In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes

Nishikawa, Hanako; Sasaki, Masaru; Nishiyama, Kenichi

doi:10.21769/bioprotoc.3626

Cited by 9 publications

(14 citation statements)

References 24 publications

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“…PL (500 μg), YidC (40 μg), and F 0 F 1 -ATPase (25 μg), solubilized in 1.5% (wt/vol) OG, were mixed and incubated on ice for 30 min, followed by dialysis against buffer A (50 mM HEPES-KOH, pH 7.5, 1 mM dithiothreitol) for at least 3 h at 4°C. Proteoliposomes, thus reconstituted, were recovered by centrifugation (160,000g, 1 h, 4°C) and suspended in buffer A. DAG-containing liposomes were formed by sonication as described (35). PL and DAG (10% amount to PL), mixed in the solvent, were dried under a nitrogen stream and then under vacuum.…”

Section: Reconstitution Of (Proteo)liposomesmentioning

confidence: 99%

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

et al. 2021

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Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

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Section: Reconstitution Of (Proteo)liposomesmentioning

confidence: 99%

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

et al. 2021

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“…MPIase was mixed with E. coli PL and dioleoylglycerol in a solvent system (chloroform/ethanol/water: 3/7/4) in the ratio of 10 : 100 : 10, dried under an N 2 gas stream and then under vacuum, and then hydrated by sonication to form liposomes. Equal amounts in phospholipids of YidC/Pm‐UncI proteoliposomes were fused to MPIase liposomes by repeated, three times, cycles of freezing and thawing [11,30], giving 5% MPIase and 5% DAG in proteoliposomes. The proteoliposomes were briefly sonicated immediately before the assay.…”

Section: Methodsmentioning

confidence: 99%

Ring assembly of c subunits of F₀F₁‐ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase‐dependent membrane insertion

et al. 2021

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The c subunits of F0F1‐ATP synthase (F0c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F0c (Pm‐F0c), of which the ring structure is resistant to SDS. Ring assembly of Pm‐F0c requires P. modestum UncI (Pm‐UncI). Ring assembly of in vitro synthesized Pm‐F0c was observed when both YidC and Pm‐UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm‐F0c insertion allowing the subsequent YidC/Pm‐UncI‐dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm‐F0c.

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“…To explore the structure-activity relationship, various MPIase analogs (Figure 2b) were synthesized and applied to the activity assay, as outlined in Figure 2c. [13] The synthesized trisaccharyl pyrophospholipid, termed mini-MPIase-3, comprises the minimal unit required for the activity (Figure 2d). [15] Mini-MPIase-3(6-OH) and Trisac-DAG, mini-MPIase-3 analogs lacking the 6-Oacetyl group on GlcNAc and pyrophosphate, respectively, exhibited lower integration activity than mini-MPIase-3.…”

Section: Membrane Protein Integrase (Mpiase)mentioning

confidence: 99%

Bacterial Glycolipid Acting on Protein Transport Across Membranes

Mori,

Shionyu,

Shimamoto

et al. 2024

ChemBioChem

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The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and chaperones have been recognized as crucial factors in this process. However, recent studies have highlighted the significant roles played by lipids and a glycolipid present in biological membranes in membrane protein transport. Membrane lipids can influence transport efficiency by altering the physicochemical properties of membranes. Notably, our studies have revealed that diacylglycerol (DAG) attenuates mobility in the membrane core region, leading to a dramatic suppression of membrane protein integration. Conversely, a glycolipid in Escherichia coli inner membranes, named membrane protein integrase (MPIase), enhances integration not only through the alteration of membrane properties but also via direct interactions with membrane proteins. This review explores the mechanisms of membrane protein integration mediated by membrane lipids, specifically DAG, and MPIase. Our results, along with the employed physicochemical analysis methods such as fluorescence measurements, nuclear magnetic resonance, surface plasmon resonance, and docking simulation, are presented to elucidate these mechanisms.

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In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes

Cited by 9 publications

References 24 publications

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

Ring assembly of c subunits of F₀F₁‐ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase‐dependent membrane insertion

Bacterial Glycolipid Acting on Protein Transport Across Membranes

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In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes

Cited by 9 publications

References 24 publications

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins

Ring assembly of c subunits of F0F1‐ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase‐dependent membrane insertion

Bacterial Glycolipid Acting on Protein Transport Across Membranes

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Ring assembly of c subunits of F₀F₁‐ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase‐dependent membrane insertion