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.
View Article and Find Full Text PDFAsymmetric 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.
View Article and Find Full Text PDFPhosphatidylserine (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.
View Article and Find Full Text PDFP-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.
View Article and Find Full Text PDFMembrane 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.
View Article and Find Full Text PDFWithin 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.
View Article and Find Full Text PDFP4-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 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.
View Article and Find Full Text PDFPhosphatidylserine (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.
View Article and Find Full Text PDFP4-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.
View Article and Find Full Text PDFGene 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 -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.
View Article and Find Full Text PDFType 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.
View Article and Find Full Text PDFType 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.
View Article and Find Full Text PDFCurr Protoc Protein Sci
August 2018
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.
View Article and Find Full Text PDFP4-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.
View Article and Find Full Text PDFMembrane 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.
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