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 via a lateral gate that opens towards 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. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration. Keywords membrane protein; Sec translocon; SecM, Xbp1, translation arrest; transmembrane helix; membrane integrationIn both prokaryotic and eukaryotic cells, most membrane proteins are co-translationally inserted into the membrane with the aid of Sec-type translocons 1 . While the energetics of membrane insertion is now rather well understood 2-4 , dynamic aspects have received little attention. We reasoned that direct dynamic information on the insertion process might be obtained if local forces acting on a hydrophobic segment in the nascent polypeptide chain could be measured as a function of the segment's location in the ribosome-translocon complex. To detect such forces during co-translational integration of membrane proteins into the inner membrane of Escherichia coli and the mammalian endoplasmic reticulum (ER) membrane, we decided to explore the possible utility of so-called translation-arrest peptides 5 as natural force sensors.Arrest peptides have been identified both in prokaryotic and eukaryotic proteins. SecM is a prokaryotic periplasmic protein harboring an arrest peptide that helps regulate the expression of the co-transcribed translocation-motor protein SecA 6 . During translation of SecM, the arrest peptide causes efficient ribosome stalling by blocking the incorporation of a critical proline residue into the elongating nascent chain 7 . There is strong support for the idea that # Corresponding author. Phone: Int+46-8-16 25 90. Fax: Int+46-8-15 36 79. gunnar@dbb.su.se.

SUMMARY 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. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the “membrane stress response” (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity—functions otherwise lethally compromised in lipid dysregulated cells.

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, which 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 co-translational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P-site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential while the second may reflect interactions between charged residues and the periplasmic membrane surface.

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the bestcharacterized 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. To understand how the UPR manages ER stress, a unique genetic approach was applied to reveal how the system corrects disequilibria. The data show that the UPR can address a wide range of dysfunctions that are otherwise lethal if not for its intervention. Transcriptional profiling of stressalleviated cells shows that the program can be modulated, not just in signal amplitude, but also through differential target gene expression depending on the stress. The breadth of the functions mitigated by the UPR further supports its role as a major mechanism maintaining systems robustness.chaperones | signal transduction | protein folding | protein degradation | glycosylation