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.

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase.

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

Reconstitution of ATP-dependent lipid transporters: gaining insight into molecular characteristics, regulation, and mechanisms.

Lipid transporters play a crucial role in supporting essential cellular processes such as organelle assembly, vesicular trafficking, and lipid homeostasis by driving lipid transport across membranes. Cryo-electron microscopy has recently resolved the structures of several ATP-dependent lipid transporters, but functional characterization remains a major challenge. Although studies of detergent-purified proteins have advanced our understanding of these transporters, in vitro evidence for lipid transport is still limited to a few ATP-dependent lipid transporters. Reconstitution into model membranes, such as liposomes, is a suitable approach to study lipid transporters in vitro and to investigate their key molecular features. In this review, we discuss the current approaches for reconstituting ATP-driven lipid transporters into large liposomes and common techniques used to study lipid transport in proteoliposomes. We also highlight the existing knowledge on the regulatory mechanisms that modulate the activity of lipid transporters, and finally, we address the limitations of the current approaches and future perspectives in this field.

Determination of membrane protein orientation upon liposomal reconstitution down to the single vesicle level.

Reconstitution of membrane proteins into liposomal membranes represents a key technique in enabling functional analysis under well-defined conditions. In this review, we provide a brief introduction to selected methods that have been developed to determine membrane protein orientation after reconstitution in liposomes, including approaches based on proteolytic digestion with proteases, site-specific labeling, fluorescence quenching and activity assays. In addition, we briefly highlight new strategies based on single vesicle analysis to address the problem of sample heterogeneity.

Functional Analysis of the P-Type ATPases Apt2-4 from Cryptococcus neoformans by Heterologous Expression in Saccharomyces cerevisiae.

Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen Cryptococcus neoformans contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient S. cerevisiae strain dnf1Δdnf2Δdrs2Δ, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the C. neoformans Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and -choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of dnf1Δdnf2Δdrs2Δ, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a ß-subunit. These results identify C. neoformans Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.

Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore.

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

CDC50A is required for aminophospholipid transport and cell fusion in mouse C2C12 myoblasts.

Myoblast fusion is essential for the formation of multinucleated muscle fibers and is promoted by transient changes in the plasma membrane lipid distribution. However, little is known about the lipid transporters regulating these dynamic changes. Here, we show that proliferating myoblasts exhibit an aminophospholipid flippase activity that is downregulated during differentiation. Deletion of the P4-ATPase flippase subunit CDC50A (also known as TMEM30A) results in loss of the aminophospholipid flippase activity and compromises actin remodeling, RAC1 GTPase membrane targeting and cell fusion. In contrast, deletion of the P4-ATPase ATP11A affects aminophospholipid uptake without having a strong impact on cell fusion. Our results demonstrate that myoblast fusion depends on CDC50A and may involve multiple CDC50A-dependent P4-ATPases that help to regulate actin remodeling.

P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity.

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a ß-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

Structural requirements for membrane binding of human guanylate-binding protein 1.

Human guanylate-binding protein 1 (hGBP1) is a key player in innate immunity and fights diverse intracellular microbial pathogens. Its antimicrobial functions depend on hGBP1's GTP binding- and hydrolysis-induced abilities to form large, structured polymers and to attach to lipid membranes. Crucial for both of these biochemical features is the nucleotide-controlled release of the C terminally located farnesyl moiety. Here, we address molecular details of the hGBP1 membrane binding mechanism by employing recombinant, fluorescently labeled hGBP1, and artificial membranes. We demonstrate the importance of the GTPase activity and the resulting structural rearrangement of the hGBP1 molecule, which we term the open state. This open state is supported and stabilized by homodimer contacts involving the middle domain of the protein and is further stabilized by binding to the lipid bilayer surface. We show that on the surface of the lipid bilayer a hGBP1 monolayer is built in a pins in a pincushion-like arrangement with the farnesyl tail integrated in the membrane and the N-terminal GTPase domain facing outwards. We suggest that similar intramolecular contacts between neighboring hGBP1 molecules are responsible for both polymer formation and monolayer formation on lipid membranes. Finally, we show that tethering of large unilamellar vesicles occurs after the vesicle surface is fully covered by the monolayer. Both hGBP1 polymer formation and hGBP1-induced vesicle tethering have implications for understanding the molecular mechanism of combating bacterial pathogens. DATABASES: Structural data are available in RCSB Protein Data Bank under the accession numbers: 6K1Z, 2D4H.

Chloroplast Ribosomes Interact With the Insertase Alb3 in the Thylakoid Membrane.

Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.

Functional Significance of Conserved Cysteines in the Extracellular Loops of the ATP Binding Cassette Transporter Pdr11p.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.

The transport mechanism of P4 ATPase lipid flippases.

P4 ATPase lipid flippases are ATP-driven transporters that translocate specific lipids from the exoplasmic to the cytosolic leaflet of biological membranes, thus establishing a lipid gradient between the two leaflets that is essential for many cellular processes. While substrate specificity, subcellular and tissue-specific expression, and physiological functions have been assigned to a number of these transporters in several organisms, the mechanism of lipid transport has been a topic of intense debate in the field. The recent publication of a series of structural models based on X-ray crystallography and cryo-EM studies has provided the first glimpse into how P4 ATPases have adapted the transport mechanism used by the cation-pumping family members to accommodate a substrate that is at least an order of magnitude larger than cations.

Agrobacterium tumefaciens Small Lipoprotein Atu8019 Is Involved in Selective Outer Membrane Vesicle (OMV) Docking to Bacterial Cells.

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra- and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.

Label-Free Fluorescence Quantification of Hydrolytic Enzyme Activity on Native Substrates Reveals How Lipase Function Depends on Membrane Curvature.

Lipases are important hydrolytic enzymes used in a spectrum of technological applications, such as the pharmaceutical and detergent industries. Because of their versatile nature and ability to accept a broad range of substrates, they have been extensively used for biotechnological and industrial applications. Current assays to measure lipase activity primarily rely on low-sensitivity measurements of pH variations or visible changes of material properties, like hydration, and often require high amounts of proteins. Fluorescent readouts, on the other hand, offer high contrast and even single-molecule sensitivity, albeit they are reliant on fluorogenic substrates that structurally resemble the native ones. Here we present a method that combines the highly sensitive readout of fluorescent techniques while reporting enzymatic lipase function on native substrates. The method relies on embedding the environmentally sensitive fluorescent dye pHrodo and native substrates into the bilayer of liposomes. The charged products of the enzymatic hydrolysis alter the local membrane environment and thus the fluorescence intensity of pHrodo. The fluorescence can be accurately quantified and directly assigned to product formation and thus enzymatic activity. We illustrated the capacity of the assay to report the function of diverse lipases and phospholipases both in a microplate setup and at the single-particle level on individual nanoscale liposomes using total internal reflection fluorescence (TIRF). The parallelized sensitive readout of microscopy combined with the inherent polydispersity in sizes of liposomes allowed us to screen the effect of membrane curvature on lipase function and identify how mutations in the lid region control the membrane curvature-dependent activity. We anticipate this methodology to be applicable for sensitive activity readouts for a spectrum of enzymes where the product of the enzymatic reaction is charged.

Short-chain lipid-conjugated pH sensors for imaging of transporter activities in reconstituted systems and living cells.

The design of ion sensors has gained importance for the study of ion dynamics in cells, with fluorescent proton nanosensors attracting particular interest because of their applicability in monitoring pH gradients in biological microcompartments and reconstituted membrane systems. In this work, we describe the improved synthesis, photophysical properties and applications of pH sensors based on amine-reactive pHrodo esters and short-chain lipid derivatives of phosphoethanolamine. The major features of these novel probes include strong fluorescence under acidic conditions, efficient partitioning into membranes, and extractability by back exchange to albumin. These features allow for the selective labeling of the inner liposomal leaflet in reconstituted membrane systems for studying proton pumping activities in a quantitative fashion, as demonstrated by assaying the activity of a plant plasma membrane H+-ATPase. Furthermore, the short-chain lipid-conjugated pH sensors enable the monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells. Collectively, our results demonstrate the applicability of short-chain lipid-conjugated sensors for in vivo and in vitro studies and thus pave the way for the design of lipid-conjugated sensors selective to other biologically relevant ions, e.g. calcium and sodium.

The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2: a phosphatidylserine flippase.

Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In the present study, we succeeded in purifying recombinant aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana, in complex with the ALA-interacting subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise, the enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.

The P5A ATPase Spf1p is stimulated by phosphatidylinositol 4-phosphate and influences cellular sterol homeostasis.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.

Novel tool to quantify cell wall porosity relates wall structure to cell growth and drug uptake.

Even though cell walls have essential functions for bacteria, fungi, and plants, tools to investigate their dynamic structure in living cells have been missing. Here, it is shown that changes in the intensity of the plasma membrane dye FM4-64 in response to extracellular quenchers depend on the nano-scale porosity of cell walls. The correlation of quenching efficiency and cell wall porosity is supported by tests on various cell types, application of differently sized quenchers, and comparison of results with confocal, electron, and atomic force microscopy images. The quenching assay was used to investigate how changes in cell wall porosity affect the capability for extension growth in the model plant Arabidopsis thaliana Results suggest that increased porosity is not a precondition but a result of cell extension, thereby providing new insight on the mechanism plant organ growth. Furthermore, it was shown that higher cell wall porosity can facilitate the action of antifungal drugs in Saccharomyces cerevisiae, presumably by facilitating uptake.

LEGO-Inspired Drug Design: Unveiling a Class of Benzo[d]thiazoles Containing a 3,4-Dihydroxyphenyl Moiety as Plasma Membrane H+ -ATPase Inhibitors.

The fungal plasma membrane H+ -ATPase (Pma1p) is a potential target for the discovery of new antifungal agents. Surprisingly, no structure-activity relationship studies for small molecules targeting Pma1p have been reported. Herein, we disclose a LEGO-inspired fragment assembly strategy for the design, synthesis, and discovery of benzo[d]thiazoles containing a 3,4-dihydroxyphenyl moiety as potential Pma1p inhibitors. A series of 2-(benzo[d]thiazol-2-ylthio)-1-(3,4-dihydroxyphenyl)ethanones was found to inhibit Pma1p, with the most potent IC50 value of 8 µm in an in vitro plasma membrane H+ -ATPase assay. These compounds were also found to strongly inhibit the action of proton pumping when Pma1p was reconstituted into liposomes. 1-(3,4-Dihydroxyphenyl)-2-((6-(trifluoromethyl)benzo[d]thiazol-2-yl)thio)ethan-1-one (compound 38) showed inhibitory activities on the growth of Candida albicans and Saccharomyces cerevisiae, which could be correlated and substantiated with the ability to inhibit Pma1p in vitro.

Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p.

The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function.