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Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against the physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC – aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with a recruited chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.

SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrelOuter-MembraneProteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperonese.g.SurA, which prevent aggregation. OMPs are then offloaded to the β-BarrelAssemblyMachinery (BAM) in the outer-membrane for insertion and folding. We show theHolo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changesviaSecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.

SummaryMany bacteria express flexible protein filaments on their surface that enable a variety of important cellular functions. Type IV pili are examples of such filaments and are comprised of a helical assembly of repeating pilin subunits. Type IV pili are involved in motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). They are therefore powerful structures that enable bacterial proliferation and genetic adaptation, potentially leading to the development of pathogenicity and antibiotic resistance. They are also targets for drug development.By a complement of experimental approaches, we show that the bacteriumThermus thermophilusproduces two different forms of type IV pilus. We have determined the structures of both and built atomic models. The structures answer key unresolved questions regarding the molecular architecture of type IV pili and identify a new type of pilin. We also delineate the roles of the two filaments in promoting twitching and natural transformation.

Viruses have developed intricate molecular machines to infect, replicate within and escape from their host cells. Perhaps one of the most intriguing of these mechanisms is the pyramidal egress structure that has evolved in archaeal viruses, such as SIRV2 or STIV1. The structure and mechanism of these virus-associated pyramids (VAPs) has been studied by cryo-electron tomography and complementary biochemical techniques, revealing that VAPs are formed by multiple copies of a virus-encoded 10-kDa protein (PVAP) that integrate into the cell membrane and assemble into hollow, sevenfold symmetric pyramids. In this process, growing VAPs puncture the protective surface layer and ultimately open to release newly replicated viral particles into the surrounding medium. PVAP has the striking capability to spontaneously integrate and self-assemble into VAPs in biological membranes of the archaea, bacteria and eukaryotes. This renders the VAP a universal membrane remodelling system. In this review, we provide an overview of the VAP structure and assembly mechanism and discuss the possible use of VAPs in nano-biotechnology.

To truly understand bioenergetic processes such as ATP synthesis, membrane-bound substrate transport or flagellar rotation, systems need to be analysed in a cellular context. Cryo-ET (cryo-electron tomography) is an essential part of this process, as it is currently the only technique which can directly determine the spatial organization of proteins at the level of both the cell and the individual protein complexes. The need to assess bioenergetic processes at a cellular level is becoming more and more apparent with the increasing interest in mitochondrial diseases. In recent years, cryo-ET has contributed significantly to our understanding of the molecular organization of mitochondria and chloroplasts. The present mini-review first describes the technique of cryo-ET and then discusses its role in membrane bioenergetics specifically in chloroplasts and mitochondrial research.

The majority of outer membrane proteins (OMPs) from Gram-negative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport–associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

For more than half a century, electron microscopy has been a main tool for investigating the complex ultrastructure and organization of chloroplast thylakoid membranes, but, even today, the three-dimensional relationship between stroma and grana thylakoids, and the arrangement of the membrane protein complexes within them are not fully understood. Electron cryo-tomography (cryo-ET) is a powerful new technique for visualizing cellular structures, especially membranes, in three dimensions. By this technique, large membrane protein complexes, such as the photosystem II supercomplex or the chloroplast ATP synthase, can be visualized directly in the thylakoid membrane at molecular (4-5 nm) resolution. This short review compares recent advances by cryo-ET of plant thylakoid membranes with earlier results obtained by conventional electron microscopy.

Cyclophilin D (CYPD) is a mitochondrial peptidyl prolyl-cis,trans-isomerase involved in opening of the mitochondrial permeability transition pore (mPTP). CYPD abundance increases during aging in mammalian tissues and in the aging model organism Podospora anserina. Here, we show that treatment of the P. anserina wild-type with low concentrations of the cyclophilin inhibitor cyclosporin A (CSA) extends lifespan. Transgenic strains overexpressing PaCypD are characterized by reduced stress tolerance, suffer from pronounced mitochondrial dysfunction and are characterized by accelerated aging and induction of cell death. Treatment with CSA leads to correction of mitochondrial function and lifespan to that of the wild-type. In contrast, PaCypD deletion strains are not affected by CSA within the investigated concentration range and show increased resistance against inducers of oxidative stress and cell death. Our data provide a mechanistic link between programmed cell death (PCD) and organismal aging and bear implications for the potential use of CSA to intervene into biologic aging.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

International audience; Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Aging is one of the most fundamental, yet least understood biological processes that affect all forms of eukaryotic life. Mitochondria are intimately involved in aging, but the underlying molecular mechanisms are largely unknown. Electron cryotomography of whole mitochondria from the aging model organism Podospora anserina revealed profound age-dependent changes in membrane architecture. With increasing age, the typical cristae disappear and the inner membrane vesiculates. The ATP synthase dimers that form rows at the cristae tips dissociate into monomers in inner-membrane vesicles, and the membrane curvature at the ATP synthase inverts. Dissociation of the ATP synthase dimer may involve the peptidyl prolyl isomerase cyclophilin D. Finally, the outer membrane ruptures near large contact-site complexes, releasing apoptogens into the cytoplasm. Inner-membrane vesiculation and dissociation of ATP synthase dimers would impair the ability of mitochondria to supply the cell with sufficient ATP to maintain essential cellular functions.

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.