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Newly synthesized proteins are targeted to the SecY protein-conducting channel for translocation across the membrane; here, cryo-electron microscopy structures of inactive and active ribosome–channel complexes are presented, revealing that ribosome binding does not result in major structural changes to transmembrane regions of the channel, and that stable channel opening requires loop insertion of the translocating nascent chain. Protein translocation channel structures Newly synthesized proteins are targeted to the SecY/Sec61 protein conducting channel for translocation across the membrane. Two papers in this issue of Nature use cryo-electron microscopy to provide insights into the mechanisms of this important protein translocation process. Eunyong Park et al . present the structures of inactive and active bacterial ribosome-channel complexes, and Marko Gogala et al . those of a mammalian ribosome-channel complex in closed and partially open configurations. The structures reveal that ribosome binding does not cause major structural changes in the channel. Instead, membrane insertion of a hydrophobic domain of the nascent protein seems to help open part of the channel while ensuring that the channel stays sealed for ion flow. Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis 1 , 2 . Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase 3 , 4 , 5 . The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways. Protein complexes at single-molecule resolution Analysis of protein interactions is crucial for understanding cellular function and regulation. Here, Taekjip Ha and colleagues develop a novel method for elucidating the identity and stoichiometry of protein complexes from cells and tissues at single-molecule resolution. The method, called single molecule pull-down or SiMPull, can discriminate between multiple association states of a protein, and simultaneously allows determination of complex stoichiometry through photobleaching step analysis. The potential of the assay is demonstrated in a variety of contexts, including endogenous proteins from tissue extracts, organelles and membrane proteins.

Histone-modification by the Polycomb system Polycomb group (PcG) proteins are transcriptional repressors that modify chromatin and regulate important developmental genes. One chromatin-modifying activity associated with Polycomb is an enzyme that ubiquitinates histone H2A. Here, Scheuermann et al . find a Drosophila PcG complex with H2A deubiquitination activity that is important for gene repression in vivo . Polycomb gene silencing may thus involve a dynamic balance between H2A ubiquitination and deubiquitination. Polycomb group (PcG) proteins are transcriptional repressors that modify chromatin and regulate important developmental genes. One PcG-associated, chromatin-modifying activity is an enzyme that ubiquitinates histone H2A of chromatin. Here, a fruitfly PcG complex that is associated with H2A deubiquitination, and thereby with gene repression, is identified. PcG-mediated gene silencing might thus involve a dynamic balance between ubiquitination and deubiquitination of H2A. Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants 1 , 2 , 3 , 4 , 5 , 6 . In Drosophila , genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive 1 , 7 , 8 . Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes 1 , 2 , 3 , 4 . Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1–4 ). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila . Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo . Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

Membrane fusion within the eukaryotic endomembrane system depends on the initial recognition of Rab GTPase on transport vesicles by multisubunit tethering complexes and subsequent coupling to SNARE-mediated fusion. The conserved vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) tethering complex combines both activities. Here we present the overall structure of the fusion-active HOPS complex. Our data reveal a flexible ≈30-nm elongated seahorse-like structure, which can adopt contracted and elongated shapes. Surprisingly, both ends of the HOPS complex contain a Rab-binding subunit: Vps41 and Vps39. The large head contains in addition to Vps41 the SNARE-interacting Vps33, whereas Vps39 is found in the bulky tip of its tail. Vps11 and Vps18 connect head and tail. Our data suggest that HOPS bridges Ypt7-positive membranes and chaperones SNAREs at fusion sites.

Xkr8–Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8–Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8–Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling. Cryo-EM and X-ray crystal structures reveal the architecture of the human Xkr8–Basigin complex, providing insights into the molecular mechanism of phospholipid scrambling.

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium. RNA binding proteins are important regulators of RNA function. Here the authors describe a method for isolation of RNA-protein complexes that does not rely on a specific RNA sequence or motif, and demonstrate the approach by providing the global RNA-bound proteomes of human HEK293 cells and Salmonella Typhimurium.

Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation–mass spectrometry, CF–MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF–MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container (https://hub.docker.com/r/baderlab/bio-epic/).

Key Points In this article, we review the current status of affinity purification and mass spectrometry (AP–MS) and its promise for better understanding protein complexes, complex structure and the dynamics of complex formation. We describe the general AP–MS strategy, with an emphasis on generic approaches (flag-tag, tandem AP) and how AP–MS of multiple components (that is, high-density AP–MS) can help to reveal the true composition of protein complexes. Recent high-throughput studies with flag-tagging or tandem AP significantly improved our understanding of protein–protein interactions in yeast. AP–MS can be combined with classical biochemical purification approaches to reveal complex composition and to resolve the problem of mutually exclusive complexes co-precipitating with the same tagged protein. Crosslinkers can contribute to AP–MS strategies by stabilizing weak or transient protein interactions and by revealing details concerning complex organization and interacting surfaces. Stoichiometry of protein complexes can be obtained using intact-complex mass measurement and absolute quantitative proteomics tools. Quantitative proteomics approaches can help to decipher the dynamics of protein-complex formation. The combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of protein complexes and large protein-interaction networks. Emerging AP–MS approaches promise a better understanding of protein-complex stoichiometry, structural organization and the dynamics of protein-complex composition. The versatile combination of affinity purification and mass spectrometry (AP–MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP–MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP–MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein–complex assembly.

Photosynthesis: a supercomplex of supercomplexes During photosynthesis, light energy is utilized by photosystems 1 (PSI) and II (PSII), located in the thylakoid membranes of chloroplasts, to establish an electron flow that ultimately results in the production of ATP and NADPH. Two modes of electron flow exist — a linear electron flow and a cyclic electron flow. The latter pathway generates more ATP but the molecular components of the supercomplex involved in the process have remained elusive. This issue is now addressed directly in the green alga Chlamydomonas reinhardtii . A combination of biochemical and spectroscopic techniques reveals the supercomplex that drives cyclic electron flow to be made up not only of the photosystem/peripheral antenna supercomplex, but also of two known redox proteins — cytochrome b 6 f complex and ferredoxin-NADPH oxidoreductase (FNR) — with a few small proteins as well. During photosynthesis, light energy is used by photosystems I and II to establish electron flow, which ultimately results in the production of ATP and NADPH. Two modes of electron flow exist, a linear electron flow and a cyclic electron flow (CEF). The latter pathway generates more ATP, but its molecular components have been elusive. Here, a combination of biochemical and spectroscopic techniques has been used to identify the supercomplex that drives CEF in the green alga Chlamydomonas reinhardtii . Photosynthetic light reactions establish electron flow in the chloroplast’s thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist—linear electron flow (LEF) from water to NADP + via photosystem (PS) II and PSI in series 1 and cyclic electron flow (CEF) around PSI (ref. 2 ). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii , the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3 ), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions 4 ). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b 6 f complex (Cyt bf ), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5 ) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf , whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf , indicating that this supercomplex is engaged in CEF ( Supplementary Fig. 1 ). Thus, formation and dissociation of the PSI–LHCI–LHCII–FNR–Cyt bf –PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

Essence of E. coli Proteomic analysis of native protein–protein interactions in E. coli combined with protein mass spectrometry has revealed an interaction network consisting of the proteins essential to bacterial life. The network is highly conserved, providing insight into core bacterial processes, the nature of evolutionary constraints, and suitable new antimicrobial drug targets. Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism 1 , no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (∼ 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged successfully, whereas 648 could be purified to homogeneity and their interacting protein partners identified by mass spectrometry. An interaction network of protein complexes involved in diverse biological processes was uncovered and validated by sequential rounds of tagging and purification. This network includes many new interactions as well as interactions predicted based solely on genomic inference or limited phenotypic data 2 . This study provides insight into the function of previously uncharacterized bacterial proteins and the overall topology of a microbial interaction network, the core components of which are broadly conserved across Prokaryota.