scholarly journals Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein

2016 ◽  
Vol 44 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Yeun Ju Kim ◽  
Maria Luisa Guzman-Hernandez ◽  
Eva Wisniewski ◽  
Nicolas Echeverria ◽  
Tamas Balla
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Lipid Transfer ◽  
Acid Transport ◽  
Contact Zones ◽  
Quiescent Cells ◽  
Associated Proteins ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of ◽  
Lateral Sclerosis

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and–B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER–PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER–PM contact zones with a possible link to a devastating human disease.

scholarly journals ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maxime Boutry ◽  
Peter K. Kim
Keyword(s):  
Lipid Transfer Protein ◽  
Super Resolution ◽  
Lipid Transfer ◽  
Direct Role ◽  
Mitochondrial Division ◽  
Transfer Protein ◽  
Division Site ◽  
Associated Proteins ◽  
Division Process ◽  
Phosphatidylinositol 4

AbstractMitochondrial division is not an autonomous event but involves multiple organelles, including the endoplasmic reticulum (ER) and lysosomes. Whereas the ER drives the constriction of mitochondrial membranes, the role of lysosomes in mitochondrial division is not known. Here, using super-resolution live-cell imaging, we investigate the recruitment of lysosomes to the site of mitochondrial division. We find that the ER recruits lysosomes to the site of division through the interaction of VAMP-associated proteins (VAPs) with the lysosomal lipid transfer protein ORP1L to induce a three-way contact between the ER, lysosome, and the mitochondrion. We also show that ORP1L might transport phosphatidylinositol-4-phosphate (PI(4)P) from lysosomes to mitochondria, as inhibiting its transfer or depleting PI(4)P at the mitochondrial division site impairs fission, demonstrating a direct role for PI(4)P in the division process. Our findings support a model where the ER recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria.

scholarly journals Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process

2015 ◽  
Vol 210 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Kirk W. Donovan ◽  
Anthony Bretscher
Keyword(s):  
Plasma Membrane ◽  
Secretory Vesicle ◽  
Regulatory Proteins ◽  
Regulatory Mechanisms ◽  
Secretory Vesicles ◽  
Myosin V ◽  
Gtp Hydrolysis ◽  
Associated Proteins ◽  
Hydrolysis Of

Post-Golgi secretory vesicle trafficking is a coordinated process, with transport and regulatory mechanisms to ensure appropriate exocytosis. While the contributions of many individual regulatory proteins to this process are well studied, the timing and dependencies of events have not been defined. Here we track individual secretory vesicles and associated proteins in vivo during tethering and fusion in budding yeast. Secretory vesicles tether to the plasma membrane very reproducibly for ∼18 s, which is extended in cells defective for membrane fusion and significantly lengthened and more variable when GTP hydrolysis of the exocytic Rab is delayed. Further, the myosin-V Myo2p regulates the tethering time in a mechanism unrelated to its interaction with exocyst component Sec15p. Two-color imaging of tethered vesicles with Myo2p, the GEF Sec2p, and several exocyst components allowed us to document a timeline for yeast exocytosis in which Myo2p leaves 4 s before fusion, whereas Sec2p and all the components of the exocyst disperse coincident with fusion.

New molecular mechanisms of inter-organelle lipid transport

2016 ◽  
Vol 44 (2) ◽  
pp. 486-492 ◽  
Author(s):  
Guillaume Drin ◽  
Joachim Moser von Filseck ◽  
Alenka Čopič
Keyword(s):  
Molecular Mechanisms ◽  
Lipid Binding ◽  
Phosphoinositide Metabolism ◽  
Lipid Transfer ◽  
Cellular Lipid ◽  
Transport Pathways ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Associated Proteins ◽  
Phosphatidylinositol 4

Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated.

scholarly journals Enforced expression of phosphatidylinositol 4-phosphate 5-kinase homolog alters PtdIns(4,5)P2 distribution and the localization of small G-proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yanbo Yang ◽  
Miriam Park ◽  
Masashi Maekawa ◽  
Gregory D. Fairn
Keyword(s):  
Plasma Membrane ◽  
Surface Charge ◽  
Electrostatic Interactions ◽  
Negative Charge ◽  
Anionic Charge ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Polybasic Domains ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

Abstract The generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) is essential for many functions including control of the cytoskeleton, signal transduction, and endocytosis. Due to its presence in the plasma membrane and anionic charge, PtdIns(4,5)P2, together with phosphatidylserine, provide the inner leaflet of the plasma membrane with a negative surface charge. This negative charge helps to define the identity of the plasma membrane, as it serves to recruit or regulate a multitude of peripheral and membrane proteins that contain polybasic domains or patches. Here, we determine that the phosphatidylinositol 4-phosphate 5-kinase homolog (PIPKH) alters the subcellular distribution of PtdIns(4,5)P2 by re-localizing the three PIP5Ks to endomembranes. We find a redistribution of the PIP5K family members to endomembrane structures upon PIPKH overexpression that is accompanied by accumulation of PtdIns(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). PIP5Ks are targeted to membranes in part due to electrostatic interactions; however, the interaction between PIPKH and PIP5K is maintained following hydrolysis of PtdIns(4,5)P2. Expression of PIPKH did not impair bulk endocytosis as monitored by FM4-64 uptake but did result in clustering of FM4-64 positive endosomes. Finally, we demonstrate that accumulation of polyphosphoinositides increases the negative surface charge of endosomes and in turn, leads to relocalization of surface charge probes as well as the polycationic proteins K-Ras and Rac1.

scholarly journals Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion

1986 ◽  
Vol 234 (1) ◽  
pp. 13-20 ◽  
Author(s):  
H M'Zali ◽  
F Giraud
Keyword(s):  
Phosphatidic Acid ◽  
Membrane Vesicles ◽  
Acid Synthesis ◽  
Cholesterol Depletion ◽  
Membrane Domains ◽  
Human Erythrocyte Membrane ◽  
Phosphatidylinositol 4 ◽  
Triton X ◽  
Hydrolysis Of ◽  
Synthesis And Degradation

The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol-depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200].

scholarly journals RdgBα reciprocally transfers PA and PI at ER–PM contact sites to maintain PI(4,5)P2 homoeostasis during phospholipase C signalling in Drosophila photoreceptors

2016 ◽  
Vol 44 (1) ◽  
pp. 286-292 ◽  
Author(s):  
Shamshad Cockcroft ◽  
Kathryn Garner ◽  
Shweta Yadav ◽  
Evelyn Gomez-Espinoza ◽  
Padinjat Raghu
Keyword(s):  
Lipid Transfer ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Hydrophobic Cavity ◽  
Dual Specificity ◽  
Lipid Kinases ◽  
Membrane Contact ◽  
Phosphatidylinositol 4 ◽  
G Protein Coupled ◽  
Hydrolysis Of

Phosphatidylinositol (PI) is the precursor lipid for the synthesis of PI 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane (PM) and is sequentially phosphorylated by the lipid kinases, PI 4-kinase and phosphatidylinositol 4-phosphate (PI4P)-5-kinase. Receptor-mediated hydrolysis of PI(4,5)P2 takes place at the PM but PI resynthesis occurs at the endoplasmic reticulum (ER). Thus PI(4,5)P2 resynthesis requires the reciprocal transport of two key intermediates, phosphatidic acid (PA) and PI between the ER and the PM. PI transfer proteins (PITPs), defined by the presence of the PITP domain, can facilitate lipid transfer between membranes; the PITP domain comprises a hydrophobic cavity with dual specificity but accommodates a single phospholipid molecule. The class II PITP, retinal degeneration type B (RdgB)α is a multi-domain protein and its PITP domain can bind and transfer PI and PA. In Drosophila photoreceptors, a well-defined G-protein-coupled phospholipase Cβ (PLCβ) signalling pathway, phototransduction defects resulting from loss of RdgBα can be rescued by expression of the PITP domain provided it is competent for both PI and PA transfer. We propose that RdgBα proteins maintain PI(4,5)P2 homoeostasis after PLC activation by facilitating the reciprocal transport of PA and PI at ER–PM membrane contact sites.

scholarly journals Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase

1997 ◽  
Vol 324 (3) ◽  
pp. 941-950 ◽  
Author(s):  
Denis ENGLISH ◽  
Margaret MARTIN ◽  
Kevin A. HARVEY ◽  
Luke P. AKARD ◽  
Ruth ALLEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Molecular Mass ◽  
Biological Systems ◽  
Short Chain ◽  
Intact Cells ◽  
Ability To Act ◽  
Phosphatidic Acids ◽  
Hydrolysis Of ◽  
Long Chain

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 °C and pH 7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1.4×10-3 M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin–Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

scholarly journals Rab5 regulates macropinosome closure through recruitment of the inositol 5-phosphatases OCRL/Inpp5b and the hydrolysis of PtdIns(4,5)P2

2020 ◽  
Author(s):  
Michelle E. Maxson ◽  
Helen Sarantis ◽  
Allen Volchuk ◽  
John H. Brumell ◽  
Sergio Grinstein
Keyword(s):  
Plasma Membrane ◽  
Mode Of Action ◽  
Dominant Negative ◽  
Circular Membrane ◽  
Fusion Event ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of

AbstractRab5 is required for macropinosome formation, but its site and mode of action remain unknown. We report that Rab5 acts at the plasma membrane, downstream of ruffling, to promote macropinosome sealing and scission. Dominant-negative Rab5, which obliterates macropinocytosis, had no effect on the development of membrane ruffles. However, Rab5-containing vesicles were recruited to circular membrane ruffles, and SNARE-dependent endomembrane fusion was necessary for completion of macropinocytosis. This fusion event coincided with the disappearance of PtdIns(4,5)P2 that accompanies macropinosome closure. Counteracting the depletion of PtdIns(4,5)P2 by expression of phosphatidylinositol-4-phosphate 5-kinase impaired macropinosome formation. Importantly, we found that removal of PtdIns(4,5)P2 is dependent on Rab5, through the Rab5-mediated recruitment of the inositol 5-phosphatases OCRL and Inpp5b, via APPL1. Knockdown of OCRL and Inpp5b, or APPL1 prevented macropinosome closure, without affecting ruffling. We therefore propose that Rab5 is essential for the clearance of PtdIns(4,5)P2 needed to complete macropinosome scission from the plasmalemma.

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