Direct evidence of lipid transport by the Drs2-Cdc50 flippase upon truncation of its terminal regions.

P4-ATPases in complex with Cdc50 subunits are lipid flippases that couple ATP hydrolysis with lipid transport to the cytoplasmic leaflet of membranes to create lipid asymmetry. Such vectorial transport has been shown to contribute to vesicle formation in the late secretory pathway. Some flippases are regulated by autoinhibitory regions that can be destabilized by protein kinase-mediated phosphorylation and possibly by binding of cytosolic proteins. In addition, the binding of lipids to flippases may also induce conformational changes required for the activity of these transporters. Here, we address the role of phosphatidylinositol-4-phosphate (PI4P) and the terminal autoinhibitory tails on the lipid flipping activity of the yeast lipid flippase Drs2-Cdc50. By functionally reconstituting the full-length and truncated forms of Drs2 in a 1:1 complex with the Cdc50 subunit, we provide compelling evidence that lipid flippase activity is exclusively detected for the truncated Drs2 variant and is dependent on the presence of the phosphoinositide PI4P. These findings highlight the critical role of phosphoinositides as lipid co-factors in the regulation of lipid transport by the Drs2-Cdc50 flippase.

Activation and substrate specificity of the human P4-ATPase ATP8B1.

Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P3 binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.

Phosphatidylserine transport in cell life and death.

Phosphatidylserine (PS) is a negatively charged glycerophospholipid found mainly in the plasma membrane (PM) and in the late secretory/endocytic compartments, where it regulates cellular activity and can mediate apoptosis. Export of PS from the endoplasmic reticulum, its site of synthesis, to other compartments, and its transbilayer asymmetry must therefore be precisely regulated. We review recent findings on nonvesicular transport of PS by lipid transfer proteins (LTPs) at membrane contact sites, on PS flip-flop between membrane leaflets by flippases and scramblases, and on PS nanoclustering at the PM. We also discuss emerging data on cooperation between scramblases and LTPs, how perturbation of PS distribution can lead to disease, and the specific role of PS in viral infection.

Fast-forward on P-type ATPases: recent advances on structure and function.

P-type ATPase are present in nearly all organisms. They maintain electrochemical gradients for many solutes, in particular ions, they control membrane lipid asymmetry, and are crucial components of intricate signaling networks. All P-type ATPases share a common topology with a transmembrane and three cytoplasmic domains and their transport cycle follows a general scheme - the Post-Albers-cycle. Recently, P-type ATPase research has been advanced most significantly by the technological advancements in cryo-EM analysis, which has elucidated many new P-type ATPase structures and mechanisms and revealed several new ways of regulation. In this review, we highlight the progress of the field and focus on special features that are present in the five subfamilies. Hence, we outline the new intersubunit transport model of KdpFABC, the ways in which heavy metal pumps have evolved to accommodate various substrates, the strategies Ca2+ pumps utilize to adapt to different environmental needs, the intricate molecular builds of the ion binding sites in Na,K- and H,K-ATPases, the remarkable hexameric assembly of fungal proton pumps, the many ways in which P4-ATPase lipid flippases are regulated, and finally the deorphanization of P5 pumps. Interestingly many of the described features are found in more than one of the five subfamilies, and mixed and matched together to provide optimal function and precise regulation.

eGFP as an All-in-One Tag for Purification of Membrane Proteins.

Within the last decade, cryo-electron microscopy has revolutionized our understanding of membrane proteins, but they still represent challenging targets for biochemical and structural studies. The first obstacle is often to obtain high production levels of correctly folded target protein. In these cases, the use of eGFP tags is an efficient strategy, as it allows rapid screenings of expression systems, constructs, and detergents for solubilization. Additionally, eGFP tags can now be used for affinity purification with recently developed nanobodies. Here we present a series of methods based on enhanced green fluorescent protein (eGFP) fluorescence to efficiently screen for production and stabilization of detergent-solubilized eGFP-tagged membrane proteins produced in S. cerevisiae via in-gel fluorescence SDS-PAGE and fluorescence-detection size-exclusion chromatography (FSEC). Additionally, we present a protocol describing the production of affinity resin based on eGFP-binding nanobodies produced in E. coli. We showcase the purification of human ATP7B, a copper transporting P-type ATPase, as an example of the applicability of the methods.

Expression in Saccharomyces cerevisiae and Purification of a Human Phospholipid Flippase.

Membrane proteins (MPs) are challenging to study from a biochemical standpoint owing to the difficulties associated with the isolation of these proteins from the membranes they are embedded in. Even for the expression of closely-related homologues, protocols often require to be adjusted. Prominently, the solubilization step and the stabilization of recombinant proteins during the purification process are key issues, and remain a serious bottleneck. Here, we present a method for the expression and the purification of the human ATP8B1/CDC50A lipid flippase complex. Selection of the right Saccharomyces cerevisiae strain proved to be a critical step for the successful purification of this complex. Likewise, the use of cholesteryl hemisuccinate, a cholesterol analogue, contributed to significantly increase the yield of purification. We hope that the simple method described here can help researchers to succeed in the expression of other mammalian difficult-to-express lipid flippases and, by extension, help in the production of other membrane proteins whose isolation has so far proven difficult.

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders.

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, for example in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 Å resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C-termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

Transport Pathways That Contribute to the Cellular Distribution of Phosphatidylserine.

Phosphatidylserine (PS) is a negatively charged phospholipid that displays a highly uneven distribution within cellular membranes, essential for establishment of cell polarity and other processes. In this review, we discuss how combined action of PS biosynthesis enzymes in the endoplasmic reticulum (ER), lipid transfer proteins (LTPs) acting within membrane contact sites (MCS) between the ER and other compartments, and lipid flippases and scramblases that mediate PS flip-flop between membrane leaflets controls the cellular distribution of PS. Enrichment of PS in specific compartments, in particular in the cytosolic leaflet of the plasma membrane (PM), requires input of energy, which can be supplied in the form of ATP or by phosphoinositides. Conversely, coupling between PS synthesis or degradation, PS flip-flop and PS transfer may enable PS transfer by passive flow. Such scenario is best documented by recent work on the formation of autophagosomes. The existence of lateral PS nanodomains, which is well-documented in the case of the PM and postulated for other compartments, can change the steepness or direction of PS gradients between compartments. Improvements in cellular imaging of lipids and membranes, lipidomic analysis of complex cellular samples, reconstitution of cellular lipid transport reactions and high-resolution structural data have greatly increased our understanding of cellular PS homeostasis. Our review also highlights how budding yeast has been instrumental for our understanding of the organization and transport of PS in cells.

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase.

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 ß-subunit.

Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium-encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.

P4-ATPases: how an old dog learnt new tricks - structure and mechanism of lipid flippases.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active, inward directed translocation (flip) of lipids in eukaryotic membranes. The resulting lipid asymmetry potentiates the membrane and is essential for a wide range of cellular processes such as vesicle biogenesis and trafficking and membrane protein regulation, whereas dissipation of lipid asymmetry is required in blood coagulation and apoptosis. Through recent advances in cryo-electron microscopy, several landmark structures of yeast and human lipid flippases have been reported, highlighting the similarities and differences they share with the cation transporting P-type ATPases. Here, we discuss the recent lipid flippase structures in the context of subunit architecture and organization, auto-regulation and lipid transport.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

Screening of Detergents for Stabilization of Functional Membrane Proteins.

Membrane protein studies usually require use of detergents to extract and isolate proteins from membranes and manipulate them in a soluble context for their functional or structural characterization. However, solubilization with detergent may interfere with MP stability and may directly affect MP function or structure. Moreover, detergent properties can be affected such as critical micellar concentration (CMC) can be affected by the experimental conditions. Consequently, the experimenter must pay attention to both the protein and the behavior of the detergent. This article provides a convenient protocol for estimating the CMC of detergents in given experimental conditions. Then, it presents two protocols aimed at monitoring the function of a membrane protein in the presence of detergent. Such experiments may help to test various detergents for their inactivating or stabilizing effects on long incubation times, ranging from few hours to some days. © 2018 by John Wiley & Sons, Inc.

High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate. Using thrombin as the protease, Cdc50p remains intact and in complex with Drs2p, which is cleaved at two positions, namely after Arg104 and after Arg 1290, resulting in a homogeneous sample lacking 104 and 65 residues from its N and C termini, respectively. Removal of the 1291-1302-amino acid region of the C-terminal extension is critical for relieving the auto-inhibition of full-length Drs2p, whereas the 1-104 N-terminal residues have an additional but more modest significance for activity. The present results therefore reveal that trimming off appropriate regions of the terminal extensions of Drs2p can greatly increase its ATPase activity in the presence of PI4P and demonstrate that relief of such auto-inhibition remains compatible with subsequent regulation by PI4P. These experiments suggest that activation of the Drs2p-Cdc50p flippase follows a multistep mechanism, with preliminary release of a number of constraints, possibly through the binding of regulatory proteins in the trans-Golgi network, followed by full activation by PI4P.

Slow Phospholipid Exchange between a Detergent-Solubilized Membrane Protein and Lipid-Detergent Mixed Micelles: Brominated Phospholipids as Tools to Follow Its Kinetics.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.