Outer Leaflet Research Articles

Utilizing asymmetric giant unilamellar vesicles to determine how low-melting phospholipids affect interleaflet coupling strength.

Model membranes that mimic cell membrane asymmetry can be used to study coupling between lateral and transverse lipid organization. Theory predicts that, in a leaflet composed of lipids that would otherwise phase separate in a symmetric bilayer, domain formation can be suppressed by strong coupling to a uniformly mixed leaflet. Conversely, when coupled to a leaflet with a tendency to demix, an otherwise miscible leaflet can be induced to demix. Each of these outcomes has been observed in model asymmetric membranes of different composition, suggesting that cells may be able to control domain formation through changes in lipid content and/or asymmetric lipid distribution. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were investigated using confocal fluorescence microscopy; the mixtures containing 14:1-PC, 16:1-PC, and 18:1-PC showed macroscopic phase separation at room temperature, while the mixtures containing 20:1-PC and 22:1-PC did not. Cryogenic electron microscopy, small-angle neutron scattering, and Förster resonance energy transfer confirmed the presence of domains in the first three systems and revealed nanoscopic heterogeneity in the mixture containing 20:1-PC, while the 22:1-PC mixture was uniform. aGUVs were then prepared by calcium-induced hemifusion of the GUVs to a supported lipid bilayer composed of the corresponding low-TM lipid and Chol (80/20 mol%). The aGUVs showed a range of outer leaflet exchange, which revealed a correlation between phase behavior and extent of asymmetry. Domains in the 16:1-PC and 18:1-PC systems were suppressed in aGUVs with more than 50 and 60% asymmetry, respectively, indicating a relatively large free energy cost for antiregistered domains.

Characterizing the interactions between the LPS transport protein LptC and the ABC transporter LptB2FG.

Synthesis of the essential glycolipid lipopolysaccharide (LPS) in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following this process, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM). LPS transport to the OM is an essential process and disruption at any stage has detrimental effects on bacterial viability. LptB2FG, the first component of the Lpt system, is a type IV ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane helix, which intercalates between the LptF and LptG transmembrane domains, and has been shown to regulate the ATPase activity of LptB2FG. Despite a high level of structural homology between the periplasmic domains of LptF and LptG, the crystal and cryo-EM structures of LptB2FG in complex with LptC show preferential binding of the periplasmic domain of LptC to the periplasmic domain of LptF. Therefore, the role of LptG in this transport process remains unknown. Additionally, we identified six partial loss of function LptC mutations during an in vivo exploratory mutagenesis study. To further investigate the LptB2FGC complex, site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy, microscale thermophoresis (MST), and an in vitro ATPase assay were employed to characterize differences between the binding partners of periplasmic LptF and LptG along with the mechanistic effects of the LptC loss of function mutants on partner binding and ATPase function or regulation. This work provides evidence suggesting a previously undefined interaction between LptC and LptG while also exploring the role of partial loss of function LptC mutants.

Asymmetric phase diagram of DPPC/DOPC to investigate inter-leaflet coupling.

Cell membranes are asymmetric, meaning that the bilayer is composed of leaflets with different lipid composition. Simplified models for the exoplasmic leaflet typically form coexisting Ld (liquid disordered) + Lo (liquid ordered) phases in symmetric bilayers. In contrast, the cytoplasmic leaflet lacks high-melting lipids, and models for this leaflet always form a single fluid phase in symmetric bilayers. Thus, among the major open questions is how the phase behavior of these two leaflets is coupled in an asymmetric bilayer. Here, we report the first experimentally determined asymmetric phase diagram for a binary lipid mixture, DPPC/DOPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine) at room temperature. Different from cholesterol-containing model membranes that separate into coexisting liquid phases, this simpler mixture exhibits gel + fluid coexistence over a range of composition and temperature. We used calcium-induced hemifusion to engineer asymmetric GUVs (aGUVs), and we measured the extent of outer leaflet exchange through quantitative analysis of fluorescent probe intensities. Our experiments can be directly compared to theoretical phase diagrams calculated from mean field theory, where phase boundaries and tieline orientations depend on the relative strength of in-plane and out-of-plane lipid interaction energies. The experimental DPPC/DOPC asymmetric phase diagram is consistent with an intermediate coupling strength whereby anti-registered domains coexist with registered domains at lower levels of asymmetry, but phase separation is completely suppressed at higher levels of asymmetry. Our results allow us to estimate a mismatch free energy of 0.2-0.3 kT per square nanometer for a gel domain directly opposite a fluid domain.

Microcontact-printed patterned supported bilayer for measurement of flow-mediated protein mobility.

Many cells respond to fluid shear stress. For example, endothelial cells produce vasodilator nitric oxide (NO) in response to blood flow. This response requires the presence of glypican-1, an extracellular GPI-anchoring protein; but the mechanism of activation is unknown. In environments like the circulatory system, there is some evidence that flow transport of extracellular lipid-anchored protein occurs. The problem is that it is unknown whether this transport contributes to flow sensing and signaling. Our lab developed a method to measure lipid-anchored protein mobility under flow in a simplified model system made up of supported bilayer patches formed from giant unilamellar vesicles (GUVs). However, physiologically interesting proteins obtained directly from cells via plasma membrane vesicles (PMVs) cannot be investigated using this method, since they end up oriented toward the solid support. Here, we demonstrate a procedure for creating finite supported membrane patches using microcontact printing with bovine serum albumin. This method results in patches with upper leaflets formed from the outer leaflets of the vesicles, making it possible to assay flow mobility of PMV-derived proteins. We show that we can measure protein flow mobility using this system. This will make it possible to compare mobility of the same protein in cells and in simplified supported membranes.

Small changes in lipid backbone linkages can lead to big changes in liquid-liquid phase separation in model membranes.

Cells can dynamically alter the spatial organization of lipids and proteins in the plasma membrane (PM) to regulate processes including cell signaling. The size and morphology of membrane domains, and how these depend on lipid composition, have drawn attention as potentially important variables in these processes. Model membrane studies have proven invaluable for elucidating the influence of phospholipid chains and headgroups, as well as cholesterol concentration, on membrane phase behavior. Following this approach, we have investigated simplified three- and four-component models for the PM outer leaflet to understand the role of the lipid backbone. Specifically, we replaced ester-linked DPPC and DOPC with their ether-linked counterparts to selectively change the membrane dipole potential of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases, while titrating DOPC with POPC to control the line tension. Fluorescence imaging of GUVs gave insight into the macroscopic phase behavior, while FRET and cryo-EM of LUVs allowed us to probe nanoscale heterogeneity. Our results can be understood in terms of a model in which the tendency to minimize line tension is counteracted by dipole repulsion, which can in some cases result in stable arrays of small domains or modulated phases. These findings suggest that relatively small changes in the structure of the lipid backbone can influence domain formation and morphology in biomimetic membranes.

Cell-sized asymmetric phospholipid-amphiphilic protein vesicles with growth, fission, and molecule transportation

Lipid vesicles, which mimic cell membranes in structure and components, have been used to study the origin of life and artificial cell construction. A different approach to developing cell-mimicking systems focuses on the formation of protein- or polypeptide-based vesicles. However, micro-sized protein vesicles that are similar in membrane dynamics to the cell and that reconstitute membrane proteins are difficult to form. In this study, we generated cell-sized asymmetric phospholipid-amphiphilic protein (oleosin) vesicles that allow the reconstitution of membrane proteins and the growth and fission of vesicles. These vesicles are composed of a lipid membrane on the outer leaflet and an oleosin membrane on the inner leaflet. Further, we elucidated a mechanism for the growth and fission of cell-sized asymmetric phospholipid-oleosin vesicles by feeding phospholipid micelles. Our asymmetric phospholipid-oleosin vesicles with the advantages of the lipid leaflet and the protein leaflet will potentially promote understanding of biochemistry and synthetic biology.

Glycolysis regulates KRAS plasma membrane localization and function through defined glycosphingolipids

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

Catanionic Vesicles as a Facile Scaffold to Display Natural N-Glycan Ligands for Probing Multivalent Carbohydrate-Lectin Interactions.

Multivalent interactions are a key characteristic of protein-carbohydrate recognition. Phospholipid-based liposomes have been explored as a popular platform for multivalent presentation of glycans, but this platform has been plagued by the instability of typical liposomal formulations in biological media. We report here the exploitation of catanionic vesicles as a stable lipid-based nanoparticle scaffold for displaying large natural N-glycans as multivalent ligands. Hydrophobic insertion of lipidated N-glycans into the catanionic vesicle bilayer was optimized to allow for high-density display of structurally diverse N-glycans on the outer membrane leaflet. In an enzyme-linked competitive lectin-binding assay, the N-glycan-coated vesicles demonstrated a clear clustering glycoside effect, with significantly enhanced affinity for the corresponding lectins including Sambucus nigra agglutinin (SNA), concanavalin A (ConA), and human galectin-3, in comparison with their respective natural N-glycan ligands. Our results showed that relatively low density of high-mannose and sialylated complex type N-glycans gave the maximal clustering effect for binding to ConA and SNA, respectively, while relatively high-density display of the asialylated complex type N-glycan provided maximal clustering effects for binding to human galectin 3. Moreover, we also observed a macromolecular crowding effect on the binding of ConA to high-mannose N-glycans when catanionic vesicles bearing mixed high-mannose and complex-type N-glycans were used. The N-glycan-coated catanionic vesicles are stable and easy to formulate with varied density of ligands, which could serve as a feasible vehicle for drug delivery and as potent inhibitors for intervening protein-carbohydrate interactions implicated in disease.

Multiscale molecular dynamics simulations predict arachidonic acid binding sites in human ASIC1a and ASIC3 transmembrane domains.

Acid-sensing ion channels (ASICs) play important roles in inflammatory pathways by conducting ions across the neuronal membrane in response to proton binding under acidic conditions. Recent studies have shown that ASICs can be modulated by arachidonic acid (AA), and, in the case of the ASIC3 subtype, even activated by AA at physiological pH. However, the mechanism by which these fatty acids act on the channel is still unknown. Here, we have used multiscale molecular dynamics simulations to predict a putative, general binding region of AA to models of the human ASIC protein. We have identified, in agreement with recent studies, residues in the outer leaflet transmembrane region which interact with AA. In addition, despite their similar modulation, we observe subtle differences in the AA interaction pattern between human ASIC1a and human ASIC3, which can be reversed by mutating three key residues at the outer leaflet portion of TM1. We further probed interactions with these residues in hASIC3 using atomistic simulations and identified possible AA coordinating interactions; salt bridge interactions of AA with R65hASIC3 and R68hASIC3 and AA tail interactions with the Y58hASIC3 aromatic ring. We have shown that longer fatty acid tails with more double bonds have increased relative occupancy in this region of the channel, a finding supported by recent functional studies. We further proposed that the modulatory effect of AA on ASIC does not result from changes in local membrane curvature. Rather, we speculate that it may occur through structural changes to the ion channel upon AA binding.

Brominated lipid probes expose structural asymmetries in constricted membranes

Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane-remodeling system composed of human CHMP1B and IST1, we observed leaflet-level and protein-localized structural lipid patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated stearoyl-docosahexanenoyl-phosphocholine showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

(p)ppGpp and moonlighting RNases influence the first step of lipopolysaccharide biosynthesis in Escherichia coli.

The outer membrane (OM) protects Gram-negative bacteria from harsh environmental conditions and provides intrinsic resistance to many antimicrobial compounds. The asymmetric OM is characterized by phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Previous reports suggested an involvement of the signaling nucleotide ppGpp in cell envelope homeostasis in Escherichia coli. Here, we investigated the effect of ppGpp on OM biosynthesis. We found that ppGpp inhibits the activity of LpxA, the first enzyme of LPS biosynthesis, in a fluorometric in vitro assay. Moreover, overproduction of LpxA resulted in elongated cells and shedding of outer membrane vesicles (OMVs) with altered LPS content. These effects were markedly stronger in a ppGpp-deficient background. We further show that RnhB, an RNase H isoenzyme, binds ppGpp, interacts with LpxA, and modulates its activity. Overall, our study uncovered new regulatory players in the early steps of LPS biosynthesis, an essential process with many implications in the physiology and susceptibility to antibiotics of Gram-negative commensals and pathogens.

Cholesterol affected dynamics of lipids in tailor-made vesicles by ArcVes software during multi micro second coarse grained molecular dynamics simulations

<abstract> <p>In biotechnology, as well as in nanomedicine, vesicular structures are essential features of the cellular life cycle. Lipid compositions define the applicability of the vesicles. Here, the dynamics of lipid molecules within vesicles of approximately 10 nm in diameter are monitored using coarse-grained molecular dynamics (CGMD) simulations (MARTINI). The vesicles consist of (i) 1-palmitoyl-2-oleoyl-<italic>sn</italic>-glycero-3-phosphocholine (POPC), as well as 1,2-dioleoyl-<italic>sn</italic>-glycero-3-phosphocholine (DOPC), each in a mixture with cholesterol (CHOL) (POPC/CHOL, DOPC/CHOL), (ii) POPC/DOPC mixture and (iii) pure POPC and DOPC. Vesicles are generated by placing the individual lipids spatially separated by an area-per-lipid based distance onto the sphere using an arc correction, implemented into ArcVes, which is an in-house developed software. This protocol leads to the generation of vesicles which remain stable during extended MD simulations. In the presence of cholesterol, the mixed vesicles show time-dependent changes such as a decrease in both radii and membrane thickness. The number of lipids in each leaflet of the mixed vesicles reveals a trend of POPC and DOPC flipping to the outer leaflet and of cholesterol to the inner leaflet. This leads to a cholesterol rich path-formation in both the inner and outer leaflets. The diffusivity of the lipids in the vesicles mixed with cholesterol is lower than the diffusivity of the pure vesicles, similar to the observation for the respective flat membrane patches. In general, the diffusivity of the lipids is larger in the outer leaflets than the inner leaflets.</p> </abstract>

Prion receptors, prion internalization, intra- and inter-cellular transport.

Cellular prion protein (PrP C ) is a membrane bound protein that undergoes several post translational modifications. It can exert pleiotropic functions either in physiological and pathological conditions. The disease-causing isoform of the prion protein, called PrP Sc , acts as corruptive seed able to establish a process of misfolding and aggregation of further PrP Sc molecules. Therefore, PrP C acts as substrate for PrP Sc formation and as receptor to convey neurotoxic functions typical of other prion-like proteins involved in several neurodegenerative conditions. PrP C is transported from the outer leaflet of the cellular membrane to the intracellular compartments through several endocytic pathways, including clathrin-dependent and independent mechanisms and interactions with different adaptor proteins. During prion infection, the pathological counterpart PrP Sc , can be uptaken by the cells with mechanisms partially overlapping the ones of the physiological form, PrP C . The intercellular routing of both prion protein isoforms involves the release through vesicles and tunneling nanotubes. From the extracellular milieu, PrP C and PrP Sc can be uptaken by adjacent cells, thus exerting physiological and pathological functions.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

Probing the elastic response of lipid bilayers and nanovesicles to leaflet tensions via volume per lipid.

Biological and biomimetic membranes are based on lipid bilayers, consisting of two monolayers or leaflets. One important but challenging physical parameter of these membranes is their tension. For a long time, this tension was explicitly or implicitly taken to be the bilayer tension, acting on the whole bilayer membrane. More recently, it has been realized that it is useful to decompose the bilayer tension into two leaflet tensions and that these tensions are accessible to molecular dynamics simulations. To divide the bilayer up into two leaflets, it is necessary to introduce a midsurface that defines the spatial extent of the two leaflets. In previous studies, this midsurface was obtained from the density profiles across the bilayer and was then used to compute the molecular area per lipid. Here, we develop an alternative approach based on three-dimensional Voronoi tessellation and molecular volume per lipid. Using this volume-based approach, we determine the reference states with tensionless leaflets as well as the optimal volumes and areas per lipid. The optimal lipid volumes have practically the same value in both leaflets, irrespective of the size and curvature of the nanovesicles, whereas the optimal lipid areas are different for the two leaflets and depend on the vesicle size. In addition, we introduce lateral volume compressibilities to describe the elastic response of the lipid volume to the leaflet tensions. We show that the outer leaflet of a nanovesicle is more densely packed and less compressible than the inner leaflet and that this difference becomes more pronounced for smaller vesicles.

A Kinetic Map of the Influence of Biomimetic Lipid Model Membranes on Aβ42 Aggregation.

The aggregation of the amyloid β (Aβ) peptide is one of the molecular hallmarks of Alzheimer's disease (AD). Although Aβ deposits have mostly been observed extracellularly, various studies have also reported the presence of intracellular Aβ assemblies. Because these intracellular Aβ aggregates might play a role in the onset and progression of AD, it is important to investigate their possible origins at different locations of the cell along the secretory pathway of the amyloid precursor protein, from which Aβ is derived by proteolytic cleavage. Senile plaques found in AD are largely composed of the 42-residue form of Aβ (Aβ42). Intracellularly, Aβ42 is produced in the endoplasmatic reticulum (ER) and Golgi apparatus. Since lipid bilayers have been shown to promote the aggregation of Aβ, in this study, we measure the effects of the lipid membrane composition on the in vitro aggregation kinetics of Aβ42. By using large unilamellar vesicles to model cellular membranes at different locations, including the inner and outer leaflets of the plasma membrane, late endosomes, the ER, and the Golgi apparatus, we show that Aβ42 aggregation is inhibited by the ER and Golgi model membranes. These results provide a preliminary map of the possible effects of the membrane composition in different cellular locations on Aβ aggregation and suggest the presence of an evolutionary optimization of the lipid composition to prevent the intracellular aggregation of Aβ.

Cellular function of (a)symmetric biological membranes.

In mammalian cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. The maintenance of asymmetric phospholipid distribution has been demonstrated to be required for a wide range of cellular functions including cell division, cell migration, and signal transduction. However, we recently reported that asymmetric phospholipid distribution is disrupted in Drosophila cell membranes, and this unique phospholipid distribution leads to the formation of highly deformable cell membranes. In addition, it has become clear that asymmetry in the trans-bilayer distribution of phospholipids is disturbed even in living mammalian cells under certain circumstances. In this article, we introduce our recent studies while focusing on the trans-bilayer distribution of phospholipids, and discuss the cellular functions of (a)symmetric biological membranes.

Targeting LPS biosynthesis and transport in gram-negative bacteria in the era of multi-drug resistance

Gram-negative bacteria pose a major threat to human health in an era fraught with multi-drug resistant bacterial infections. Despite extensive drug discovery campaigns over the past decades, no new antibiotic target class effective against gram-negative bacteria has become available to patients since the advent of the carbapenems in 1985. Antibiotic discovery efforts against gram-negative bacteria have been hampered by limited intracellular accumulation of xenobiotics, in large part due to the impermeable cell envelope comprising lipopolysaccharide (LPS) in the outer leaflet of the outer membrane, as well as a panoply of efflux pumps. The biosynthesis and transport of LPS are essential to the viability and virulence of most gram-negative bacteria. Thus, both LPS biosynthesis and transport are attractive pathways to target therapeutically. In this review, we summarize the LPS biosynthesis and transport pathways and discuss efforts to find small molecule inhibitors against targets within these pathways.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1–16 mol % smooth LPS (corresponding to 16–79 wt %), while vesicles with inner leaflet LPS reach coverages of 2–7 mol % smooth LPS (28–60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Two distinct lipid transporters together regulate invasive filamentous growth in the human fungal pathogen Candida albicans.

Flippases transport lipids across the membrane bilayer to generate and maintain asymmetry. The human fungal pathogen Candida albicans has 5 flippases, including Drs2, which is critical for filamentous growth and phosphatidylserine (PS) distribution. Furthermore, a drs2 deletion mutant is hypersensitive to the antifungal drug fluconazole and copper ions. We show here that such a flippase mutant also has an altered distribution of phosphatidylinositol 4-phosphate [PI(4)P] and ergosterol. Analyses of additional lipid transporters, i.e. the flippases Dnf1-3, and all the oxysterol binding protein (Osh) family lipid transfer proteins, i.e. Osh2-4 and Osh7, indicate that they are not critical for filamentous growth. However, deletion of Osh4 alone, which exchanges PI(4)P for sterol, in a drs2 mutant can bypass the requirement for this flippase in invasive filamentous growth. In addition, deletion of the lipid phosphatase Sac1, which dephosphorylates PI(4)P, in a drs2 mutant results in a synthetic growth defect, suggesting that Drs2 and Sac1 function in parallel pathways. Together, our results indicate that a balance between the activities of two putative lipid transporters regulates invasive filamentous growth, via PI(4)P. In contrast, deletion of OSH4 in drs2 does not restore growth on fluconazole, nor on papuamide A, a toxin that binds PS in the outer leaflet of the plasma membrane, suggesting that Drs2 has additional role(s) in plasma membrane organization, independent of Osh4. As we show that C. albicans Drs2 localizes to different structures, including the Spitzenkörper, we investigated if a specific localization of Drs2 is critical for different functions, using a synthetic physical interaction approach to restrict/stabilize Drs2 at the Spitzenkörper. Our results suggest that the localization of Drs2 at the plasma membrane is critical for C. albicans growth on fluconazole and papuamide A, but not for invasive filamentous growth.