Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis

Eaglesfield, Ross; Madsen, Mary Ann; Sanyal, Suparna; Reboud, Julien; Amtmann, Anna

doi:10.1016/j.isci.2021.102429

Cited by 14 publications

(18 citation statements)

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“…For inspiration of suitable N-terminal sequences, we investigated naturally occurring sequences that have strong selection pressure against aggregation, which is generally toxic to cells. Initially, we again focused on MscL, which inserts co-translationally in bacteria, without assistance of the Sec translocon, [38], [39] similar to the cell-free expression system studied here. We hypothesized that as different bacterial species have large differences in their membrane composition, there might be a species with membrane compositions which better reflect the properties of simple synthetic membranes used in our cell-free reactions.…”

Section: Diverse Bacterial N-terminal Domain Sequences Balance Aggreg...mentioning

confidence: 99%

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Steinkühler¹,

Peruzzi²,

Krüger³

et al. 2023

Preprint

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Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

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Section: Diverse Bacterial N-terminal Domain Sequences Balance Aggreg...mentioning

confidence: 99%

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Steinkühler¹,

Peruzzi²,

Krüger³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Alternatively a cell-free system composed of recombinantly expressed and purified transcription/translation components (e.g. PURExpress, [ 99 ]) can be used [ 100 – 108 ]. By using cell-free expression, co-translational folding can be followed, and unnatural amino acids or fluorescent probes can be incorporated into the protein as it is synthesized, or the rate of folding can be altered ( figure 5 ).…”

Section: Methods To Investigate Co-translational Foldingmentioning

confidence: 99%

Methods to study folding of alpha-helical membrane proteins in lipids

et al. 2022

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How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages. This review will discuss folding methods which can be used to study alpha-helical membrane proteins in bicelles, liposomes, nanodiscs or native membranes. These folding methods include in vitro folding methods in liposomes such as denaturant unfolding studies, and single-molecule force spectroscopy studies in bicelles, liposomes and native membranes. This review will also discuss recent advances in co-translational folding studies, which use cell-free expression with liposomes or nanodiscs or are performed in vivo with native membranes.

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“…Furthermore, the C-terminal domain of Oxa1 was found to act as a binding site for the mitochondrial ribosome (Szyrach et al, 2003) suggesting that localising the translation of certain hydrophobic proteins to the membrane in close proximity to Oxa1 is key to their correct insertion and assembly. The membrane proximity of translation was also recently shown to be crucial for the thermodynamically driven assembly of a bacterial α-helical membrane protein in an entirely in vitro artificial system (Eaglesfield et al, 2021). The yeast protein Mba1 was also initially identified as a component of the respiratory chain assembly pathway acting independently of Oxa1 for the insertion of Cox2, cytochrome b and Cox1 (Preuss et al, 2001).…”

Section: Oxa1 and Respiratory Chain Assemblymentioning

confidence: 99%

Targeting and Insertion of Membrane Proteins in Mitochondria

Eaglesfield

Tokatlidis

2021

Front. Cell Dev. Biol.

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Mitochondrial membrane proteins play an essential role in all major mitochondrial functions. The respiratory complexes of the inner membrane are key for the generation of energy. The carrier proteins for the influx/efflux of essential metabolites to/from the matrix. Many other inner membrane proteins play critical roles in the import and processing of nuclear encoded proteins (∼99% of all mitochondrial proteins). The outer membrane provides another lipidic barrier to nuclear-encoded protein translocation and is home to many proteins involved in the import process, maintenance of ionic balance, as well as the assembly of outer membrane components. While many aspects of the import and assembly pathways of mitochondrial membrane proteins have been elucidated, many open questions remain, especially surrounding the assembly of the respiratory complexes where certain highly hydrophobic subunits are encoded by the mitochondrial DNA and synthesised and inserted into the membrane from the matrix side. This review will examine the various assembly pathways for inner and outer mitochondrial membrane proteins while discussing the most recent structural and biochemical data examining the biogenesis process.

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Cotranslational recruitment of ribosomes in protocells recreates a translocon-independent mechanism of proteorhodopsin biogenesis

Abstract: Generated a simple artificial cell model for membrane protein insertionWe identified proteininherent control of translational targeting without chaperones Ribosomes, artificially tethered to GUVs increased membrane protein insertion

Cited by 14 publications

References 50 publications

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Methods to study folding of alpha-helical membrane proteins in lipids

Targeting and Insertion of Membrane Proteins in Mitochondria

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