Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins

Rigaud, Jean‐Louis; Pitard, Bruno; Lévy, Daniel

doi:10.1016/0005-2728(95)00091-v

Cited by 466 publications

(507 citation statements)

References 112 publications

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“…The increase in the value of (n×η) caused a decrease in the half times for onset and passive reequilibration, which was re-adjusted by reducing the membrane permeabilities of the ions. These results are consistent with the fact that it is not possible to control protein orientation in the membrane of proteoliposomes prepared by sonication, while PSI proteoliposomes prepared by detergent mediated reconstitution have good asymmetric protein orientation and low permeability values [42]. In addition, vesicles with iontight membranes have also reduced vesicle permeability to both PMS and ascorbate, which increases the efficiency of the light-induced proton transfer [4].…”

Section: Simulation Of An Increased Transmembrane Proton Gradientsupporting

confidence: 81%

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Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

Pennisi

Greenbaum

Yoshida

2010

Biophysical Chemistry

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

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Section: Simulation Of An Increased Transmembrane Proton Gradientsupporting

confidence: 81%

“…Sonication is generally chosen due to its inherent simplicity, but the mechanical energy delivered during sonication may cause disruption of membrane proteins [42].…”

Section: Characterization Of the Psi Preparationmentioning

confidence: 99%

Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

Pennisi

Greenbaum

Yoshida

2010

Biophysical Chemistry

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“…Here we show that effects of phospholipid structure on accumulation of Ca# + are different from those on ATP hydrolysis. To measure accumulation of Ca# + , the ATPase has been reconstituted into large sealed vesicles, using a procedure based on that of Rigaud et al [18], which gives reconstituted vesicles with very low ionic permeabilities [14,[19][20][21][22].…”

Section: Methodsmentioning

confidence: 99%

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Dalton

Pilot

Mall

et al. 1999

Biochemical Journal

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Accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H+ transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca2+ showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (DOPS)] led to higher levels of accumulation of Ca2+, 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca2+. Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca2+ from the reconstituted vesicles were slow. The Ca2+-accumulation process was simulated assuming either simple passive leak of Ca2+ from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca2+ on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.

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“…(17,18)). Mixed lipid liposomes containing detergents constitute a more permeable bilayer and we expected that such structures would effect the in vitro reconstitution of MprF activity.…”

Section: Use Of Detergentmentioning

confidence: 99%

Monitoring Lys-tRNALys phosphatidylglycerol transferase activity

Roy

Ibba

2008

Methods

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In some bacteria Lys-tRNA Lys is used both in translation and for the specific addition of Lys to phosphatidylglycerol in the cytoplasmic membrane. This reaction is catalyzed by the membrane protein MprF, and the lysyl-phosphatidylglycerol formed contributes to the resistance of these bacteria to various cationic antibacterial molecules. Obtaining proteins and reconstituting an in vitro system mimicking membrane conditions is a major challenge to studying the function of membrane proteins, especially when labile substrates such as Lys-tRNA Lys are required. Here we report methods to obtain a stable enriched membrane fraction containing MprF, and the techniques necessary to quantitatively monitor its activity in vitro and in vivo.

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Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins

Cited by 466 publications

References 112 publications

Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Monitoring Lys-tRNALys phosphatidylglycerol transferase activity

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