A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles

Paweletz, Laura Charlotte; Veit, Sarina; Pomorski, Thomas

doi:10.21769/bioprotoc.4542

Cited by 5 publications

(3 citation statements)

References 20 publications

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“…Proteoliposomes (10 µl, ~ 500 ng AHA2) were incubated for 2 h either sequentially, first with 20 pmol membrane impermeable SNAP dye (10 µM SNAP-Surface® 488, New England BioLabs Inc.) followed by 20 pmol membrane permeable SNAP dye (10 µM SNAP-Cell® 647-SiR, New England BioLabs Inc.) in reconstitution buffer supplemented with 1 mM DTT or with each dye separately. Samples were analyzed by SDS-PAGE using 12% gels and visualized on a ChemiDoc XRS Imaging System (Bio-Rad Laboratories GmbH, München, Germany) using the Image Lab™ software and preprogrammed option for Coomassie stained gels or Alexa488/Alexa647 fluorophores, respectively (51).…”

Section: 𝑦 = 𝐵𝑜𝑡𝑡𝑜𝑚 + 𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚 1 + 𝑒mentioning

confidence: 99%

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase

Paweletz,

Holtbrügge,

Löb

et al. 2023

Preprint

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The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase AHA2 upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partly conserved across functionally different P-type ATPases from several organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

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Section: 𝑦 = 𝐵𝑜𝑡𝑡𝑜𝑚 + 𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚 1 + 𝑒mentioning

confidence: 99%

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase

Paweletz,

Holtbrügge,

Löb

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Membrane proteins reconstituted in liposomal model membrane systems have either the cytosolic side (inside-out orientation) or the extracellular side (outside-out orientation) exposed. ATP-dependent transporters appear to prefer an inside-out orientation in the vesicle, due to bulky domains and the curvature of the vesicle [ 78 ] but the exact protein orientation is hardly predictable, which limits the quantitative and functional analysis of vesicle preparations [ 79 , 80 ].…”

Section: Studying Lipid Transporter Activities: From Cell-based Assay...mentioning

confidence: 99%

Reconstitution of ATP-dependent lipid transporters: gaining insight into molecular characteristics, regulation, and mechanisms

Herrera

Pomorski

2023

Bioscience Reports

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Lipid transporters play a crucial role in supporting essential cellular processes such as organelle assembly, vesicular trafficking, and lipid homeostasis by driving lipid transport across membranes. Cryo-electron microscopy has recently resolved the structures of several ATP-dependent lipid transporters, but functional characterization remains a major challenge. Although studies of detergent-purified proteins have advanced our understanding of these transporters, in vitro evidence for lipid transport is still limited to a few ATP-dependent lipid transporters. Reconstitution into model membranes, such as liposomes, is a suitable approach to study lipid transporters in vitro and to investigate their key molecular features. In this review, we discuss the current approaches for reconstituting ATP-driven lipid transporters into large liposomes and common techniques used to study lipid transport in proteoliposomes. We also highlight the existing knowledge on the regulatory mechanisms that modulate the activity of lipid transporters, and finally, we address the limitations of the current approaches and future perspectives in this field.

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“…Proteoliposomes (10 µL, ~500 ng AHA2) were incubated for 2 h either sequentially, first with 20 pmol membrane impermeable SNAP dye (10 µM SNAP-Surface ® 488, New England BioLabs Inc., Ipswich, MA, USA) followed by 20 pmol membrane permeable SNAP dye (10 µM SNAP-Cell ® 647-SiR, New England BioLabs Inc.) in reconstitution buffer supplemented with 1 mM DTT or with each dye separately. Samples were analyzed by SDS-PAGE using 12% gels and visualized on a ChemiDoc XRS Imaging System (Bio-Rad Laboratories GmbH, München, Germany) using the Image Lab™ software (https://www.bio-rad.com/zh-cn/product/image-lab-software?ID=KRE6P5E8Z& WT_mc_id=211202033049&WT_srch=1&WT_knsh_id=cr180107&gclid=EAIaIQobChMI0e KynPLxgAMVlZhmAh2HUArSEAAYASABEgJPdvD_BwE) and pre-programmed option for Coomassie stained gels or Alexa488/Alexa647 fluorophores, respectively [55].…”

Section: Protein Orientation Assaymentioning

confidence: 99%

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase

Paweletz,

Holtbrügge,

Löb

et al. 2023

IJMS

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The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

show abstract

A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles

Cited by 5 publications

References 20 publications

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase

Reconstitution of ATP-dependent lipid transporters: gaining insight into molecular characteristics, regulation, and mechanisms

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase

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A Fluorescence-based Approach Utilizing Self-labeling Enzyme Tags to Determine Protein Orientation in Large Unilamellar Vesicles

Cited by 5 publications

References 20 publications

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H+-ATPase

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H+-ATPase

Reconstitution of ATP-dependent lipid transporters: gaining insight into molecular characteristics, regulation, and mechanisms

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase

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Product

Resources

About

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase

Anionic phospholipids stimulate the proton pumping activity of the plant plasma membrane P-type H⁺-ATPase