Studies on novel clathrin adaptor-related proteins

Thesis (Ph. D. in Sciene)--University of Tsukuba, (A), no.2513, 2001.3.23Includes bibliographical reference

Mechanisms of Membrane Curvature Generation in Membrane Traffic

During the vesicular trafficking process, cellular membranes undergo dynamic morphological changes, in particular at the vesicle generation and fusion steps. Changes in membrane shape are regulated by small GTPases, coat proteins and other accessory proteins, such as BAR domain-containing proteins. In addition, membrane deformation entails changes in the lipid composition as well as asymmetric distribution of lipids over the two leaflets of the membrane bilayer. Given that P4-ATPases, which catalyze unidirectional flipping of lipid molecules from the exoplasmic to the cytoplasmic leaflets of the bilayer, are crucial for the trafficking of proteins in the secretory and endocytic pathways, changes in the lipid composition are involved in the vesicular trafficking process. Membrane remodeling is under complex regulation that involves the composition and distribution of lipids as well as assembly of proteins

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

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

Phospholipid‐flipping activity of P4‐ATPase drives membrane curvature

リン脂質の移動によって細胞膜が変形するメカニズムを解明 --ウイルス・細菌の細胞への侵入を制御の可能性--. 京都大学プレスリリース. 2018-03-30.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

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

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