Regeneration of Streptavidin-coated Paramagnetic Beads for Multiple uses in Inter-membrane Lipid Transfer Assays

2016 ◽  
Vol 1 (1) ◽  
pp. 13-18
Keyword(s):  
Membrane Lipid ◽  
Lipid Transfer ◽  
Multiple Uses ◽  
Paramagnetic Beads

scholarly journals The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Author(s):  
Juan Luis Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Cell Function ◽  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cellular Environment ◽  
Synaptotagmin 1

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER.

scholarly journals The type 2 diabetes gene product STARD10 is a phosphoinositide binding protein that controls insulin secretory granule biogenesis

2020 ◽  
Author(s):  
Gaelle R. Carrat ◽  
Elizabeth Haythorne ◽  
Alejandra Tomas ◽  
Leena Haataja ◽  
Andreas Müller ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Cell Function ◽  
Phosphatidyl Inositol ◽  
Binding Pocket ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Β Cell ◽  
Lipid Kinase ◽  
Insulin Granule

AbstractObjectiveRisk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids, and thus the pathways through which STARD10 regulates β-cell function, are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and its effect on proinsulin processing and insulin granule biogenesis and maturation.MethodsWe used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStarD10KO) and performed electron microscopy, pulse-chase, RNA sequencing and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay were performed on purified STARD10 protein.ResultsβStarD10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of “rod-like” dense cores. Correspondingly, basal secretion of proinsulin was increased. Amongst the differentially expressed genes in βStarD10KO islets, expression of the phosphoinositide binding proteins Pirt and Synaptotagmin 1 were decreased while lipidomic analysis demonstrated changes in phosphatidyl inositol levels. The inositol lipid kinase PIP4K2C was also identified as a STARD10 binding partner. STARD10 bound to inositides phosphorylated at the 3’ position and solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides.ConclusionOur data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis and insulin processing.

scholarly journals Lipids and membrane-associated proteins in autophagy

2020 ◽  
Author(s):  
Linsen Li ◽  
Mindan Tong ◽  
Yuhui Fu ◽  
Fang Chen ◽  
Shen Zhang ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Lipid Composition ◽  
Membrane Lipid ◽  
Membrane Properties ◽  
Lipid Transfer ◽  
Membrane Remodeling ◽  
Membrane Lipid Composition ◽  
Future Studies ◽  
Associated Proteins ◽  
Lysosome Membrane

Abstract Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.

Phospholipid modifications influence acyl-CoA:1-acyl-glycero-3-phosphocholine O-acyltransferase in rat liver plasma membranes

1991 ◽  
Vol 69 (9) ◽  
pp. 643-648 ◽  
Author(s):  
Albena B. Momchilova ◽  
Tania T. Markovska ◽  
Svetlana E. Koshlukova ◽  
Kamen S. Koumanov ◽  
Roumen G. Pankov
Keyword(s):  
Membrane Fluidity ◽  
Rat Liver ◽  
Lipid Composition ◽  
Plasma Membranes ◽  
Physical State ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Acyltransferase Activity ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

The influence of the membrane lipid composition and physical state on the activity of acyl-CoA: 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase in rat liver plasma membranes has been investigated. The membrane's lipid composition has been modified either by lipid transfer proteins or by partial delipidation with exogenous phospholipases. The results indicate that membrane fluidity is of particular importance for membrane-bound palmitoyl-CoA: and oleoyl-CoA: 1-acyl-glycero-3-phosphocholine acyltransferase. The incorporation of phospholipids that induce membrane fluidization such as dioleoylphosphatidylcholine, egg yolk phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine was accompanied by an elevation of acyltransferase activity. On the contrary, the phospholipids causing augmentation of membrane rigidity induced a decrease of this activity. A suggestion is made concerning the possible role of the membrane physical state for the deacylation–reacylation cycle in rat liver plasma membranes.Key words: acyltransferase, plasma membranes, membrane fluidity, phospholipids.

scholarly journals MICOS and phospholipid transfer by Ups2–Mdm35 organize membrane lipid synthesis in mitochondria

2016 ◽  
Vol 213 (5) ◽  
pp. 525-534 ◽  
Author(s):  
Mari J. Aaltonen ◽  
Jonathan R. Friedman ◽  
Christof Osman ◽  
Bénédicte Salin ◽  
Jean-Paul di Rago ◽  
...  
Keyword(s):  
Lipid Synthesis ◽  
Membrane Lipid ◽  
Intermembrane Space ◽  
Lipid Transfer ◽  
Inner Mitochondrial Membrane ◽  
Phospholipid Transfer ◽  
Mitochondrial Intermembrane Space ◽  
In Trans ◽  
And Function ◽  
Specific Lipid

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2–Mdm35 complexes (SLMO2–TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2–Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2–Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function.

scholarly journals The structure and flexibility analysis of the Arabidopsis synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Vol 4 (10) ◽  
pp. e202101152
Author(s):  
Juan L Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Variable Number ◽  
Lipid Transfer ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Synaptotagmin 1 ◽  
Hinge Points ◽  
A Site

Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communication in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.

scholarly journals Novel Peroxisome–Vacuole Contacts in Yeast

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641982962 ◽  
Author(s):  
Huala Wu ◽  
Ida J. van der Klei
Keyword(s):  
Hansenula Polymorpha ◽  
Membrane Lipid ◽  
Eukaryotic Cells ◽  
Peroxisome Proliferation ◽  
Lipid Transfer ◽  
Cell Compartments ◽  
Almost All

Peroxisomes are important organelles and present in almost all eukaryotic cells. Close associations between peroxisomes and other cell compartments are known for several decades. The first molecular details of physical contacts between peroxisomes and various other organelles are now beginning to emerge. We recently described a novel contact between peroxisomes and vacuoles in the yeast Hansenula polymorpha, which develops during conditions of strong peroxisome proliferation. At such conditions, Pex3-GFP forms focal patches at the peroxisome–vacuole contacts, while overproduction of Pex3 promotes their formation. These results reveal a novel function for Pex3 in the formation of these contacts, where it might act as a tethering protein. We speculate that the peroxisome–vacuole contact is important for membrane lipid transfer at conditions of strong organellar expansion.

Lipid Exchange Rates of Conventional and Polymer Stabilized Liposomes

2002 ◽  
Vol 724 ◽  
Author(s):  
Awad Ahmed ◽  
Nicole Heldt ◽  
Gregory Slack ◽  
Yuzhuo Li
Keyword(s):  
Exchange Rates ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Migration Rates ◽  
Lipid Exchange ◽  
Bound Lipids ◽  
Hydrophobic Tail ◽  
Exchange Kinetics ◽  
Liposome System ◽  
Polymer Stabilized

AbstractPolymer-stabilized liposome systems consisting of polyethylene glycol bound lipids (PEG-lipids) and conventional (nonpolymer stabilized) liposomes were compared in terms of their inter-membrane lipid migration rates. In order to monitor the exchange of lipids between the membranes, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PY-PC), a phospholipid with pyrene attached to the hydrophobic tail, was used to label the liposome. Labeled and unlabeled liposome systems were mixed and fluorescence spectroscopy was used to examine the lipid transfer. More specifically, the relative employed to deduce the exchange kinetics. After labeled and unlabeled liposome systems were mixed, the E/M ratio for PY-PC in a polymer stabilized liposome system decreased by 66% over a period of 80 minutes, while the E/M for PY-PC in a conventional liposome system decreased 70% in less than 2 minutes. This suggests that the exchange rate for lipids in polymer stabilized liposome systems is much slower than that of conventional liposome systems. In addition, the exchange rates for both conventional and polymer stabilized liposome systems are accelerated at an elevated temperature.

scholarly journals Transport of a fluorescent phosphatidylcholine analog from the plasma membrane to the Golgi apparatus.

1984 ◽  
Vol 99 (2) ◽  
pp. 742-751 ◽  
Author(s):  
R G Sleight ◽  
R E Pagano
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Fluorescent Protein ◽  
Chinese Hamster ◽  
Membrane Lipid ◽  
Lipid Transfer ◽  
Outer Leaflet ◽  
Membrane Lipid Bilayer ◽  
Fluorescent Lipid

We have examined the internalization and degradation of a fluorescent analog of phosphatidylcholine after its insertion into the plasma membrane of cultured Chinese hamster fibroblasts. 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC) was incorporated into the cell surface by liposome-cell lipid transfer at 2 degrees C. The fluorescent lipid remained localized at the plasma membrane as long as the cells were kept at 2 degrees C; however, when the cells were warmed to 37 degrees C, internalization of some of the fluorescent lipid occurred. Most of the internalized C6-NBD-PC accumulated in the Golgi apparatus although a small amount was found randomly distributed throughout the cytoplasm in punctate fluorescent structures. Internalization of the fluorescent lipid at 37 degrees C was blocked by the presence of inhibitors of endocytosis. Incubation of cells containing C6-NBD-PC at 37 degrees C resulted in a rapid degradation of the fluorescent lipid. This degradation occurred predominantly at the plasma membrane. The degradation of C6-NBD-PC resulted in the release of NBD-fatty acid into the medium. We have compared the internalization of the fluorescent lipid with that of a fluorescent protein bound to the cell surface. Both fluorescent lipid and protein remained at the plasma membrane at 2 degrees C and neither were internalized at 37 degrees C in the presence of inhibitors of endocytosis. However, when incubated at 37 degrees C under conditions that permit endocytosis, the two fluorescent species appeared at different intracellular sites. Our data suggest that there is no transmembrane movement of C6-NBD-PC and that the fluorescent probe reflects the internalization of the outer leaflet of the plasma membrane lipid bilayer. The results are consistent with the Golgi apparatus as being the primary delivery site of phospholipid by bulk membrane movement from the plasma membrane.

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