CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

Waugh, Mark G.; Minogue, Shane; Clayton, Emma L.; Hsuan, J. Justin

doi:10.1194/jlr.m017814

Cited by 14 publications

(15 citation statements)

References 63 publications

(67 reference statements)

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“…Hence, the % value for cholesterol determined here is not the physiological proportion of cholesterol in TGN/endosomal membranes but rather, the amount present in the membrane vesicles after the extensive membrane disruption and isolation procedures used in this study. In concordance with this explanation, we have previously shown that membrane fractions prepared in the presence of carbonate are subject to substantial depletion of non-integral proteins (Waugh, 2013;Waugh et al, 2011a;Waugh et al, 2011b). As proteins have a density of 1.35 mg/mL (Chick and Martin, 1913;Fischer et al, 2004) and other membrane components such as lipids tend to have much lower densities, the value of 1.134 ± 0.005 mg/mL calculated for the density of the residual component seems reasonable.…”

Section: [10]supporting

confidence: 71%

“…The use of this assay to measure membrane cholesterol mass has been previously validated (Bate et al, 2008;Minogue et al, 2010;Nicholson and Ferreira, 2009). Ganglioside glycosphingolipids were detected by dot blotting of membrane fractions (Ilangumaran et al, 1996) and probing with HRP-conjugated cholera toxin B subunit as described previously (Ilangumaran et al, 1996;Mazzone et al, 2006;Waugh, 2013;Waugh et al, 2011a;Waugh et al, 2011b). Membranebound cholera toxin was visualized by incubation with chemiluminescence detection reagents and spots were quantified as described for the analysis of immunoblotting data (Waugh, 2013).…”

Section: Measurements Of Membrane Lipid Levelsmentioning

confidence: 99%

“…Manuscript to be reviewed Uesaka et al, 1994). Bearing in mind these limitations, we used this well established technique (Clarke et al, 2007;Correa et al, 2007;Domon et al, 2011;Ersek et al, 2015;Ilangumaran et al, 1996;Liu et al, 2013;Liu et al, 2015;Mazzone et al, 2006;Nguyen et al, 2007;Pang et al, 2004;Pristera et al, 2012;Russelakis-Carneiro et al, 2004;Tauzin et al, 2008;Waugh, 2013;Waugh et al, 2011a;Waugh et al, 2011b) to generate a composite yet simple signal to assess if there was any redistribution of these structurally related non-sterol molecules in the density gradient following cyclodextrin addition (figure 4). We observed that the ganglioside content of the buoyant fractions was decreased by about 50% following cyclodextrin treatment and this is consistent with mathematical analysis that vesicle size reduction is due to the non-selective desorption of membrane lipids.…”

Section: [11]mentioning

confidence: 99%

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Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

2015

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Cholesterol has important functions in the organization of membrane structure and this may be mediated via the formation of cholesterol-rich, liquid-ordered membrane microdomains often referred to as lipid rafts. Methyl-beta-cyclodextrin (cyclodextrin) is commonly used in cell biology studies to extract cholesterol and therefore disrupt lipid rafts. However, in this study we reassessed this experimental strategy and investigated the effects of cyclodextrin on the physical properties of sonicated and carbonate-treated intracellular membrane vesicles isolated from Cos-7 fibroblasts. We treated these membranes, which mainly originate from the trans-Golgi network and endosomes, with cyclodextrin and measured the effects on their equilibrium buoyant density, protein content, represented by the palmitoylated protein phosphatidylinositol 4-kinase type IIalpha, and cholesterol. Despite the reduction in mass stemming from cholesterol removal, the vesicles became denser, indicating a possible large volumetric decrease, and this was confirmed by measurements of hydrodynamic vesicle size. Subsequent mathematical analyses demonstrated that only half of this change in membrane size was attributable to cholesterol loss. Hence, the non-selective desorption properties of cyclodextrin are also involved in membrane size and density changes. These findings may have implications for preceding studies that interpreted cyclodextrin-induced changes to membrane biochemistry in the context of lipid raft disruption without taking into account our finding that cyclodextrin treatment also reduces membrane size.

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Section: [10]supporting

confidence: 71%

Section: Measurements Of Membrane Lipid Levelsmentioning

confidence: 99%

Section: [11]mentioning

confidence: 99%

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Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

2015

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“…Although I describe here a detergent-free, carbonate-based method, which enables researchers to achieve the cofractionation of phosphoinositides, sterols and ganglioside, it is technically feasible to substitute detergents for carbonate, to add both reagents together 68,72 or, alternatively, to omit both reagents and sonicate in neutral buffer so as to preserve noncovalent protein interactions with the membrane. Another published variation is to use Optiprep as the density-gradient medium for the ultracentrifugation step of the protocol, at the same concentration (wt/vol) as sucrose 71 .…”

Section: Experimental Designmentioning

confidence: 99%

“…This feature is particularly advantageous for downstream lipidomic and proteomic analyses, as the exclusion of detergents minimizes the potential for losses of some phospholipid species and palmitoylated proteins 68,72 .…”

Section: Advantages Of the Protocolmentioning

confidence: 99%

Raft-like membranes from the trans-Golgi network and endosomal compartments

Waugh

2013

Nat Protoc

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This article describes a procedure to prepare a raft-like intracellular membrane fraction enriched for the trans-Golgi network (TGN) and endosomal compartments. The initial step in this technique involves cell disruption by homogenization, followed by clearance of the plasma membrane, late endosomes, mitochondria and the endoplasmic reticulum by differential sedimentation. Carbonate treatment, sonication and sucrose density-gradient ultracentrifugation are subsequently used to isolate the target membranes. The isolated subcellular fraction contains less than 1% of the total cellular proteins, but it is highly enriched for syntaxin-6 and Rab11. Typically, 40-60% of the cellular pool of GM1 glycosphingolipid and 10-20% of the total cellular cholesterol cofractionate with this buoyant membrane fraction. Given the role of GM1 as a cell-surface receptor for the cholera toxin and that levels of both GM1 and cholesterol in the TGN-endosomal compartment are upregulated in some inherited diseases, this protocol can potentially be applied to the analysis of disease-associated changes to GM1-enriched intracellular membranes. The isolated membranes are very well separated from caveolin-rich domains of the plasma membrane, the TGN and recycling endosomes. The entire protocol can be completed in as little as 1 d.

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Measuring Phosphatidylinositol Generation on Biological Membranes

Waugh

2016

Methods in Molecular Biology

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Phosphatidylinositol (PI) is a phospholipid molecule required for the generation of seven different phosphoinositide lipids which have a diverse range of signaling and trafficking functions. The precise mechanism of phosphatidylinositol supply during receptor activated signaling and the cellular compartmentation of the synthetic process are still incompletely understood and remain controversial despite several decades of research in this area. The synthesis of phosphatidylinositol requires the activity of an enzyme called phosphatidylinositol synthase, also known as CDIPT, which catalyzes a reversible headgroup exchange reaction on its substrate liponucleotide CDP-diacylglycerol resulting in the incorporation of inositol to generate phosphatidylinositol and the release of CMP. This protocol describes a method for locating PI synthase activity in isolated, intact biological membranes and vesicles.

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CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

Cited by 14 publications

References 63 publications

Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

Peer Review #3 of "Modeling the effects of cyclodextrin on intracellular membrane vesicles from Cos-7 cells prepared by sonication and carbonate treatment (v0.1)"

Raft-like membranes from the trans-Golgi network and endosomal compartments

Measuring Phosphatidylinositol Generation on Biological Membranes

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