Insertion Of Membrane Proteins Research Articles

Deciphering the Interdomain Coupling in a Gram-Negative Bacterial Membrane Insertase.

YidC is a membrane protein that plays an important role in inserting newly generated proteins into lipid membranes. The Sec-dependent complex is responsible for inserting proteins into the lipid bilayer in bacteria. YidC facilitates the insertion and folding of membrane proteins, both in conjunction with the Sec complex and independently. Additionally, YidC acts as a chaperone during the folding of proteins. Multiple investigations have conclusively shown that Gram-positive bacterial YidC has Sec-independent insertion mechanisms. Through the use of microsecond-level all-atom molecular dynamics (MD) simulations, we have carried out an in-depth investigation of the YidC protein originating from Gram-negative bacteria. This research sheds light on the significance of multiple domains of the YidC structure at a detailed molecular level by utilizing equilibrium MD simulations. Specifically, multiple models of YidC embedded in the lipid bilayer were constructed to characterize the critical role of the C2 loop and the periplasmic domain (PD) present in Gram-negative YidC, which is absent in its Gram-positive counterpart. Based on our results, the C2 loop plays a role in the overall stabilization of the protein, most notably in the transmembrane (TM) region, and it also has an allosteric influence on the PD region. We have found critical inter- and intradomain interactions that contribute to the stability of the protein and its function. Finally, our study provides a hypothetical Sec-independent insertion mechanism for Gram-negative bacterial YidC.

The Dsc complex and its role in Golgi quality control.

Membrane proteins play crucial roles in cellular functions. However, processes such as the insertion of membrane proteins into the endoplasmic reticulum (ER), their folding into native structures, the assembly of multi-subunit membrane protein complexes, and their targeting from the ER to specific organelles are prone to errors and have a relatively high failure rate. To prevent the accumulation of defective or orphaned membrane proteins, quality control mechanisms assess folding, quantity, and localization of these proteins. This quality control is vital for preserving organelle integrity and maintaining cellular health. In this mini-review, we will focus on how selective membrane protein quality control at the Golgi apparatus, particularly through the defective for SREBP cleavage (Dsc) ubiquitin ligase complex, detects orphaned proteins and prevents their mis-localization to other organelles.

Outer membrane protein assembly mediated by BAM-SurA complexes

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Exploring the Darobactin Class of Antibiotics: A Comprehensive Review from Discovery to Recent Advancements.

The prevalence of antimicrobial resistance in Gram-negative bacteria poses a greater challenge due to their intrinsic resistance to many antibiotics. Recently, darobactins have emerged as a novel class of antibiotics originating from previously unexplored Gram-negative bacterial species such as Photorhabdus, Vibrio, Pseudoalteromonas and Yersinia. Darobactins belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) class of antibiotics, exhibiting selective activity against Gram-negative bacteria. They target the β-barrel assembly machinery (BAM), which is crucial for the maturation and insertion of outer membrane proteins in Gram-negative bacteria. The dar operon in the producer's genome encodes for the synthesis of darobactins, which are characterized by a fused ring system connected via an alkyl-aryl ether linkage (C-O-C) and a C-C cross-link. The enzyme DarE, using the radical S-adenosyl-l-methionine (rSAM), facilitates the formation of these bonds. Biosynthetic manipulation of the darobactin gene cluster, along with its expression in a surrogate host, has enabled access to diverse darobactin analogues with variable antibiotic activities. Recently, two independent research groups successfully achieved the total synthesis of darobactin, employing Larock heteroannulation to construct the bicyclic structure. This paper presents a comprehensive review of darobactins, encompassing their discovery through to the most recent advancements.

Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

The SNX17-Retriever endosomal recycling pathway is a key regulator of synaptic function and plasticity in hippocampal neurons

The insertion and removal of integral membrane proteins is a key mechanism for the regulation of synaptic function. Once surface proteins are internalized, they can either be routed to lysosomes for degradation or can be recycled back to the cell surface. Two recycling protein complexes - the SNX27-Retromer complex and the recently discovered SNX17-Retriever complex - are the main drivers in recycling internalized cargoes in non-neuronal cells. In neurons, the SNX27-Retromer pathway has been shown to regulate surface expression of AMPA-type glutamate receptors (AMPARs) during long-term potentiation (LTP), but almost nothing is known regarding the role of the SNX17-Retriever pathway at synapses. Here, using cultured hippocampal neurons, we demonstrate that the SNX17-Retriever pathway localizes to both synaptic and extra-synaptic sites and that RNAi-mediated disruption of this pathway leads to the functional and structural loss of excitatory synapses. Moreover, we find that synaptic stimulation leads to the recruitment of SNX17 to synaptic sites in a manner that depends on Ca2+ influx through NMDARs, activation of CaMKII, and Retriever complex binding. Knockdown of either SNX17 or the Retriever subunit DSCR3 prevents structural plasticity of dendritic spines during LTP. Finally, this loss of structural plasticity is associated with altered traffcking of the cell adhesion molecule β1 Integrin, which is a SNX17 cargo in non-neuronal cells and as we show, in neurons as well. Additionally, SNX17-Retriever is shown to not play a role in AMPAR recycling. Preliminary studies suggest that SNX27 and SNX17 sort distinct cargos in neurons, suggesting that these two recycling pathways each play important, but unique, roles at synaptic sites. National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) under award numbers R01-NS129198 to L.S. Weisman and M.A. Sutton, R01-NS099340 and R01- NS064015 to L.S. Weisman, R01-NS097498 to M.A. Sutton, and R21-NS125449, and R01-NS116008 to S. Iwase, by the National Institute of Mental Health (NIMH) of the NIH under award number R21-MH127485 to S. Iwase. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Experimental and computational approaches for membrane protein insertion and topology determination

Membrane proteins play pivotal roles in a wide array of cellular processes and constitute approximately a quarter of the protein-coding genes across all organisms. Despite their ubiquity and biological significance, our understanding of these proteins remains notably less comprehensive compared to their soluble counterparts. This disparity in knowledge can be attributed, in part, to the inherent challenges associated with employing specialized techniques for the investigation of membrane protein insertion and topology. This review will center on a discussion of molecular biology methodologies and computational prediction tools designed to elucidate the insertion and topology of helical membrane proteins.

B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Roles of a Glycolipid MPIase in Sec-Independent Membrane Protein Insertion.

Membrane protein integrase (MPIase), an endogenous glycolipid in Escherichia coli (E. coli) membranes, is essential for membrane protein insertion in E. coli. We have examined Sec-independent membrane protein insertion mechanisms facilitated by MPIase using physicochemical analytical techniques, namely solid-state nuclear magnetic resonance, fluorescence measurements, and surface plasmon resonance. In this review, we outline the physicochemical characteristics of membranes that may affect membrane insertion of proteins. Subsequently, we introduce our results verifying the effects of membrane lipids on insertion and estimate the impact of MPIase. Although MPIase is a minor component of E. coli membranes, it regulates insertion by altering the physicochemical properties of the membrane. In addition, MPIase promotes insertion by interacting with substrate proteins. We propose comprehensive mechanisms for the membrane insertion of proteins involving MPIase, which provide a physicochemical basis for understanding the roles of glycolipids in protein translocation.

Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response.

The endoplasmic reticulum (ER) is the key organelle for membrane biogenesis. Most lipids are synthesized in the ER, and most membrane proteins are first inserted into the ER membrane before they are transported to their target organelle. The composition and properties of the ER membrane must be carefully controlled to provide a suitable environment for the insertion and folding of membrane proteins. The unfolded protein response (UPR) is a powerful signaling pathway that balances protein and lipid production in the ER. Here, we summarize our current knowledge of how aberrant compositions of the ER membrane, referred to as lipid bilayer stress, trigger the UPR.

The contribution of mRNA targeting to spatial protein localization in bacteria.

About 30% of all bacterial proteins execute their function outside of the cytosol and must be inserted into or translocated across the cytoplasmic membrane. This requires efficient targeting systems that recognize N-terminal signal sequences in client proteins and deliver them to protein transport complexes in the membrane. While the importance of these protein transport machineries for the spatial organization of the bacterial cell is well documented in multiple studies, the contribution of mRNA targeting and localized translation to protein transport is only beginning to emerge. mRNAs can exhibit diverse subcellular localizations in the bacterial cell and can accumulate at sites where new protein is required. This is frequently observed for mRNAs encoding membrane proteins, but the physiological importance of membrane enrichment of mRNAs and the consequences it has for the insertion of the encoded protein have not been explored in detail. Here, we briefly highlight some basic concepts of signal sequence-based protein targeting and describe in more detail strategies that enable the monitoring of mRNA localization in bacterial cells and potential mechanisms that route mRNAs to particular positions within the cell. Finally, we summarize some recent developments that demonstrate that mRNA targeting and localized translation can sustain membrane protein insertion under stress conditions when the protein-targeting machinery is compromised. Thus, mRNA targeting likely acts as a back-up strategy and complements the canonical signal sequence-based protein targeting.

YajC, a predicted membrane protein, promotes Enterococcus faecium biofilm formation in vitro and in a rat endocarditis model.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed ΔyajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium, we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Modular Assembly of Mitochondrial β-Barrel Proteins.

Mitochondrial β-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All β-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The β-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of β-barrel proteins, the folding of the β-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial β-barrel proteins.

The intricate link between membrane lipid structure and composition and membrane structural properties in bacterial membranes.

It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.

In Vitro Reconstruction of Bacterial β-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of β-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli(E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented β-barrel membrane proteins via intact β-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled β-barrel membrane proteins by demonstrating four representative β-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Stress-Based Screening for Compounds That Inhibit β-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of β-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.

Phospholipid dependency of membrane protein insertion by the Sec translocon

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli.

Quantitative Mass Spectrometry Characterizes Client Spectra of Components for Targeting of Membrane Proteins to and Their Insertion into the Membrane of the Human ER.

To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.