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Abstract Gram-negative bacteria assemble many proteins into the inner and outer membranes and export a large number of proteins to the periplasm or to the extracellular medium. During the billions of years bacteria have been around, they have evolved a number of different pathways with sophisticated machines to accurately and efficiently move proteins from one location to another. In this review, we first introduce specific proteins that are representative substrates of the protein transport pathways and describe their function. Then, their specific routes from synthesis to their destinations are described mentioning the signal peptide that may initiate their export and discuss what is known about the folding state of the substrates during transport. The membrane translocation device involved, the energy source required for transport, and whether a chaperone is needed will be discussed. In this review, we summarize the current knowledge by which proteins are exported, secreted and assembled into membranes in Gram-negative bacteria.

Antimicrobial peptides (AMPs), as a key component of the immune defense systems of organisms, are a promising solution to the serious threat of drug-resistant bacteria to public health. As one of the most representative and extensively studied AMPs, melittin has exceptional broad-spectrum activities against microorganisms, including both Gram-positive and Gram-negative bacteria. Unfortunately, the action mechanism of melittin with bacterial membranes, especially the underlying physics of peptide-induced membrane poration behaviors, is still poorly understood, which hampers efforts to develop melittin-based drugs or agents for clinical applications. In this mini-review, we focus on recent advances with respect to the membrane insertion behavior of melittin mostly from a computational aspect. Membrane insertion is a prerequisite and key step for forming transmembrane pores and bacterial killing by melittin, whose occurrence is based on overcoming a high free-energy barrier during the transition of melittin molecules from a membrane surface-binding state to a transmembrane-inserting state. Here, intriguing simulation results on such transition are highlighted from both kinetic and thermodynamic aspects. The conformational changes and inter-peptide cooperation of melittin molecules, as well as melittin-induced disturbances to membrane structure, such as deformation and lipid extraction, are regarded as key factors influencing the insertion of peptides into membranes. The associated intermediate states in peptide conformations, lipid arrangements, membrane structure, and mechanical properties during this process are specifically discussed. Finally, potential strategies for enhancing the poration ability and improving the antimicrobial performance of AMPs are included as well.

The most simple membrane protein insertion catalyst known so far is the universal YidC/Oxa/Alb insertase that is composed of a single multi-spanning protein present in archaea, bacteria and in eukaryotic organelles. In bacteria, YidC is known to integrate small membrane proteins on its own and more complex proteins in conjunction with the SecYEG translocase. In mitochondria, the YidC homologue Oxa is responsible for the insertion of all membrane proteins synthesized in the matrix since no Sec homologues are present in the mitochondrial inner membrane. This is tantamount to the observation that YidC is able to operate also independently of SecYEG in bacteria. Reconstituted into liposomes, YidC rapidly and efficiently binds to substrate proteins and leads to their integration into the bilayer. Additionally, single molecule force spectroscopy experiments show that YidC binds to unfolded membrane proteins and promotes their folding into the membrane bilayer. To achieve membrane insertion and the correct folding, the periplasmic regions of the substrate have to cross the membrane with the help of YidC by a mechanism that is presently explored.

Rhamnolipids (RLs) are potential biocontrol agents for crop culture protection. Their mode of action has been proposed as dual, combining plant protection activation and antifungal activities. The present work focuses on the interaction of natural RLs with plant and fungi membrane models at the molecular scale. Representative models were constructed and the interaction with RLs was studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (2H NMR) spectroscopic measurements. Molecular dynamic (MD) simulations were performed to investigate RL insertion in lipid bilayers. Our results showed that the RLs fit into the membrane models and were located near the lipid phosphate group of the phospholipid bilayers, nearby phospholipid glycerol backbones. The results obtained with plant plasma membrane models suggest that the insertion of RLs inside the lipid bilayer did not significantly affect lipid dynamics. Oppositely, a clear fluidity increase of fungi membrane models was observed. This effect was related to the presence and the specific structure of ergosterol. The nature of the phytosterols could also influence the RL effect on plant plasma membrane destabilization. Subtle changes in lipid dynamics could then be linked with plant defense induction and the more drastic effects associated with fungal membrane destabilization.

The applications of the pH low insertion peptide (pHLIP) in cancer diagnosis and cross-membrane cargo delivery have drawn increasing attention in the past decade. With its origin as the transmembrane (TM) helix C of bacteriorhodopsin, pHLIP is also an important model for understanding how pH can affect the folding and topogenesis of a TMα-helix. Protonations of multiple D/E residues transform pHLIP from an unstructured coil at membrane surface (known as state II, at pH ≥ 7) to a TM α-helix (state III, pH ≤ 5.3). While these initial and end states of pHLIP insertion have been firmly established, what happens at the intervening pH values is less clear. However, the intervening pH range is most relevant to pHLIP−cell interactions in the acidic extracellular tumor environment (and in the endosomes within cells). Here, using advanced solid-state NMR spectroscopy with palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine unilamellar vesicles as the model membrane, we systematically examined the state of pHLIP−membrane interactions (in terms of the membrane locations of D/E residues, as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate states distinct from the initial and end states were discovered to exist at each of the intervening pH examined. They support a multistage model of pHLIP insertion in which the D/E titrations occur in a defined sequence at distinct intermediate pH values. This multistage model has important ramifications in pHLIP applications.

Classical dynamins are best understood for their ability to generate vesicles by membrane fission. During clathrin-mediated endocytosis (CME), dynamin is recruited to the membrane through multivalent protein and lipid interactions between its proline-rich domain (PRD) with SRC Homology 3 (SH3) domains in endocytic proteins and its pleckstrin-homology domain (PHD) with membrane lipids. Variable loops (VL) in the PHD bind lipids and partially insert into the membrane thereby anchoring the PHD to the membrane. Recent molecular dynamics (MD) simulations reveal a novel VL4 that interacts with the membrane. Importantly, a missense mutation that reduces VL4 hydrophobicity is linked to an autosomal dominant form of Charcot-Marie-Tooth (CMT) neuropathy. We analyzed the orientation and function of the VL4 to mechanistically link data from simulations with the CMT neuropathy. Structural modeling of PHDs in the cryo-electron microscopy (cryo-EM) cryoEM map of the membrane-bound dynamin polymer confirms VL4 as a membrane-interacting loop. In assays that rely solely on lipid-based membrane recruitment, VL4 mutants with reduced hydrophobicity showed an acute membrane curvature-dependent binding and a catalytic defect in fission. Remarkably, in assays that mimic a physiological multivalent lipid- and protein-based recruitment, VL4 mutants were completely defective in fission across a range of membrane curvatures. Importantly, expression of these mutants in cells inhibited CME, consistent with the autosomal dominant phenotype associated with the CMT neuropathy. Together, our results emphasize the significance of finely tuned lipid and protein interactions for efficient dynamin function.

Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent–membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

The current best membrane-protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of membrane proteins. However, because the insertion of transmembrane helices into the membrane is the outcome of molecular interactions among protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of membrane-protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.

BAR (Bin/Amphiphysin/Rvs) domains and amphipathic α‐helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent‐shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed that membrane curvature sensing critically depends on the N‐terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains emerge as an important means for a protein to sense membrane curvature. Measurements on single liposomes allowed us to document heterogeneous binding behaviour within the ensemble and quantify the influence of liposome polydispersity on bulk membrane curvature sensing experiments. The latter results suggest that bulk liposome‐binding experiments should be interpreted with great caution.

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.