Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae.

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii, uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors.

Structural insights into the chloroplast protein import in land plants.

Chloroplast proteins are imported via the translocon at the outer chloroplast membrane (TOC)-translocon at the inner chloroplast membrane (TIC) supercomplex, driven by an ATPase motor. The Ycf2-FtsHi complex has been identified as the chloroplast import motor. However, its assembly and cooperation with the TIC complex during preprotein translocation remain unclear. Here, we present the structures of the Ycf2-FtsHi and TIC complexes from Arabidopsis and an ultracomplex formed between them from Pisum. The Ycf2-FtsHi structure reveals a heterohexameric AAA+ ATPase motor module with characteristic features. Four previously uncharacterized components of Ycf2-FtsHi were identified, which aid in complex assembly and anchoring of the motor module at a tilted angle relative to the membrane. When considering the structures of the TIC complex and the TIC-Ycf2-FtsHi ultracomplex together, it becomes evident that the tilted motor module of Ycf2-FtsHi enables its close contact with the TIC complex, thereby facilitating efficient preprotein translocation. Our study provides valuable structural insights into the chloroplast protein import process in land plants.

Nucleic acid-binding KH domain proteins influence a spectrum of biological pathways including as part of membrane-localized complexes.

K-Homology domain (KH domain) proteins bind single-stranded nucleic acids, influence protein-protein interactions of proteins that harbor them, and are found in all kingdoms of life. In concert with other functional protein domains KH domains contribute to a variety of critical biological activities, often within higher order machineries including membrane-localized protein complexes. Eukaryotic KH domain proteins are linked to developmental processes, morphogenesis, and growth regulation, and their aberrant expression is often associated with cancer. Prokaryotic KH domain proteins are involved in integral cellular activities including cell division and protein translocation. Eukaryotic and prokaryotic KH domains share structural features, but are differentiated based on their structural organizations. In this review, we explore the structure/function relationships of known examples of KH domain proteins, and highlight cases in which they function within or at membrane surfaces. We also summarize examples of KH domain proteins that influence bacterial virulence and pathogenesis. We conclude the article by discussing prospective research avenues that could be pursued to better investigate this largely understudied protein category.

The F plasmid conjutome: the repertoire of E. coli proteins translocated through an F-encoded type IV secretion system.

Bacterial conjugation systems pose a major threat to human health through their widespread dissemination of mobile genetic elements (MGEs) carrying cargoes of antibiotic resistance genes. Using the Cre Recombinase Assay for Translocation (CRAfT), we recently reported that the IncFV pED208 conjugation system also translocates at least 16 plasmid-encoded proteins to recipient bacteria. Here, we deployed a high-throughput CRAfT screen to identify the repertoire of chromosomally encoded protein substrates of the pED208 system. We identified 32 substrates encoded by the Escherichia coli W3110 genome with functions associated with (i) DNA/nucleotide metabolism, (ii) stress tolerance/physiology, (iii) transcriptional regulation, or (iv) toxin inhibition. The respective gene deletions did not impact pED208 transfer proficiencies, nor did Group 1 (DNA/nucleotide metabolism) mutations detectably alter the SOS response elicited in new transconjugants upon acquisition of pED208. However, MC4100(pED208) donor cells intrinsically exhibit significantly higher SOS activation than plasmid-free MC4100 cells, and this plasmid carriage-induced stress response is further elevated in donor cells deleted of several Group 1 genes. Among 10 characterized substrates, we gained evidence of C-terminal or internal translocation signals that could function independently or synergistically for optimal protein transfer. Remarkably, nearly all tested proteins were also translocated through the IncN pKM101 and IncP RP4 conjugation systems. This repertoire of E. coli protein substrates, here termed the F plasmid "conjutome," is thus characterized by functions of potential benefit to new transconjugants, diverse TSs, and the capacity for promiscuous transfer through heterologous conjugation systems. IMPORTANCE: Conjugation systems comprise a major subfamily of the type IV secretion systems (T4SSs) and are the progenitors of a second large T4SS subfamily dedicated to translocation of protein effectors. This study examined the capacity of conjugation machines to function as protein translocators. Using a high-throughput reporter screen, we determined that 32 chromosomally encoded proteins are delivered through an F plasmid conjugation system. The translocated proteins potentially enhance the establishment of the co-transferred F plasmid or mitigate mating-induced stresses. Translocation signals located C-terminally or internally conferred substrate recognition by the F system and, remarkably, many substrates also were translocated through heterologous conjugation systems. Our findings highlight the plasticity of conjugation systems in their capacities to co-translocate DNA and many protein substrates.

Plastid Molecular Chaperone HSP90C Interacts with the SecA1 Subunit of Sec Translocase for Thylakoid Protein Transport.

The plastid stroma-localized chaperone HSP90C plays a crucial role in maintaining optimal proteostasis within chloroplasts and participates in protein translocation processes. While existing studies have revealed HSP90C's direct interaction with the Sec translocase-dependent client pre-protein PsbO1 and the SecY1 subunit of the thylakoid membrane-bound Sec1 translocase channel system, its direct involvement with the extrinsic homodimeric Sec translocase subunit, SecA1, remains elusive. Employing bimolecular fluorescence complementation (BiFC) assay and other in vitro analyses, we unraveled potential interactions between HSP90C and SecA1. Our investigation revealed dynamic interactions between HSP90C and SecA1 at the thylakoid membrane and stroma. The thylakoid membrane localization of this interaction was contingent upon active HSP90C ATPase activity, whereas their stromal interaction was associated with active SecA1 ATPase activity. Furthermore, we observed a direct interaction between these two proteins by analyzing their ATP hydrolysis activities, and their interaction likely impacts their respective functional cycles. Additionally, using PsbO1, a model Sec translocase client pre-protein, we studied the intricacies of HSP90C's possible involvement in pre-protein translocation via the Sec1 system in chloroplasts. The results suggest a complex nature of the HSP90C-SecA1 interaction, possibly mediated by the Sec client protein. Our studies shed light on the nuanced aspects of HSP90C's engagement in orchestrating pre-protein translocation, and we propose a potential collaborative role of HSP90C with SecA1 in actively facilitating pre-protein transport across the thylakoid membrane.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

New insights into the structure and dynamics of the TOM complex in mitochondria.

To date, there is no general physical model of the mechanism by which unfolded polypeptide chains with different properties are imported into the mitochondria. At the molecular level, it is still unclear how transit polypeptides approach, are captured by the protein translocation machinery in the outer mitochondrial membrane, and how they subsequently cross the entropic barrier of a protein translocation pore to enter the intermembrane space. This deficiency has been due to the lack of detailed structural and dynamic information about the membrane pores. In this review, we focus on the recently determined sub-nanometer cryo-EM structures and our current knowledge of the dynamics of the mitochondrial two-pore outer membrane protein translocation machinery (TOM core complex), which provide a starting point for addressing the above questions. Of particular interest are recent discoveries showing that the TOM core complex can act as a mechanosensor, where the pores close as a result of interaction with membrane-proximal structures. We highlight unusual and new correlations between the structural elements of the TOM complexes and their dynamic behavior in the membrane environment.

Discontinuous Translocation of a Luciferase Protein beyond Graft Junction in Tobacco.

Transgrafting, a grafting technique that uses both genetically modified (GM) and non-GM plants, is a novel plant breeding technology that can be used to improve the efficiency of crop cultivation without introducing foreign genes into the edible parts of non-GM plants. This technique can facilitate the acquisition of disease resistance and/or increased yield. However, the translocation of low-molecular-weight compounds, ribonucleic acid (RNA), and proteins through graft junctions raises a potential safety risk for food crops. Here, we used a transgenic tobacco plant expressing a firefly luciferase gene (LUC) to examine the translocation of the LUC protein beyond the graft junction in grafted plants. We observed the bi-directional translocation of LUC proteins in transgrafted tobacco plants, i.e., from the rootstock to scion and vice versa. Transcriptomic analysis revealed that transcripts of the LUC gene were undetectable in non-GM plant bodies, indicating that the LUC protein itself was translocated. Moreover, the movement of the LUC protein is an episodic (i.e., non-continuous) event, since non-GM samples showing high LUC activity were flanked by non-GM samples showing no apparent LUC activity. Translocation from the GM to non-GM part depends on the characteristics of GM plant bodies; here, the enhanced translocation of the LUC protein into the non-GM scion was observed when LUC-expressing rootstocks with hairy roots were used. Moreover, the quantity of translocated LUC protein was far below the level that is generally required to induce an allergenic response. Finally, since the LUC protein levels of plants used for transgrafting are moderate and the LUC protein itself is relatively unstable, further investigation is necessary regarding whether the newly expressed protein in GM plants is highly stable, easily translocated, and/or highly expressed.

KIF5B plays important roles in dendritic spine plasticity and dendritic localization of PSD95 and FMRP in the mouse cortex in vivo.

Kinesin 1 (KIF5) is one major type of motor protein in neurons, but its members' function in the intact brain remains less studied. Using in vivo two-photon imaging, we find that conditional knockout of Kif5b (KIF5B cKO) in CaMKIIα-Cre-expressing neurons shows heightened turnover and lower stability of dendritic spines in layer 2/3 pyramidal neurons with reduced spine postsynaptic density protein 95 acquisition in the mouse cortex. Furthermore, the RNA-binding protein fragile X mental retardation protein (FMRP) is translocated to the proximity of newly formed spines several hours before the spine formation events in vivo in control mice, but this preceding transport of FMRP is abolished in KIF5B cKO mice. We further find that FMRP is localized closer to newly formed spines after fear extinction, but this learning-dependent localization is disrupted in KIF5B cKO mice. Our findings provide the crucial in vivo evidence that KIF5B is involved in the dendritic targeting of synaptic proteins that underlies dendritic spine plasticity.

Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.

Tracking the Activity and Position of Mitochondrial ß-Barrel Proteins.

Total interference reflection fluorescence (TIRF) microscopy of lipid bilayers is an effective technique for studying the lateral movement and ion channel activity of single integral membrane proteins. Here we describe how to integrate the mitochondrial outer membrane preprotein translocase TOM-CC and its ß-barrel protein-conducting channel Tom40 into supported lipid bilayers to identify possible relationships between movement and channel activity. We propose that our approach can be readily applied to membrane protein channels where transient tethering to either membrane-proximal or intramembrane structures is accompanied by a change in channel permeation.

TransGCN: a semi-supervised graph convolution network-based framework to infer protein translocations in spatio-temporal proteomics.

Protein subcellular localization (PSL) is very important in order to understand its functions, and its movement between subcellular niches within cells plays fundamental roles in biological process regulation. Mass spectrometry-based spatio-temporal proteomics technologies can help provide new insights of protein translocation, but bring the challenge in identifying reliable protein translocation events due to the noise interference and insufficient data mining. We propose a semi-supervised graph convolution network (GCN)-based framework termed TransGCN that infers protein translocation events from spatio-temporal proteomics. Based on expanded multiple distance features and joint graph representations of proteins, TransGCN utilizes the semi-supervised GCN to enable effective knowledge transfer from proteins with known PSLs for predicting protein localization and translocation. Our results demonstrate that TransGCN outperforms current state-of-the-art methods in identifying protein translocations, especially in coping with batch effects. It also exhibited excellent predictive accuracy in PSL prediction. TransGCN is freely available on GitHub at https://github.com/XuejiangGuo/TransGCN.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

Cellular forgetting, desensitisation, stress and ageing in signalling networks. When do cells refuse to learn more?

Recent findings show that single, non-neuronal cells are also able to learn signalling responses developing cellular memory. In cellular learning nodes of signalling networks strengthen their interactions e.g. by the conformational memory of intrinsically disordered proteins, protein translocation, miRNAs, lncRNAs, chromatin memory and signalling cascades. This can be described by a generalized, unicellular Hebbian learning process, where those signalling connections, which participate in learning, become stronger. Here we review those scenarios, where cellular signalling is not only repeated in a few times (when learning occurs), but becomes too frequent, too large, or too complex and overloads the cell. This leads to desensitisation of signalling networks by decoupling signalling components, receptor internalization, and consequent downregulation. These molecular processes are examples of anti-Hebbian learning and 'forgetting' of signalling networks. Stress can be perceived as signalling overload inducing the desensitisation of signalling pathways. Ageing occurs by the summative effects of cumulative stress downregulating signalling. We propose that cellular learning desensitisation, stress and ageing may be placed along the same axis of more and more intensive (prolonged or repeated) signalling. We discuss how cells might discriminate between repeated and unexpected signals, and highlight the Hebbian and anti-Hebbian mechanisms behind the fold-change detection in the NF-κB signalling pathway. We list drug design methods using Hebbian learning (such as chemically-induced proximity) and clinical treatment modalities inducing (cancer, drug allergies) desensitisation or avoiding drug-induced desensitisation. A better discrimination between cellular learning, desensitisation and stress may open novel directions in drug design, e.g. helping to overcome drug resistance.

Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon.

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

Quantitative image analysis of intracellular protein translocation in 3-dimensional tissues for pharmacodynamic studies of immunogenic cell death.

A recent development in cancer chemotherapy is to use cytotoxics to induce tumor-specific immune response through immunogenic cell death (ICD). In ICD, calreticulin is translocated from endoplasmic reticulum to cell membrane (ecto-CRT) which serves as the 'eat-me-signal' to antigen-presenting cells. Ecto-CRT measurements, e.g., by ecto-CRT immunostaining plus flow cytometry, can be used to study the pharmacodynamics of ICD in single cells, whereas ICD studies in intact 3-dimensional tissues such as human tumors require different approaches. The present study described a method that used (a) immunostaining with fluorescent antibodies followed by confocal microscopy to obtain the spatial locations of two molecules-of-interest (CRT and a marker protein WGA), and (b) machine-learning (trainable WEKA segmentation) and additional image processing tools to locate the target molecules, remove the interfering signals in the nucleus, cytosol and extracellular space, enable the distinction of the inner and outer edges of the cell membrane and thereby identify the cells with ecto-CRT. This method, when applied to 3-dimensional human bladder cancer cell spheroids, yielded drug-induced ecto-CRT measurements that were qualitatively comparable to the flow cytometry results obtained with single cells disaggregated from spheroids. This new method was applied to study drug-induced ICD in short-term cultures of surgical specimens of human patient bladder tumors.

YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes.

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Molecular view of ER membrane remodeling by the Sec61/TRAP translocon.

Protein translocation across the endoplasmic reticulum (ER) membrane is an essential step during protein entry into the secretory pathway. The conserved Sec61 protein-conducting channel facilitates polypeptide translocation and coordinates cotranslational polypeptide-processing events. In cells, the majority of Sec61 is stably associated with a heterotetrameric membrane protein complex, the translocon-associated protein complex (TRAP), yet the mechanism by which TRAP assists in polypeptide translocation remains unknown. Here, we present the structure of the core Sec61/TRAP complex bound to a mammalian ribosome by cryogenic electron microscopy (cryo-EM). Ribosome interactions anchor the Sec61/TRAP complex in a conformation that renders the ER membrane locally thinner by significantly curving its lumenal leaflet. We propose that TRAP stabilizes the ribosome exit tunnel to assist nascent polypeptide insertion through Sec61 and provides a ratcheting mechanism into the ER lumen mediated by direct polypeptide interactions.

Chloroplast protein translocation pathways and ubiquitin-dependent regulation at a glance.

Chloroplasts conduct photosynthesis and numerous metabolic and signalling processes that enable plant growth and development. Most of the ∼3000 proteins in chloroplasts are nucleus encoded and must be imported from the cytosol. Thus, the protein import machinery of the organelle (the TOC-TIC apparatus) is of fundamental importance for chloroplast biogenesis and operation. Cytosolic factors target chloroplast precursor proteins to the TOC-TIC apparatus, which drives protein import across the envelope membranes into the organelle, before various internal systems mediate downstream routing to different suborganellar compartments. The protein import system is proteolytically regulated by the ubiquitin-proteasome system (UPS), enabling centralized control over the organellar proteome. In addition, the UPS targets a range of chloroplast proteins directly. In this Cell Science at a Glance article and the accompanying poster, we present mechanistic details of these different chloroplast protein targeting and translocation events, and of the UPS systems that regulate chloroplast proteins.

Towards solving the mystery of peroxisomal matrix protein import.

Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even import folded proteins, but the underlying mechanism has been a mystery. Recent results reveal how import receptors shuttle cargo into peroxisomes. The cargo-bound receptors move from the cytosol across the peroxisomal membrane completely into the matrix by a mechanism that resembles transport through the nuclear pore. The receptors then return to the cytosol through a separate retrotranslocation channel, leaving the cargo inside the organelle. This cycle concentrates imported proteins within peroxisomes, and the energy for cargo import is supplied by receptor export. Peroxisomal protein import thus fundamentally differs from other previously known mechanisms for translocating proteins across membranes.