Enzymatic trans-bilayer lipid transport: Mechanisms, efficiencies, slippage, and membrane curvature

Shukla, Sankalp; Baumgart, Tobias

doi:10.1016/j.bbamem.2020.183534

Cited by 17 publications

(21 citation statements)

References 147 publications

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“…With the large range of fluorophores commercially available, we anticipate that our approach can be expanded from dual to multi-color setup to enable quantitative, parallel analyses including determination of membrane compositional heterogeneity and physical properties. The versatile strategy presented here is a powerful tool for optimization of reconstitution conditions and should benefit further investigation of many types of membrane proteins, including analysis of thus far under-studied lipid flippases at the single vesicle level (Thomsen et al, 2019; Shukla and Baumgart, 2021; López-Marqués et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

Veit

Paweletz

Bohr

et al. 2022

Preprint

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Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analyses of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole (NBD) dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

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Section: Discussionmentioning

confidence: 99%

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

Veit

Paweletz

Bohr

et al. 2022

Preprint

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“…Four models were built and simulated with the C36 lipid FF to investigate the influences of PI vs PS and diabetes on membrane properties. The diabetic models contain significantly less cholesterol (13 lateral compressibility and a looser lipid packing which was reflected by both the component surface area and S CD . The increased fluidity of the diabetic membrane agrees with the electron spin resonance spectroscopy study by Zuvic-Butorac et al 159 and corresponds well to reports of the diabetic myelin sheath being more prone to damage.…”

Section: ■ Mammalian Membrane Modelsmentioning

confidence: 99%

“…The cell membrane is a complex mixture of lipids and proteins that can provide protection to the cell as a plasma membrane (PM) or define cellular organelles, e.g., the endoplasmic reticulum (ER), trans-Golgi network (TGN), and mitochondria . Although membrane-associated proteins have been a strong focus in research on cell signaling, − molecular transport across cell membranes, − and shuttles for lipid movement within/between organelles, − proteins do not reside in a homogeneous sea of lipids that form a bilayer. These cellular membranes are quite dynamic and can have hundreds of varied lipids that can phase separate into liquid-ordered and -disordered domains. ,, These varied domains within a given organelle are believed to be important to certain functions of membrane-associated proteins.…”

Section: Introductionmentioning

confidence: 99%

All-Atom Modeling of Complex Cellular Membranes

Hsieh

Klauda

2021

Langmuir

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Cell membranes are composed of a variety of lipids and proteins where they interact with each other to fulfill their roles. The first step in modeling these interactions in molecular simulations is to have reliable mimetics of the membrane’s lipid environment. This Feature Article presents our recent efforts to model complex cellular membranes using all-atom force fields. A short review of the CHARMM36 (C36) lipid force field and its recent update to incorporate the long-range dispersion is presented. Key examples of model membranes mimicking various species and organelles are given. These include single-celled organisms such as bacteria (E. coli., chlamydia, and P. aeruginosa) and yeast (plasma membrane, endoplasmic reticulum, and trans-Golgi network) and more advanced ones such as plants (soybean and Arabidopsis thaliana) and mammals (ocular lens, stratum corneum, and peripheral nerve myelin). Leaflet asymmetry in composition has also been applied to some of these models. With the increased lipid diversity in the C36 lipid FF, these complex models can better reflect the structural, mechanical, and dynamic properties of realistic membranes and open an opportunity to study biological processes involving other molecules.

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“…Our work sets the stage for future experiments where the critical question of the effect of membrane lipid composition on scramblase activity can be examined by generating GUVs using a combination of proteoliposomes reconstituted with purified scramblases such as members of the GPCR and TMEM16 families, and liposomes of the desired composition 29 , 30 . In contrast to single vesicle studies using LUVs, GUVs with diameters of a few micrometers are comparable to the size of eukaryotic cells, thus mimicking the same lipid reservoir and membrane curvature 31 , 32 . Of practical importance is the fact that they can be easily observed without the need of advanced optical microscopy, e.g ., total internal reflection fluorescence microscopy, which allows investigations on spatial membrane organization 33 .…”

Section: Introductionmentioning

confidence: 99%

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

Mathiassen

Menon

Pomorski

2021

Sci Rep

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Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.

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Enzymatic trans-bilayer lipid transport: Mechanisms, efficiencies, slippage, and membrane curvature

Cited by 17 publications

References 147 publications

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

All-Atom Modeling of Complex Cellular Membranes

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

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