Takuma Kishimoto

Dissecting BAR domain function in the yeast Amphiphysins Rvs161 and Rvs167 during endocytosis.

Using a structure–function analysis, we find that Rvs proteins are initially recruited to sites of endocytosis through their curvature-sensing and membrane-binding ability in a manner dependent on complex sphingolipids.

Determinants of endocytic membrane geometry, stability, and scission.

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

Protein Kinases Fpk1p and Fpk2p are Novel Regulators of Phospholipid Asymmetry

Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50Delta mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3Delta/dnf1Delta dnf2Delta mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1Delta fpk2Delta mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.

Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM‐binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze‐fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution‐sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase‐deficient Niemann‐Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.—Makino, A., Abe, M., Murate, M., Inaba, T., Yilmaz, N., Hullin‐Matsuda, F., Kishimoto, T., Schieber, N. L., Taguchi, T., Arai, H., Anderluh, G., Parton, R. G., Kobayashi, T. Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB J. 29, 477‐493 (2015). www.fasebj.org

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.

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Background: Little is known about how mammalian cells modulate retrograde sterol transport for cellular cholesterol homeostasis. Results: ABCA1 deficiency impairs endocytic retrograde sterol transport to the endoplasmic reticulum and activates the SREBP-2 pathway. Conclusion: ABCA1 participates in bidirectional sterol movement at the plasma membrane to regulate cellular cholesterol homeostasis. Significance: A novel function of ABCA1 is identified. Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom‐derived proteins of the aegerolysin family—pleurotolysin A2 (PlyA2; KD = 12nM), ostreolysin (Oly; KD = 1.3 nM), and erylysin A (EryA; KD = 1.3 nM)—strongly associated with CPE/cholesterol (Chol)‐containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid‐phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross‐reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (KD = 41 μM), rendering it a suitable tool for visualizing CPE in lipid‐blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.—Bhat, H. B., Ishitsuka, R., Inaba, T., Murate, M., Abe, M., Makino, A., Kohyama‐Koganeya, A., Nagao, K., Kurahashi, A., Kishimoto, T., Tahara, M., Yamano, A., Nagamune, K., Hirabayashi, Y., Juni, N., Umeda, M., Fujimori, F., Nishibori, K., Yamaji‐Hasegawa, A., Greimel, P., Kobayashi, T. Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates. FASEB J. 29, 3920‐3934 (2015). www.fasebj.org

Detectors for evaluating the cellular landscape of sphingomyelin- and cholesterol-rich membrane domains.

Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Targeting Cholesterol in a Liquid-Disordered Environment by Theonellamides Modulates Cell Membrane Order and Cell Shape

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3β-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.