Multivalent interactions facilitate motor-dependent protein accumulation at growing microtubule plus-ends.

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

Reconstitution of Ultrawide DNA Origami Pores in Liposomes for Transmembrane Transport of Macromolecules.

Molecular traffic across lipid membranes is a vital process in cell biology that involves specialized biological pores with a great variety of pore diameters, from fractions of a nanometer to >30 nm. Creating artificial membrane pores covering similar size and complexity will aid the understanding of transmembrane molecular transport in cells, while artificial pores are also a necessary ingredient for synthetic cells. Here, we report the construction of DNA origami nanopores that have an inner diameter as large as 30 nm. We developed methods to successfully insert these ultrawide pores into the lipid membrane of giant unilamellar vesicles (GUVs) by administering the pores concomitantly with vesicle formation in an inverted-emulsion cDICE technique. The reconstituted pores permit the transmembrane diffusion of large macromolecules, such as folded proteins, which demonstrates the formation of large membrane-spanning open pores. The pores are size selective, as dextran molecules with a diameter up to 28 nm can traverse the pores, whereas larger dextran molecules are blocked. By FRAP measurements and modeling of the GFP influx rate, we find that up to hundreds of pores can be functionally reconstituted into a single GUV. Our technique bears great potential for applications across different fields from biomimetics, to synthetic biology, to drug delivery.

A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex.

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.

Publisher Correction: The condensin holocomplex cycles dynamically between open and collapsed states.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The condensin holocomplex cycles dynamically between open and collapsed states.

Structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open 'O' shapes and collapsed 'B' shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.

Nonspherical Coacervate Shapes in an Enzyme-Driven Active System.

Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient nonspherical shapes, and we systematically study how PNPase concentration, UDP concentration, and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of nonspherical coacervate shapes may have broader implications for the relationship between (bio)chemical activity and coacervate biology.

Structural Dynamics of the YidC:Ribosome Complex during Membrane Protein Biogenesis.

Members of the YidC/Oxa1/Alb3 family universally facilitate membrane protein biogenesis, via mechanisms that have thus far remained unclear. Here, we investigated two crucial functional aspects: the interaction of YidC with ribosome:nascent chain complexes (RNCs) and the structural dynamics of RNC-bound YidC in nanodiscs. We observed that a fully exposed nascent transmembrane domain (TMD) is required for high-affinity YidC:RNC interactions, while weaker binding may already occur at earlier stages of translation. YidC efficiently catalyzed the membrane insertion of nascent TMDs in both fluid and gel phase membranes. Cryo-electron microscopy and fluorescence analysis revealed a conformational change in YidC upon nascent chain insertion: the essential TMDs 2 and 3 of YidC were tilted, while the amphipathic helix EH1 relocated into the hydrophobic core of the membrane. We suggest that EH1 serves as a mechanical lever, facilitating a coordinated movement of YidC TMDs to trigger the release of nascent chains into the membrane.

Parallel Structural Evolution of Mitochondrial Ribosomes and OXPHOS Complexes.

The five macromolecular complexes that jointly mediate oxidative phosphorylation (OXPHOS) in mitochondria consist of many more subunits than those of bacteria, yet, it remains unclear by which evolutionary mechanism(s) these novel subunits were recruited. Even less well understood is the structural evolution of mitochondrial ribosomes (mitoribosomes): while it was long thought that their exceptionally high protein content would physically compensate for their uniquely low amount of ribosomal RNA (rRNA), this hypothesis has been refuted by structural studies. Here, we present a cryo-electron microscopy structure of the 73S mitoribosome from Neurospora crassa, together with genomic and proteomic analyses of mitoribosome composition across the eukaryotic domain. Surprisingly, our findings reveal that both structurally and compositionally, mitoribosomes have evolved very similarly to mitochondrial OXPHOS complexes via two distinct phases: A constructive phase that mainly acted early in eukaryote evolution, resulting in the recruitment of altogether approximately 75 novel subunits, and a reductive phase that acted during metazoan evolution, resulting in gradual length-reduction of mitochondrially encoded rRNAs and OXPHOS proteins. Both phases can be well explained by the accumulation of (slightly) deleterious mutations and deletions, respectively, in mitochondrially encoded rRNAs and OXPHOS proteins. We argue that the main role of the newly recruited (nuclear encoded) ribosomal- and OXPHOS proteins is to provide structural compensation to the mutationally destabilized mitochondrially encoded components. While the newly recruited proteins probably provide a selective advantage owing to their compensatory nature, and while their presence may have opened evolutionary pathways toward novel mitochondrion-specific functions, we emphasize that the initial events that resulted in their recruitment was nonadaptive in nature. Our framework is supported by population genetic studies, and it can explain the complete structural evolution of mitochondrial ribosomes and OXPHOS complexes, as well as many observed functions of individual proteins.

A structural model of the active ribosome-bound membrane protein insertase YidC.

The integration of most membrane proteins into the cytoplasmic membrane of bacteria occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or in concert with the SecY complex. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid-versus-protein-exposure and molecular dynamics simulations. The model suggests a distinctive arrangement of the conserved five transmembrane domains and a helical hairpin between transmembrane segment 2 (TM2) and TM3 on the cytoplasmic membrane surface. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure reveals how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and identifies a site for membrane protein insertion at the YidC protein-lipid interface. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.

Visualization of a polytopic membrane protein during SecY-mediated membrane insertion.

The biogenesis of polytopic membrane proteins occurs co-translationally on ribosomes that are tightly bound to a membrane-embedded protein-conducting channel: the Sec-complex. The path that is followed by nascent proteins inside the ribosome and the Sec-complex is relatively well established; however, it is not clear what the fate of the N-terminal transmembrane domains (TMDs) of polytopic membrane proteins is when the C-terminal TMDs domains are not yet synthesized. Here, we present the sub-nanometer cryo-electron microscopy structure of an in vivo generated ribosome-SecY complex that carries a membrane insertion intermediate of proteorhodopsin (PR). The structure reveals a pre-opened Sec-complex and the first two TMDs of PR already outside the SecY complex directly in front of its proposed lateral gate. Thus, our structure is in agreement with positioning of N-terminal TMDs at the periphery of SecY, and in addition, it provides clues for the molecular mechanism underlying membrane protein topogenesis.

Signal sequence-independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle.

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence-independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.

Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts.

Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.

Identification of two interaction sites in SecY that are important for the functional interaction with SecA.

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.

Stepwise evolution of the Sec machinery in Proteobacteria.

The Sec machinery facilitates the translocation of proteins across and into biological membranes. In several of the Proteobacteria, this machinery contains accessory features that are not present in any other bacterial division. The genomic distribution of these features in the context of bacterial phylogeny suggests that the Sec machinery has evolved in discrete steps. The canonical Sec machinery was initially supplemented with SecB; subsequently, SecE was extended with two transmembrane segments and, finally, SecM was introduced. The Sec machinery of Escherichia coli and other Enterobacteriales represents the end product of this stepwise evolution.

Topologically fixed SecG is fully functional.

It has been proposed that the bitopic membrane protein SecG undergoes topology inversion during translocation of (pre)proteins via SecYEG. Here we show that SecG covalently cross-linked to SecY cannot invert its topology while remaining fully functional in protein translocation. Our results strongly disfavor topology inversion of SecG during protein translocation.

Covalently dimerized SecA is functional in protein translocation.

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.

SecY-SecY and SecY-SecG contacts revealed by site-specific crosslinking.

Protein translocation across the cytoplasmic membrane of Escherichia coli is mediated by the integral membrane complex SecYEG and the peripherally bound ATPase SecA. To probe the environment of the cytoplasmic domains of SecY within the SecYEG complex, we introduced single cysteine residues in each of the six cytoplasmic domains. Neighbouring SecY molecules with a single cysteine residue in cytoplasmic domains C1, C2 or C6 formed a disulfide bond upon oxidation. The presence of the disulfide bond between two C2 domains reversibly inhibited protein translocation. Chemical crosslinking showed that the C2 and C3 domains are in close proximity of SecG and chemical modification of the cysteine residue in the C5 domain with N-ethyl-maleimide or fluorescein-5-maleimide inactivates the SecYEG complex. Taken together, our data give novel insights in the interactions between subunits of the SecYEG complex and emphasise the importance of cytoplasmic domain C5 for SecY functioning.