Nir Fluman

Integral membrane proteins comprise at least a quarter of every proteome. To fold and function properly, these proteins must insert into the correct membrane with the proper topology and orientation. Although their transmembrane helices are chemically compatible with the bilayer, insertion inside cells is not a simple spontaneous event. It must occur in a crowded environment, at the correct membrane, and it often involves challenges such as transferring hydrophilic segments across the bilayer or accommodating helices that are only marginally compatible with it. Cells therefore rely on dedicated systems that direct membrane proteins to the membrane, mediate their insertion, and monitor the process to ensure that it occurs correctly. This review outlines the principles that govern membrane protein insertion in cells and explains how transmembrane sequence features interact with the machineries that mediate their entry into the bilayer. We highlight the major insertion systems of bacteria and eukaryotes and the auxiliary factors that support them, and describe how these pathways accommodate the broad range of membrane protein architectures found in cells, from single-pass proteins to complex multispanning transporters. We also discuss how cells maintain accuracy when insertion fails, through mechanisms that detect and resolve misinsertion. Together, these concepts present membrane protein insertion as a coordinated, adaptable, and safeguarded process, shaped by the interplay between sequence properties, membrane environments, and the machinery responsible for building the membrane proteome.

The biogenesis of membrane proteins (MPs) is inherently error-prone, and is therefore monitored by quality control mechanisms that remove faulty MPs. A key challenge for this surveillance is to recognize misfolded MPs, but how this is achieved remains poorly understood. Here we reveal how FtsH, the main MP quality control protease in Escherichia coli , specifically targets faulty MPs. By analyzing the in vivo degradation of two substrates, we show that lipid-facing polar residues trigger FtsH-mediated degradation. In folded MPs, such polar residues are usually buried in the protein core. Their exposure to the membrane can therefore signal misfolding and promote degradation. Strikingly, lipid-facing polar residues can even trigger degradation of a folded protein, and do not require the extended cytosolic regions typically needed for other FtsH substrates. Recognition depends on the FtsH transmembrane domain and on specific polar residues within it. Thus, sensing misfolding within the membrane helps maintain the integrity of the membrane proteome.

The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of the C-terminal tails of these proteins to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in both E. coli and human, leading to protein defects, loss of function, and genetic diseases. In E. coli, we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.

The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of these proteins’ C-terminal tails to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in both E. coli and human, leading to protein defects, loss of function, and genetic diseases. In E. coli , we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.

Membrane proteins (MPs) navigate challenging biogenesis. Errors in this process are rigorously surveilled by cellular quality control to eliminate faulty MPs. The first critical challenge of this surveillance is the accurate recognition of misfolded proteins. However, how this recognition is achieved for MPs remains poorly defined. Here we reveal the specificity mechanism of FtsH, the major quality control protease clearing faulty MPs in Escherichia coli . Analyzing the in vivo degradation of two substrates, we show that lipid-facing polar residues direct substrates to FtsH-mediated degradation. Such polar residues are typically buried in the structural cores of folded MPs, and their exposure to the membrane may thus signify misfolding and flag proteins for degradation. Remarkably, lipid-facing polar residues are sufficient for recognition and can target even folded MPs for degradation. The recognition depends on the FtsH transmembrane domain. Thus, MP misfolding is sensed within the membrane to maintain a healthy membrane proteome.

Helical membrane proteins are typically assumed to attain stable transmembrane topologies immediately upon co-translational membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transmembrane helix flips in and out of the membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, membrane topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.

Helical membrane proteins constitute roughly a quarter of all proteomes and perform diverse biological functions. To avoid aggregation, they undergo cotranslational membrane insertion and are typically assumed to attain stable transmembrane topologies immediately upon insertion. To what extent post-translational changes in topology are possible in-vivo and how they may affect biogenesis is incompletely understood. Here, we show that monomeric forms of Small Multidrug Resistance (SMR) proteins display topological dynamics, where the N-terminal transmembrane helix equilibrates between membrane-inserted and non-inserted states. We characterize the kinetics of the process and show how the composition of the helix regulates the topological dynamics. We further show that topological dynamics is a property of the unassembled monomeric protein, as the N-terminal helix becomes fixed in a transmembrane disposition upon dimerization. Membrane protein topology can thus remain dynamic long after cotranslational membrane insertion, and can be regulated by later assembly processes.

Significance: The orientations of multispanning membrane proteins in the membrane are initially determined when they are cotranslationally inserted into the membrane. However, it is not always clear whether these initial orientations may be dynamic after the insertion step or not. The present study shows that even in the case of small membrane proteins that have only four transmembrane helices and have little bias to be inserted in one or the other orientation, the initially inserted orientation remains stable and is not dynamic, at least with respect to the orientation of the entire protein. This finding suggests that membrane proteins become correctly oriented already during or immediately after synthesis, as they are inserted into the membrane.

The topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on anti-parallel homo-or hetero-dimeric Small Multidrug Resistance proteins, and examine whether the individual monomers can undergo reversible topological inversion (flip-flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane topology of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final topologies independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo .

In all living organisms, ribosomes translating membrane proteins are targeted to membrane translocons early in translation, by the ubiquitous signal recognition particle (SRP) system. In eukaryotes, the SRP Alu domain arrests translation elongation of membrane proteins until targeting is complete. Curiously, however, the Alu domain is lacking in most eubacteria. In this study, by analyzing genome-wide data on translation rates, we identified a potential compensatory mechanism in E. coli that serves to slow down the translation during membrane protein targeting. The underlying mechanism is likely programmed into the coding sequence, where Shine–Dalgarno-like elements trigger elongation pauses at strategic positions during the early stages of translation. We provide experimental evidence that slow translation during targeting and improves membrane protein production fidelity, as it correlates with better folding of overexpressed membrane proteins. Thus, slow elongation is important for membrane protein targeting in E. coli, which utilizes mechanisms different from the eukaryotic one to control the translation speed.