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Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion.

ABSTRACT: The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5K?-mediated PI(4,5)P2 production in Rab7 positive endosomes leading to impaired Rab7 inactivation and increased number of LC3 positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2-dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.

SUBMITTER: Dr. Takashi Baba

PROVIDER: S-SCDT-EMBOJ-2018-100312 | biostudies-other |

REPOSITORIES: biostudies-other

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Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion.

Project description:The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.

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Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion.

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Project description:During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 ? (PI4KII?) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KII? to the PM. We conclude that by directing the trafficking of PI4KII?-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

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Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D.

Project description:Macroautophagy (in this paper referred to as autophagy) and the ubiquitin-proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane-bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and in inducing phospholipase D (PLD) activity. PIP(2) and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.

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Phosphatidylinositol(4,5)bisphosphate: diverse functions at the plasma membrane.

Project description:Phosphatidylinositol(4,5) bisphosphate (PI(4,5)P2) has become a major focus in biochemistry, cell biology and physiology owing to its diverse functions at the plasma membrane. As a result, the functions of PI(4,5)P2 can be explored in two separate and distinct roles - as a substrate for phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K) and as a primary messenger, each having unique properties. Thus PI(4,5)P2 makes contributions in both signal transduction and cellular processes including actin cytoskeleton dynamics, membrane dynamics and ion channel regulation. Signalling through plasma membrane G-protein coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immune receptors all use PI(4,5)P2 as a substrate to make second messengers. Activation of PI3K generates PI(3,4,5)P3 (phosphatidylinositol(3,4,5)trisphosphate), a lipid that recruits a plethora of proteins with pleckstrin homology (PH) domains to the plasma membrane to regulate multiple aspects of cellular function. In contrast, PLC activation results in the hydrolysis of PI(4,5)P2 to generate the second messengers, diacylglycerol (DAG), an activator of protein kinase C and inositol(1,4,5)trisphosphate (IP3/I(1,4,5)P3) which facilitates an increase in intracellular Ca2+. Decreases in PI(4,5)P2 by PLC also impact on functions that are dependent on the intact lipid and therefore endocytosis, actin dynamics and ion channel regulation are subject to control. Spatial organisation of PI(4,5)P2 in nanodomains at the membrane allows for these multiple processes to occur concurrently.

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Phosphatidylinositol 4,5-bisphosphate (PIP(2)) controls magnesium gatekeeper TRPM6 activity.

Project description:TRPM6 is crucial for human Mg2+ homeostasis as patients carrying TRPM6 mutations develop hypomagnesemia and secondary hypocalcemia (HSH). However, the activation mechanism of TRPM6 has remained unknown. Here we demonstrate that phosphatidylinositol-4,5-bisphophate (PIP2) controls TRPM6 activation and Mg2+ influx. Stimulation of PLC-coupled M1-receptors to deplete PIP2 potently inactivates TRPM6. Translocation of over-expressed 5-phosphatase to cell membrane to specifically hydrolyze PIP2 also completely inhibits TRPM6. Moreover, depolarization-induced-activation of the voltage-sensitive-phosphatase (Ci-VSP) simultaneously depletes PIP2 and inhibits TRPM6. PLC-activation induced PIP2-depletion not only inhibits TRPM6, but also abolishes TRPM6-mediated Mg2+ influx.Furthermore, neutralization of basic residues in the TRP domain leads to nonfunctional or dysfunctional mutants with reduced activity by PIP2, suggesting that they are likely to participate in interactions with PIP2.Our data indicate that PIP2 is required for TRPM6 channel function; hydrolysis of PIP2 by PLC-coupled hormones/agonists may constitute an important pathway for TRPM6 gating, and perhaps Mg2+ homeostasis.

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Plasma membrane phosphatidylinositol (4,5)-bisphosphate promotes Weibel-Palade body exocytosis.

Project description:Weibel-Palade bodies (WPB) are specialized secretory organelles of endothelial cells that control vascular hemostasis by regulated, Ca2+-dependent exocytosis of the coagulation-promoting von-Willebrand factor. Some proteins of the WPB docking and fusion machinery have been identified but a role of membrane lipids in regulated WPB exocytosis has so far remained elusive. We show here that the plasma membrane phospholipid composition affects Ca2+-dependent WPB exocytosis and von-Willebrand factor release. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] becomes enriched at WPB-plasma membrane contact sites at the time of fusion, most likely downstream of phospholipase D1-mediated production of phosphatidic acid (PA) that activates phosphatidylinositol 4-phosphate (PI4P) 5-kinase γ. Depletion of plasma membrane PI(4,5)P2 or down-regulation of PI4P 5-kinase γ interferes with histamine-evoked and Ca2+-dependent WPB exocytosis and a mutant PI4P 5-kinase γ incapable of binding PA affects WPB exocytosis in a dominant-negative manner. This indicates that a unique PI(4,5)P2-rich environment in the plasma membrane governs WPB fusion possibly by providing interaction sites for WPB-associated docking factors.

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Hepatitis B virus is degraded by autophagosome-lysosome fusion mediated by Rab7 and related components.

Project description: Not available

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Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

Project description:The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

| S-EPMC3769341 | biostudies-literature

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