2 and SMS1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS‐1 cells. The use of the two SM‐binding probes may provide more insights into various sphingomyelin‐mediated processes in different topological domains.

LYCAT, a homologue of C. elegans acl-8, acl-9 and acl-10, determines the fatty acid composition of phosphatidylinositol in mice

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 identified 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 identified 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-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient 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.

Impaired retrograde membrane traffic through endosomes in a mutant CHO cell defective in phosphatidylserine synthesis

Phosphatidylserine (PS), a relatively minor constituent in the plasma membrane (PM), participates in various cellular processes such as clearance of apoptotic cells and recruitment of signaling molecules. PS also localizes in the membranes of endocytic organelles, such as recycling endosomes (REs). We recently showed that in REs, PS binds to the pleckstrin homology (PH) domain of evectin‐2, thereby regulating retrograde traffic from REs to the Golgi. However, direct evidence that PS has a role in retrograde traffic is lacking. Here, we examined the contribution of PS to endosomal membrane traffic by exploiting a mutant CHO cell line (PSA‐3) that is defective in PS synthesis. In PSA‐3 cells, the Golgi localization of TGN38, a protein that circulates between the Golgi and the PM through endosomes by retrograde traffic, was abolished, whereas the localizations of other organelle markers remained unchanged. Increasing the cellular PS level by adding ethanolamine to the culture medium restored the Golgi localization of TGN38. Tracking the endocytic fate of cell surface TGN38 that was labeled by anti‐TGN38 antibody showed that retrograde transport of TGN38 was impaired at endosomes, not at the PM. These findings provide direct evidence that intracellular PS is required for retrograde traffic through endosomes.

Oligo-astheno-teratozoospermia in mice lacking ORP4, a sterol-binding protein in the OSBP-related protein family.

Oligo‐astheno‐teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol‐binding protein in the oxysterol‐binding protein (OSBP)‐related protein family, causes male infertility due to severe OAT in mice. In ORP4‐deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round‐shaped head, curled back head or symplast. Spermatozoa derived from ORP4‐deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4‐deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

Oxysterol-binding protein (OSBP) is required for the perinuclear localization of intra-Golgi v-SNAREs

OSBP regulates the Golgi cholesterol level. This study demonstrates that OSBP and cholesterol are essential for localization of Golgi v-SNAREs. Knockdown of ArfGAP1 restores v-SNARE localization in OSBP-depleted cells, suggesting that OSBP-regulated cholesterol ensures proper COP-I vesicle transport.

SMAP2 Regulates Retrograde Transport from Recycling Endosomes to the Golgi

Retrograde transport is where proteins and lipids are transported back from the plasma membrane (PM) and endosomes to the Golgi, and crucial for a diverse range of cellular functions. Recycling endosomes (REs) serve as a sorting station for the retrograde transport and we recently identified evection-2, an RE protein with a pleckstrin homology (PH) domain, as an essential factor of this pathway. How evection-2 regulates retrograde transport from REs to the Golgi is not well understood. Here, we report that evection-2 binds to SMAP2, an Arf GTPase-activating protein. Endogenous SMAP2 localized mostly in REs and to a lesser extent, the trans-Golgi network (TGN). SMAP2 binds evection-2, and the RE localization of SMAP2 was abolished in cells depleted of evection-2. Knockdown of SMAP2, like that of evection-2, impaired the retrograde transport of cholera toxin B subunit (CTxB) from REs. These findings suggest that evection-2 recruits SMAP2 to REs, thereby regulating the retrograde transport of CTxB from REs to the Golgi.

Structural basis of the strict phospholipid binding specificity of the pleckstrin homology domain of human evectin-2

Evectin-2 is a recycling endosomal protein involved in retrograde transport. Its primary sequence contains an N-terminal pleckstrin homology (PH) domain and a C-terminal hydrophobic region. The PH domain of evectin-2 can specifically bind phosphatidylserine, which is enriched in recycling endosomes, and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The structure of human evectin-2 PH domain in complex with O-phospho-L-serine has recently been reported and demonstrates how the head group of phosphatidylserine is recognized. However, it was not possible to elucidate from the structure why evectin-2 cannot bind phosphatidic acid or phosphatidylethanolamine, which share a common moiety with phosphatidylserine. Here, the crystal structure at 1.75 Å resolution of an apo form of human evectin-2 PH domain, in which the ligand-binding site is free from crystal packing and is thus appropriate for comparison with the structure of the complex, is reported. Comparison between the structures of the apo form and the O-phospho-L-serine complex revealed ligand-induced conformational change evoked by interaction between the carboxyl moiety of the head group of phosphatidylserine and the main-chain N atom of Thr14. This structural change effectively explains the strict ligand specificity of the PH domain of human evectin-2.