Molecular basis of accessible plasma membrane cholesterol recognition by the GRAM domain of GRAMD1b.

Cholesterol is essential for cell physiology. Transport of the "accessible" pool of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER) by ER-localized GRAMD1 proteins (GRAMD1a/1b/1c) contributes to cholesterol homeostasis. However, how cells detect accessible cholesterol within the PM remains unclear. We show that the GRAM domain of GRAMD1b, a coincidence detector for anionic lipids, including phosphatidylserine (PS), and cholesterol, possesses distinct but synergistic sites for sensing accessible cholesterol and anionic lipids. We find that a mutation within the GRAM domain of GRAMD1b that is associated with intellectual disability in humans specifically impairs cholesterol sensing. In addition, we identified another point mutation within this domain that enhances cholesterol sensitivity without altering its PS sensitivity. Cell-free reconstitution and cell-based assays revealed that the ability of the GRAM domain to sense accessible cholesterol regulates membrane tethering and determines the rate of cholesterol transport by GRAMD1b. Thus, cells detect the codistribution of accessible cholesterol and anionic lipids in the PM and fine-tune the non-vesicular transport of PM cholesterol to the ER via GRAMD1s.

Phospholipid-flipping activity of P4-ATPase drives membrane curvature.

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 cytoplasmic C-terminal region of the ATP11C variant determines its localization at the polarized plasma membrane.

ATP11C, a member of the P4-ATPase family, is a major phosphatidylserine (PS)-flippase located at the plasma membrane. ATP11C deficiency causes a defect in B-cell maturation, anemia and hyperbilirubinemia. Although there are several alternatively spliced variants derived from the ATP11C gene, the functional differences between them have not been considered. Here, we compared and characterized three C-terminal spliced forms (we designated as ATP11C-a, ATP11C-b and ATP11C-c), with respect to their expression patterns in cell types and tissues, and their subcellular localizations. We had previously shown that the C-terminus of ATP11C-a is critical for endocytosis upon PKC activation. Here, we found that ATP11C-b and ATP11C-c did not undergo endocytosis upon PKC activation. Importantly, we also found that ATP11C-b localized to a limited region of the plasma membrane in polarized cells, whereas ATP11C-a was distributed on the entire plasma membrane in both polarized and non-polarized cells. Moreover, we successfully identified LLXY residues within the ATP11C-b C-terminus as a critical motif for the polarized localization. These results suggest that the ATP11C-b regulates PS distribution in distinct regions of the plasma membrane in polarized cells.

Yeast and human P4-ATPases transport glycosphingolipids using conserved structural motifs.

Lipid transport is an essential process with manifest importance to human health and disease. Phospholipid flippases (P4-ATPases) transport lipids across the membrane bilayer and are involved in signal transduction, cell division, and vesicular transport. Mutations in flippase genes cause or contribute to a host of diseases, such as cholestasis, neurological deficits, immunological dysfunction, and metabolic disorders. Genome-wide association studies have shown that ATP10A and ATP10D variants are associated with an increased risk of diabetes, obesity, myocardial infarction, and atherosclerosis. Moreover, ATP10D SNPs are associated with elevated levels of glucosylceramide (GlcCer) in plasma from diverse European populations. Although sphingolipids strongly contribute to metabolic disease, little is known about how GlcCer is transported across cell membranes. Here, we identify a conserved clade of P4-ATPases from Saccharomyces cerevisiae (Dnf1, Dnf2), Schizosaccharomyces pombe (Dnf2), and Homo sapiens (ATP10A, ATP10D) that transport GlcCer bearing an sn2 acyl-linked fluorescent tag. Further, we establish structural determinants necessary for recognition of this sphingolipid substrate. Using enzyme chimeras and site-directed mutagenesis, we observed that residues in transmembrane (TM) segments 1, 4, and 6 contribute to GlcCer selection, with a conserved glutamine in the center of TM4 playing an essential role. Our molecular observations help refine models for substrate translocation by P4-ATPases, clarify the relationship between these flippases and human disease, and have fundamental implications for membrane organization and sphingolipid homeostasis.

Phospholipid Flippase ATP10A Translocates Phosphatidylcholine and Is Involved in Plasma Membrane Dynamics.

We showed previously that ATP11A and ATP11C have flippase activity toward aminophospholipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE)) and ATP8B1 and that ATP8B2 have flippase activity toward phosphatidylcholine (PC) (Takatsu, H., Tanaka, G., Segawa, K., Suzuki, J., Nagata, S., Nakayama, K., and Shin, H. W. (2014) J. Biol. Chem. 289, 33543-33556). Here, we show that the localization of class 5 P4-ATPases to the plasma membrane (ATP10A and ATP10D) and late endosomes (ATP10B) requires an interaction with CDC50A. Moreover, exogenous expression of ATP10A, but not its ATPase-deficient mutant ATP10A(E203Q), dramatically increased PC flipping but not flipping of PS or PE. Depletion of CDC50A caused ATP10A to be retained at the endoplasmic reticulum instead of being delivered to the plasma membrane and abrogated the increased PC flipping activity observed by expression of ATP10A. These results demonstrate that ATP10A is delivered to the plasma membrane via its interaction with CDC50A and, specifically, flips PC at the plasma membrane. Importantly, expression of ATP10A, but not ATP10A(E203Q), dramatically altered the cell shape and decreased cell size. In addition, expression of ATP10A, but not ATP10A(E203Q), delayed cell adhesion and cell spreading onto the extracellular matrix. These results suggest that enhanced PC flipping activity due to exogenous ATP10A expression alters the lipid composition at the plasma membrane, which may in turn cause a delay in cell spreading and a change in cell morphology.

Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites.

Abnormal distribution of cellular cholesterol is associated with numerous diseases, including cardiovascular and neurodegenerative diseases. Regulated transport of cholesterol is critical for maintaining its proper distribution in the cell, yet the underlying mechanisms remain unclear. Here, we show that lipid transfer proteins, namely ORP9, OSBP, and GRAMD1s/Asters (GRAMD1a/GRAMD1b/GRAMD1c), control non-vesicular cholesterol transport at points of contact between the ER and the trans-Golgi network (TGN), thereby maintaining cellular cholesterol distribution. ORP9 localizes to the TGN via interaction between its tandem α-helices and ORP10/ORP11. ORP9 extracts PI4P from the TGN to prevent its overaccumulation and suppresses OSBP-mediated PI4P-driven cholesterol transport to the Golgi. By contrast, GRAMD1s transport excess cholesterol from the Golgi to the ER, thereby preventing its build-up. Cells lacking ORP9 exhibit accumulation of cholesterol at the Golgi, which is further enhanced by additional depletion of GRAMD1s with major accumulation in the plasma membrane. This is accompanied by chronic activation of the SREBP-2 signalling pathway. Our findings reveal the importance of regulated lipid transport at ER-Golgi contacts for maintaining cellular cholesterol distribution and homeostasis.

Visualization of accessible cholesterol using a GRAM domain-based biosensor.

Cholesterol is important for membrane integrity and cell signaling, and dysregulation of the distribution of cellular cholesterol is associated with numerous diseases, including neurodegenerative disorders. While regulated transport of a specific pool of cholesterol, known as "accessible cholesterol", contributes to the maintenance of cellular cholesterol distribution and homeostasis, tools to monitor accessible cholesterol in live cells remain limited. Here, we engineer a highly sensitive accessible cholesterol biosensor by taking advantage of the cholesterol-sensing element (the GRAM domain) of an evolutionarily conserved lipid transfer protein, GRAMD1b. Using this cholesterol biosensor, which we call GRAM-W, we successfully visualize in real time the distribution of accessible cholesterol in many different cell types, including human keratinocytes and iPSC-derived neurons, and show differential dependencies on cholesterol biosynthesis and uptake for maintaining levels of accessible cholesterol. Furthermore, we combine GRAM-W with a dimerization-dependent fluorescent protein (ddFP) and establish a strategy for the ultrasensitive detection of accessible plasma membrane cholesterol. These tools will allow us to obtain important insights into the molecular mechanisms by which the distribution of cellular cholesterol is regulated.

GRAMD1-mediated accessible cholesterol sensing and transport.

Cholesterol, an essential lipid for cell signaling and structural integrity of cellular membranes, is highly enriched in the plasma membrane (PM). However, the regulatory mechanisms that control its biosynthesis and uptake both reside in the endoplasmic reticulum (ER). Thus, the ER needs to constantly monitor the levels of PM cholesterol. This is in part mediated by regulated transport of a biochemically defined pool of cholesterol, termed "accessible" cholesterol, from the PM to the ER via evolutionarily conserved ER-anchored lipid transfer proteins, the GRAMD1s/Asters (GRAMD1a/1b/1c) (Lam/Ltc proteins in yeast). GRAMD1s possess cytosolically exposed GRAM domain and StART-like domain followed by a transmembrane ER anchor. They form homo- and hetero-meric complexes and move to the contacts formed between the ER and the PM by sensing a transient expansion of the accessible pool of cholesterol in the PM via the GRAM domain and facilitate its extraction and transport to the ER via the StART-like domain. The GRAMD1b GRAM domain possesses distinct, but synergistic sites, for recognizing accessible cholesterol and anionic lipids, including phosphatidylserine, within the PM. This property of the GRAM domain contributes to regulated tethering of the PM to ER membrane where GRAMD1s are anchored and fine-tunes StART-like domain-dependent accessible cholesterol transport. Thus, cells use GRAMD1s to sense the levels of cholesterol in the PM and regulate transport of accessible PM cholesterol to the ER in order to maintain cholesterol homeostasis.

INPP5K and Atlastin-1 maintain the nonuniform distribution of ER-plasma membrane contacts in neurons.

In neurons, the ER extends throughout all cellular processes, forming multiple contacts with the plasma membrane (PM) to fine-tune neuronal physiology. However, the mechanisms that regulate the distribution of neuronal ER-PM contacts are not known. Here, we used the Caenorhabditis elegans DA9 motor neuron as our model system and found that neuronal ER-PM contacts are enriched in soma and dendrite and mostly absent in axons. Using forward genetic screen, we identified that the inositol 5-phosphatase, CIL-1 (human INPP5K), and the dynamin-like GTPase, ATLN-1 (human Atlastin-1), help to maintain the non-uniform, somatodendritic enrichment of neuronal ER-PM contacts. Mechanistically, CIL-1 acts upstream of ATLN-1 to maintain the balance between ER tubules and sheets. In mutants of CIL-1 or ATLN-1, ER sheets expand and invade into the axon. This is accompanied by the ectopic formation of axonal ER-PM contacts and defects in axon regeneration following laser-induced axotomy. As INPP5K and Atlastin-1 have been linked to neurological disorders, the unique distribution of neuronal ER-PM contacts maintained by these proteins may support neuronal resilience during the onset and progression of these diseases.

PDZD-8 and TEX-2 regulate endosomal PI(4,5)P2 homeostasis via lipid transport to promote embryogenesis in C. elegans.

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis.

Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

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.

Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex.

Cholesterol is a major structural component of the plasma membrane (PM). The majority of PM cholesterol forms complexes with other PM lipids, making it inaccessible for intracellular transport. Transition of PM cholesterol between accessible and inaccessible pools maintains cellular homeostasis, but how cells monitor the accessibility of PM cholesterol remains unclear. We show that endoplasmic reticulum (ER)-anchored lipid transfer proteins, the GRAMD1s, sense and transport accessible PM cholesterol to the ER. GRAMD1s bind to one another and populate ER-PM contacts by sensing a transient expansion of the accessible pool of PM cholesterol via their GRAM domains. They then facilitate the transport of this cholesterol via their StART-like domains. Cells that lack all three GRAMD1s exhibit striking expansion of the accessible pool of PM cholesterol as a result of less efficient PM to ER transport of accessible cholesterol. Thus, GRAMD1s facilitate the movement of accessible PM cholesterol to the ER in order to counteract an acute increase of PM cholesterol, thereby activating non-vesicular cholesterol transport.

Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation.

We and others showed that ATP11A and ATP11C, members of the P4-ATPase family, translocate phosphatidylserine (PS) and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflets at the plasma membrane. PS exposure on the outer leaflet of the plasma membrane in activated platelets, erythrocytes, and apoptotic cells was proposed to require the inhibition of PS-flippases, as well as activation of scramblases. Although ATP11A and ATP11C are cleaved by caspases in apoptotic cells, it remains unclear how PS-flippase activity is regulated in non-apoptotic cells. Here we report that the PS-flippase ATP11C, but not ATP11A, is sequestered from the plasma membrane via clathrin-mediated endocytosis upon Ca2+-mediated PKC activation. Importantly, we show that a characteristic di-leucine motif (SVRPLL) in the C-terminal cytoplasmic region of ATP11C becomes functional upon PKC activation. Moreover endocytosis of ATP11C is induced by Ca2+-signaling via Gq-coupled receptors. Our data provide the first evidence for signal-dependent regulation of mammalian P4-ATPase.

ARF1 and ARF4 regulate recycling endosomal morphology and retrograde transport from endosomes to the Golgi apparatus.

Small GTPases of the ADP-ribosylation factor (ARF) family, except for ARF6, mainly localize to the Golgi apparatus, where they trigger formation of coated carrier vesicles. We recently showed that class I ARFs (ARF1 and ARF3) localize to recycling endosomes, as well as to the Golgi, and are redundantly required for recycling of endocytosed transferrin. On the other hand, the roles of class II ARFs (ARF4 and ARF5) are not yet fully understood, and the complementary or overlapping functions of class I and class II ARFs have been poorly characterized. In this study, we find that simultaneous depletion of ARF1 and ARF4 induces extensive tubulation of recycling endosomes. Moreover, the depletion of ARF1 and ARF4 inhibits retrograde transport of TGN38 and mannose-6-phosphate receptor from early/recycling endosomes to the trans-Golgi network (TGN) but does not affect the endocytic/recycling pathway of transferrin receptor or inhibit retrograde transport of CD4-furin from late endosomes to the TGN. These observations indicate that the ARF1+ARF4 and ARF1+ARF3 pairs are both required for integrity of recycling endosomes but are involved in distinct transport pathways: the former pair regulates retrograde transport from endosomes to the TGN, whereas the latter is required for the transferrin recycling pathway from endosomes to the plasma membrane.