The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network.

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

Atp8a1 deletion increases the proliferative activity of hematopoietic stem cells by impairing PTEN function.

PURPOSE: The eukaryotic cell plasma membrane contains several asymmetrically distributed phospholipids, which is maintained by the P4-ATPase flippase complex. Herein, we demonstrated the biological effects and mechanisms of asymmetrical loss in hematopoietic stem cells (HSCs). METHODS: An Atp8a1 knockout mouse model was employed, from which the HSC (long-term HSCs and short-term HSCs) population was analyzed to assess their abundance and function. Additionally, competitive bone marrow transplantation and 5-FU stress assays were performed. RNA sequencing was performed on Hematopoietic Stem and Progenitor Cells, and DNA damage was assayed using immunofluorescence staining and comet electrophoresis. The protein abundance for members of key signaling pathways was confirmed using western blotting. RESULTS: Atp8a1 deletion resulted in slight hyperleukocytosis, associated with the high proliferation of HSCs and BCR/ABL1 transformed leukemia stem cells (LSCs). Atp8a1 deletion increased the repopulation capability of HSCs with a competitive advantage in reconstitution assay. HSCs without Atp8a1 were more sensitive to 5-FU-induced apoptosis. Moreover, Atp8a1 deletion prevented HSC DNA damage and facilitated DNA repair processes. Genes involved in PI3K-AKT-mTORC1, DNA repair, and AP-1 complex signaling were enriched and elevated in HSCs with Atp8a1 deletion. Furthermore, Atp8a1 deletion caused decreased PTEN protein levels, resulting in the activation of PI3K-AKT-mTORC1 signaling, further increasing the activity of JNK/AP-1 signaling and YAP1 phosphorylation. CONCLUSION: We identified the role of Atp8a1 on hematopoiesis and HSCs. Atp8a1 deletion resulted in the loss of phosphatidylserine asymmetry and intracellular signal transduction chaos.

Exploring the Phospholipid Transport Mechanism of ATP8A1-CDC50.

P4-ATPase translocates lipids from the exoplasmic to the cytosolic plasma membrane leaflet to maintain lipid asymmetry distribution in eukaryotic cells. P4-ATPase is associated with severe neurodegenerative and metabolic diseases such as neurological and motor disorders. Thus, it is important to understand its transport mechanism. However, even with progress in X-ray diffraction and cryo-electron microscopy techniques, it is difficult to obtain the dynamic information of the phospholipid transport process in detail. There are still some problems required to be resolved: (1) when does the lipid transport happen? (2) How do the key residues on the transmembrane helices contribute to the free energy of important states? In this work, we explore the phospholipid transport mechanism using a coarse-grained model and binding free energy calculations. We obtained the free energy landscape by coupling the protein conformational changes and the phospholipid transport event, taking ATP8A1-CDC50 (the typical subtype of P4-ATPase) as the research object. According to the results, we found that the phospholipid would bind to the ATP8A1-CDC50 at the early stage when ATP8A1-CDC50 changes from E2P to E2Pi-PL state. We also found that the electrostatic effects play crucial roles in the phospholipid transport process. The information obtained from this work could help us in designing novel drugs for P-type flippase disorders.

The role of ATP8A1 in non-small cell lung cancer.

The study objective was to investigate the expression of ATP8A1 in non-small cell lung cancer (NSCLC) and to discover the role of ATP8A1 in the carcinogenesis of NSCLC. We collected 25 cases of tumor tissues and the adjacent normal tissues from surgeries of NSCLC patients in our hospital, among which 15 cases were found with lymph node metastasis while the other 10 were not. Immunohistochemical staining was performed to compare the expression level of TAP8A1 protein in NSCLC tissues with or without lymph node metastasis and the adjacent normal tissues. Transwell and scratch assay were used to test the invasion/migration capacity of different types of NSCLC. PCR and Western Blots were performed to detect the expression of ATP8A1 and epithelial-mesenchymal transition (EMT) markers in different cells. The percentage of ATP8A1 positive cells was (39.2±8.6)% in NSCLC tissues without lymph node metastasis, which was significantly lower than that in NSCLC tissues with lymph node metastasis ((74.7±11.0)%, P<0.05) as wells as remarkably higher than that in adjacent normal tissues with no ATP8A1 expression (P<0.05). When compared with normal H1299 cells, the invasion ability of ATP8A1 knock-down cells (si-H1299) was down-regulated by (31.2±5.7)%, the migration ability was down-regulated by (23.4±7.1)%, and the gene expression level of MMP and Vimentin was significantly reduced (P<0.05) while the expression of E-cadherin was remarkably increased (P<0.05). ATP8A1 was overexpressed in NSCLC tissues which promoted the expression of MMP-9 and Vimentin as well as suppressed the expression of E-cadherin thus resulting in the elevated invasion/migration ability of NSCLC cells.

Aberrant hippocampal Atp8a1 levels are associated with altered synaptic strength, electrical activity, and autistic-like behavior.

Type IV ATPases are putative aminophospholipid translocases (APLTs), more commonly known as flippases. A pronounced induction of the flippase Atp8a1 was observed in post-mortem tissue homogenates from the hippocampus and temporal lobe of juvenile autistic subjects compared to age-matched controls. In order to simulate the human data, C57BL/6 mice were allowed to develop after intra-hippocampal injection of recombinant lentivirus expressing Atp8a1 at the early developmental stage of postnatal day 6 (P6). Transmission electron microscopy (TEM) analysis of the lentivirus-Atp8a1 treated (Atp8a1+) mice in adulthood revealed fewer and weaker excitatory synapses in the hippocampal CA1 region compared to mice injected with empty virus. Significant inhibition of the Schaffer collateral pathway was observed in the Atp8a1+ mice in paired-pulse recording (PPR) at 20-ms inter-stimulus interval. In the three-chambered sociability test, the Atp8a1+ mice displayed no preference for an encaged stranger mouse over a novel object, which is a characteristic autistic-like behavior. In sharp contrast, Atp8a1 (-/-) mice displayed a preference for a stranger mouse over the novel object, which is characteristic of neurotypical mouse behavior. However, similar to the Atp8a1+ mice, the Atp8a1 (-/-) mice harbored fewer and weaker excitatory synapses in CA1 compared to wild-type controls, and displayed inhibition at 20-ms inter-stimulus interval in PPR. These findings suggest that both elevated and diminished levels of Atp8a1 during early development are detrimental to brain connectivity, but only elevated Atp8a1 is associated with aberrant social behavior. Mice with augmented levels of Atp8a1 may therefore serve as a potential model in autism research.

MiR-140-3p suppressed cell growth and invasion by downregulating the expression of ATP8A1 in non-small cell lung cancer.

MicroRNAs (miRNAs) as a class of small noncoding RNA molecules regulate the expression of targeted gene. The dysregulation of microRNAs is reported to be involved in carcinogenesis and tumor progression. Here, we identified miR-140-3p as a downregulated microRNA in most cancer tissues including lung cancer tissues, compared with their normal counterparts. MiR-140-3p was observed to perform its tumor suppressor function via its inhibition on cell growth, migration and invasion but its induction of cell apoptosis. Furthermore, the growth of non-small-cell lung cancer (NSCLC) cells in nude mouse models were suppressed by overexpression of miR-140-3p. ATP8A1 was demonstrated as a novel direct target of miR-140-3p using a luciferase assay. The increased level of intracellular ATP8A1 protein attenuated the inhibitor role of miR-140-3p in the growth and mobility of NSCLC cell. A regulation mechanism of miR-140-3p for the development and progression of NSCLC through downregulating the ATP8A1 expression was first discovered in the present study.

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.