During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Lipid biosensors are molecular tools used both in vivo and in vitro applications, capable of selectively detecting specific types of lipids in biological membranes. However, despite their extensive use, there is a lack of systematic characterization of their binding properties in various membrane conditions. The purpose of this study was to investigate the impact of membrane properties, such as fluidity and membrane charge, on the sensitivity of two lipid biosensors, LactC2 and P4M, to their target lipids, phosphatidylserine (PS) or phosphatidylinositol 4-phosphate (PI4P), respectively. Dual-color fluorescence cross-correlation spectroscopy, employed in this study, provided a useful technique to investigate interactions of these recombinant fluorescent biosensors with liposomes of varying compositions. The results of the study demonstrate that the binding of the LactC2 biosensor to low levels of PS in the membrane is highly supported by the presence of anionic lipids or membrane fluidity. However, at high PS levels, the presence of anionic lipids does not further enhance binding of LactC2. In contrast, neither membrane charge, nor membrane fluidity significantly affect the binding affinity of P4M to PI4P. These findings provide valuable insights into the role of membrane properties on the binding properties of lipid biosensors.

OBJECTIVE: Tuberculosis still remains a global burden and is one of the top infectious diseases from a single pathogen. Mycobacterium tuberculosis, the causative agent, has perfected many ways to replicate and persist within its host. While mycobacteria induce vacuole damage to evade the toxic environment and eventually escape into the cytosol, the host recruits repair machineries to restore the MCV membrane. However, how lipids are delivered for membrane repair is poorly understood. Using advanced fluorescence imaging and volumetric correlative approaches, we demonstrate that this involves the recruitment of the endoplasmic reticulum (ER)-Golgi lipid transfer protein OSBP8 in the Dictyostelium discoideum/Mycobacterium marinum system. Strikingly, depletion of OSBP8 affects lysosomal function accelerating mycobacterial growth. This indicates that an ER-dependent repair pathway constitutes a host defense mechanism against intracellular pathogens such as M. tuberculosis.

Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.

Phosphatidylinositol lipids regulate key processes, including vesicle trafficking and cell polarity. A recent study identified novel roles for phosphatidylinositol 4-phosphate (PI4P) in the plasma membrane of the fungal pathogen Candida albicans, including polarized hyphal growth and cell wall organization. Studies in other organisms were not able to separate the roles of PI4P in the plasma membrane and Golgi, but the C. albicans plasma membrane pool of PI4P could be selectively eliminated by deleting the STT4 kinase, which creates PI4P. Interestingly, stt4Δ mutants were strongly defective in disseminated candidiasis in mice but were not defective in an oral infection. This suggested that abnormal exposure of β-glucan in the mutant cell walls increased recruitment of innate immune cells during disseminated infection, which is not expected to impact oral infection. These results highlight novel roles of PI4P and reinforce the need to test the virulence of C. albicans mutants at different host sites.

Drug resistance mutations of hepatitis C virus (HCV) negatively impact viral replicative fitness. RNA viruses are known to change their replication behaviour when subjected to suboptimal selection pressure. Here, we assess whether mutation supply in HCV is sufficiently large to allow the selection of its variants during dual or triple direct-acting antiviral (DAA) treatment associated with augmented virus fitness or impairment. We engineered randomly mutagenized full-genome libraries to create a highly diverse population of replication-competent HCV variants in cell culture. These variants exhibited escape when treated with NS5A/NS5B inhibitors (daclatasvir/sofosbuvir), and relapse on treatment with a combination of NS3/NS5A/NS5B inhibitors (simeprevir or paritaprevir/daclatasvir/sofosbuvir). Analysis of the relationship between virus fitness and drug resistance of JFH1-derived NS5A-5B variants showed a significant positive correlation (P=0.003). At the earliest time points, intracellular RNA levels remain unchanged in both the subgenomic replicon and infection assays, whereas extracellular RNA levels increased upto ten-fold compared to wild-type JFH1. Beneficial substitutions hyperstimulated phosphatidylinositol 4-phosphate during DAA treatment, and showed decreased dependence on cyclophilins during cyclosporine A treatment, indicating an interplay of virus-host molecular mechanisms in beneficial substitution selection that may necessitate infectious virus production. This comprehensive study demonstrates a possible role for HCV fitness of overcoming drug-mediated selection pressure.

Osh6, a member of the oxysterol-binding protein-related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4-phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella-containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3-kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3-phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P-binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.