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.

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

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.

Lipid distribution in the eukaryotic cells depends on tight couplings between lipid transfer and lipid metabolism. Yet these couplings remain poorly described. Notably, it is unclear to what extent lipid exchangers of the OSBP-related proteins (ORPs) family, coupled to PI(4)P metabolism, contribute to the formation of sterol and phosphatidylserine gradient between the endoplasmic reticulum (ER) and other cell regions. To address this question, we have examined in vitro the activity of Osh4p, a representative ORP, between Golgi mimetic membranes in which PI(4)P is produced by a PI 4-kinase and ER mimetic membranes in which PI(4)P is hydrolyzed by the phosphatase Sac1p. Using quantitative, real-time assays, we demonstrate that Osh4p creates a sterol gradient between the two membranes by sterol/PI(4)P exchange as soon as a PI(4)P gradient is generated at this interface following ATP addition, and define how much PI(4)P must be synthesized for this process. Then, using a kinetic model supported by our in vitro data, we estimate to what extent PI(4)P metabolism can drive lipid transfer in cells. Finally, we show that Sec14p, by transferring phosphatidylinositol between membranes, can support the synthesis of PI(4)P and the creation of a sterol gradient by Osh4p. These results indicate to what extent ORPs, under the control of PI(4)P metabolism, can distribute lipids in the cell.

Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to β-adrenergic receptor (β-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. β1-AR and β2-ARs are the two major subtypes of β-ARs present in the human heart, however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of β1ARs drives detrimental cardiac remodeling while β2AR signaling is protective. The underlying molecular mechanisms for cardiac protection through β2ARs remain unclear. Here we show that β2-AR protects against hypertrophy through inhibition of PLCε signaling at the Golgi apparatus. The mechanism for β2AR-mediated PLC inhibition requires internalization of β2AR, activation of Gi and Gβγ subunit signaling at endosomes and ERK activation. This pathway inhibits both angiotensin II and Golgi-β1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD and HDAC5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for β2-AR antagonism of the PLCε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure.

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.