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Pietro De Camilli - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Xin Bian, Pietro De CamilliAbstract:The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial Lipid-binding Protein (SMP) domain, a Lipid-harboring module through which they mediate Lipid Transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and Lipid Transport functions of E-Syt1. Its membrane tethering activity is monitored through a turbidity-based assay, and its Lipid Transport property is analyzed via fluorescence resonance energy transfer (FRET)-based assay. These in vitro methods have enabled us to gain insight into the mechanism of action and regulation of E-Syt1, such as the role of Ca2+ in releasing E-Syt1 from an autoinhibitory conformation. The same methods could be adapted to the study of other Lipid Transport proteins that function at membrane contact sites.
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vps13a and vps13c are Lipid Transport proteins differentially localized at er contact sites
Journal of Cell Biology, 2018Co-Authors: Nikit Kumar, Marianna Leonzino, William Hancockcerutti, Florian A Horenkamp, Joshua A Lees, Heather E Wheeler, Karin M Reinisch, Pietro De CamilliAbstract:Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson's disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in Lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and Transport glyceroLipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to Lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a Lipid Transporter between the ER and other organelles, implicating defects in membrane Lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest Lipid Transport roles for these proteins as well.
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ca2 releases e syt1 autoinhibition to couple er plasma membrane tethering with Lipid Transport
The EMBO Journal, 2018Co-Authors: Xin Bian, Yasunori Saheki, Pietro De CamilliAbstract:The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and Transport Lipids between them via SMP domains. E-Syt1 tethers and Transports Lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables Lipid Transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM Lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with Lipid Transport.
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Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with Lipid Transport
The EMBO journal, 2017Co-Authors: Xin Bian, Yasunori Saheki, Pietro De CamilliAbstract:The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and Transport Lipids between them via SMP domains. E-Syt1 tethers and Transports Lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables Lipid Transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM Lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with Lipid Transport.
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Lipid Transport by tmem24 at er plasma membrane contacts regulates pulsatile insulin secretion
Science, 2017Co-Authors: Joshua A Lees, Pietro De Camilli, Heather E Wheeler, Mirko Messa, Federico Torta, Markus R Wenk, Karin M ReinischAbstract:INTRODUCTION Insulin is secreted by pancreatic β cells in response to glucose stimulation. Its release is controlled by the interplay of calcium and phosphoinositide signaling pathways. A rapid release phase, in which insulin containing granules that are already docked and primed at the plasma membrane (PM) undergo exocytosis, is followed by slow release. In this second phase, granules are docked and primed and then released in a series of bursts, each triggered by a spike in cytosolic Ca 2+ . RATIONALE To better understand the molecular basis underlying insulin secretion, we characterized TMEM24, a protein enriched in neuroendocrine cells previously suggested to be required for a normal secretory response. RESULTS We found that TMEM24 is an endoplasmic reticulum (ER) protein that concentrates at ER-PM contact sites, where it tethers the two bilayers. TMEM24 binding “in trans” to the PM is negatively regulated by phosphorylation in response to elevation of cytosolic Ca 2+ , so that TMEM24 transiently dissociates from the PM as Ca 2+ concentration spikes and then reassociates with this membrane upon dephosphorylation. Additionally, TMEM24 contains a Lipid Transport module of the synaptotagmin-like, mitochondrial and Lipid-binding protein (SMP) family, which we structurally characterized and showed to bind glyceroLipids with a preference for phosphatidylinositol (PI). Thus, TMEM24 helps deliver PI, which is synthesized in the ER, to the PM, where it is converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] to replenish pools of this Lipid hydrolyzed during glucose-stimulated signaling. Supporting a key role of TMEM24 in the coordination of Ca 2+ and phosphoinositide signaling, the Lipid Transport function of TMEM24 is essential for sustaining the intracellular Ca 2+ oscillations that trigger bursts of insulin granule release and hence insulin secretion. PI(4,5)P 2 is required for Ca 2+ -dependent exocytosis. It also controls the activity of PM ion channels that regulate cytosolic Ca 2+ levels and is the precursor of IP 3 , which also helps to modulate cytosolic Ca 2+ by triggering Ca 2+ release from the ER. Thus, in insulin-secreting cells, TMEM24 participates in coordinating Ca 2+ and phosphoinositide signaling pathways to cause pulsatile insulin secretion (see the figure). CONCLUSIONS Our findings implicate ER-PM contact sites and an ER resident Lipid-transfer protein in the direct regulation of PM phosphoinositide pools, offering fresh insights into the mechanisms of cellular phosphoinositide dynamics. More specifically, they elaborate the mechanisms underlying insulin secretion, which is impaired in patients with type II diabetes and may ultimately have therapeutic ramifications.
Xin Bian - One of the best experts on this subject based on the ideXlab platform.
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In Vitro Assays to Measure the Membrane Tethering and Lipid Transport Activities of the Extended Synaptotagmins.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Xin Bian, Pietro De CamilliAbstract:The three extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-localized membrane proteins that mediate tethering of the ER to the plasma membrane (PM) via C2 domain-dependent interactions regulated by Ca2+ and/or PI(4,5)P2. The E-Syts also contains a Synaptotagmin-like Mitochondrial Lipid-binding Protein (SMP) domain, a Lipid-harboring module through which they mediate Lipid Transport between the two adjacent membranes. Here, we describe in vitro liposome-based methods to study the membrane tethering and Lipid Transport functions of E-Syt1. Its membrane tethering activity is monitored through a turbidity-based assay, and its Lipid Transport property is analyzed via fluorescence resonance energy transfer (FRET)-based assay. These in vitro methods have enabled us to gain insight into the mechanism of action and regulation of E-Syt1, such as the role of Ca2+ in releasing E-Syt1 from an autoinhibitory conformation. The same methods could be adapted to the study of other Lipid Transport proteins that function at membrane contact sites.
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ca2 releases e syt1 autoinhibition to couple er plasma membrane tethering with Lipid Transport
The EMBO Journal, 2018Co-Authors: Xin Bian, Yasunori Saheki, Pietro De CamilliAbstract:The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and Transport Lipids between them via SMP domains. E-Syt1 tethers and Transports Lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables Lipid Transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM Lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with Lipid Transport.
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Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with Lipid Transport
The EMBO journal, 2017Co-Authors: Xin Bian, Yasunori Saheki, Pietro De CamilliAbstract:The extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and Transport Lipids between them via SMP domains. E-Syt1 tethers and Transports Lipids in a Ca2+-dependent manner, but the role of Ca2+ in this regulation is unclear. Of the five C2 domains of E-Syt1, only C2A and C2C contain Ca2+-binding sites. Using liposome-based assays, we show that Ca2+ binding to C2C promotes E-Syt1-mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P2-rich membranes, as previously suggested by studies in semi-permeabilized cells. Importantly, Ca2+ binding to C2A enables Lipid Transport by releasing a charge-based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E-Syt1 constructs defective in Ca2+ binding in either C2A or C2C failed to rescue two defects in PM Lipid homeostasis observed in E-Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca2+-triggered phosphatidylserine scrambling. Thus, a main effect of Ca2+ on E-Syt1 is to reverse an autoinhibited state and to couple membrane tethering with Lipid Transport.
Christoph Benning - One of the best experts on this subject based on the ideXlab platform.
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mechanisms of Lipid Transport involved in organelle biogenesis in plant cells
Annual Review of Cell and Developmental Biology, 2009Co-Authors: Christoph BenningAbstract:Chloroplasts are the defining organelle of photoautotrophic plant cells. Photosynthetic light reactions and electron Transport are the functions of an elaborate thylakoid membrane system inside chloroplasts. The Lipid composition of photosynthetic membranes is characterized by a substantial fraction of nonphosphorous galactoglyceroLipids reflecting the need of sessile plants to conserve phosphorus. Lipid Transport and assembly of glyceroLipids play an essential role in the biogenesis of the photosynthetic apparatus in developing chloroplasts. During chloroplast biogenesis, fatty acids are synthesized in the plastid and are exported to the endoplasmic reticulum, where they are incorporated into membrane Lipids. Alternatively, Lipids can also be assembled de novo at the inner envelope membrane of plastids in many plants. A rich repertoire of Lipid exchange mechanisms involving the thylakoid membranes, the chloroplast inner and outer envelope membranes, and the endoplasmic reticulum is emerging. Studies of t...
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Mechanisms of Lipid Transport Involved in Organelle Biogenesis in Plant Cells
Annual review of cell and developmental biology, 2009Co-Authors: Christoph BenningAbstract:Chloroplasts are the defining organelle of photoautotrophic plant cells. Photosynthetic light reactions and electron Transport are the functions of an elaborate thylakoid membrane system inside chloroplasts. The Lipid composition of photosynthetic membranes is characterized by a substantial fraction of nonphosphorous galactoglyceroLipids reflecting the need of sessile plants to conserve phosphorus. Lipid Transport and assembly of glyceroLipids play an essential role in the biogenesis of the photosynthetic apparatus in developing chloroplasts. During chloroplast biogenesis, fatty acids are synthesized in the plastid and are exported to the endoplasmic reticulum, where they are incorporated into membrane Lipids. Alternatively, Lipids can also be assembled de novo at the inner envelope membrane of plastids in many plants. A rich repertoire of Lipid exchange mechanisms involving the thylakoid membranes, the chloroplast inner and outer envelope membranes, and the endoplasmic reticulum is emerging. Studies of thylakoid biogenesis provide new insights into the general mechanisms of intermembrane Lipid transfer.
Mahmood M Hussain - One of the best experts on this subject based on the ideXlab platform.
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prap1 is a novel Lipid binding protein that promotes Lipid absorption by facilitating mttp mediated Lipid Transport
Journal of Biological Chemistry, 2021Co-Authors: Hubert W Peng, Tzuyuan Chiu, Yujen Liang, Chiajen Lee, Chihsyuan Liu, Chingshu Suen, Jeffrey J Y Yen, Hungta Chen, Mingjing Hwang, Mahmood M HussainAbstract:Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers Lipids to Lipid acceptors and whether there are other biomolecules involved in MTTP-mediated Lipid Transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated Lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated Lipid Transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to Lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing Lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated Lipid Transport and Lipid absorption.
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prap1 is a novel Lipid binding protein that promotes Lipid absorption by facilitating mttp mediated Lipid Transport
Journal of Biological Chemistry, 2020Co-Authors: Hubert W Peng, Tzuyuan Chiu, Yujen Liang, Chiajen Lee, Chihsyuan Liu, Chingshu Suen, Jeffrey J Y Yen, Hungta Chen, Mingjing Hwang, Mahmood M HussainAbstract:Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum (ER) resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers Lipids to Lipid acceptors and whether there are other biomolecules involved in MTTP-mediated Lipid Transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially co-localized in the ER. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated Lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated Lipid Transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to Lipid acceptors. Both PRAP1 deficient and the E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing Lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high fat diets compared to littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated Lipid Transport and Lipid absorption.
Barbara J. Clark - One of the best experts on this subject based on the ideXlab platform.
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The START-domain proteins in intracellular Lipid Transport and beyond.
Molecular and cellular endocrinology, 2020Co-Authors: Barbara J. ClarkAbstract:The Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) domain is a ~210 amino acid sequence that folds into an α/β helix-grip structure forming a hydrophobic pocket for Lipid binding. The helix-grip fold structure defines a large superfamily of proteins, and this review focuses on the mammalian START domain family members that include single START domain proteins with identified ligands, and larger multi-domain proteins that may have novel roles in metabolism. Much of our understanding of the mammalian START domain proteins in Lipid Transport and changes in metabolism has advanced through studies using knockout mouse models, although for some of these proteins the identity and/or physiological role of ligand binding remains unknown. The findings that helped define START domain Lipid-binding specificity, Lipid Transport, and changes in metabolism are presented to highlight that fundamental questions remain regarding the biological function(s) for START domain-containing proteins.
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the mammalian start domain protein family in Lipid Transport in health and disease
Journal of Endocrinology, 2012Co-Authors: Barbara J. ClarkAbstract:Lipid transfer proteins of the steroidogenic acute regulatory protein-related Lipid transfer (START) domain family are defined by the presence of a conserved w210 amino acid sequence that folds into an a/b helix-grip structure forming a hydrophobic pocket for ligand binding. The mammalian START proteins bind diverse ligands, such as cholesterol, oxysterols, phosphoLipids, sphingoLipids, and possibly fatty acids, and have putative roles in non-vesicular Lipid Transport, thioesterase enzymatic activity, and tumor suppression. However, the biological functions of many members of the START domain protein family are not well established. Recent research has focused on characterizing the cell-type distribution and regulation of the START proteins, examining the specificity and directionality of Lipid Transport, and identifying disease states associated with dysregulation of START protein expression. This review summarizes the current concepts of the proposed physiological and pathological roles for the mammalian START domain proteins in cholesterol and Lipid trafficking.