Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli.

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E.

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

Background: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme.

Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) or pReX and GFP-Fusions.

Optimizing the conditions for the production of membrane proteins in E. coli is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain or the pReX T7-based expression vector with membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the optimization of membrane protein production yields.

Exploration of the arrest peptide sequence space reveals arrest-enhanced variants.

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution.

Charge-driven dynamics of nascent-chain movement through the SecYEG translocon.

On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon.

Weak pulling forces exerted on Nin-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli.

Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (Nin), or facing the non-cytoplasmic side (Nout). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of Nin- and Nout-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion.

A biphasic pulling force acts on transmembrane helices during translocon-mediated membrane integration.

Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane α-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon.

The membrane stress response buffers lethal effects of lipid disequilibrium by reprogramming the protein homeostasis network.

Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood.

The unfolded protein response supports cellular robustness as a broad-spectrum compensatory pathway.

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the unfolded protein response (UPR), is responsible for maintaining endoplasmic reticulum (ER) homeostasis. The highly conserved Ire1 branch regulates hundreds of gene targets by activating a UPR-specific transcription factor.

Specific transmembrane segments are selectively delayed at the ER translocon during opsin biogenesis.

A site-specific cross-linking approach was used to study the integration of TM (transmembrane) segments 4-7 of the polytopic membrane protein, opsin, at the ER (endoplasmic reticulum). We found that although TM4 exits the ER translocon rapidly, TM segments 5, 6 and 7 are all retained at the translocon until opsin biosynthesis is terminated. Furthermore, although artificial extension of the nascent chain is not sufficient to release the C-terminal region of opsin from the translocon, substitution of the native TM segment 7 with a more hydrophobic TM segment results in its rapid lateral exit into the lipid bilayer.

Have you HRD? Understanding ERAD is DOAble!

Numerous factors are involved in the eradication of misfolded proteins, yet how these factors achieve substrate specificity remains unclear. In this issue of Cell, Denic et al. (2006) and Carvalho et al.

Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon.

We used a site-specific crosslinking approach to study the membrane integration of the polytopic protein opsin at the endoplasmic reticulum. We show that transmembrane domain 1 occupies two distinct Sec61-based environments during its integration. However, transmembrane domains 2 and 3 exit the Sec61 translocon more rapidly in a process that suggests a displacement model for their integration where the biosynthesis of one transmembrane domain would facilitate the exit of another.

Ribophorin I associates with a subset of membrane proteins after their integration at the sec61 translocon.

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide.