Protein Translocation Process Research Articles

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.

Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

MoSec61β, the beta subunit of Sec61, is involved in fungal development and pathogenicity, plant immunity, and ER-phagy in Magnaporthe oryzae

ABSTRACT The process of protein translocation into the endoplasmic reticulum (ER) is the initial and decisive step in the biosynthesis of all secretory proteins and many soluble organelle proteins. In this process, the Sec61 complex is the protein-conducting channel for transport. In this study, we identified and characterized the β subunit of the Sec61 complex in Magnaporthe oryzae (MoSec61β). Compared with the wild-type strain Guy11, the ΔMosec61β mutant exhibited highly branched mycelial morphology, reduced conidiation, high sensitivity to cell wall integrity stress, severely reduced virulence to rice and barley, and restricted biotrophic invasion. The turgor pressure of ΔMosec61β was notably reduced, which affected the function of appressoria. Moreover, ΔMosec61β was also sensitive to oxidative stress and exhibited a reduced ability to overcome plant immunity. Further examination demonstrated that MoSec61β affected the normal secretion of the apoplastic effectors Bas4 and Slp1. In addition, ΔMosec61β upregulated the level of ER-phagy. In conclusion, our results demonstrate the importance of the roles played by MoSec61β in the fungal development and pathogenesis of M. oryzae.

Structural and Functional Heat Stress Responses of Chloroplasts of Arabidopsis thaliana.

Temperature elevations constitute a major threat to plant performance. In recent years, much was learned about the general molecular mode of heat stress reaction of plants. The current research focuses on the integration of the knowledge into more global networks, including the reactions of cellular compartments. For instance, chloroplast function is central for plant growth and survival, and the performance of chloroplasts is tightly linked to the general status of the cell and vice versa. We examined the changes in photosynthesis, chloroplast morphology and proteomic composition posed in Arabidopsis thaliana chloroplasts after a single or repetitive heat stress treatment over a period of two weeks. We observed that the acclimation is potent in the case of repetitive application of heat stress, while a single stress results in lasting alterations. Moreover, the physiological capacity and its adjustment are dependent on the efficiency of the protein translocation process as judged from the analysis of mutants of the two receptor units of the chloroplast translocon, TOC64, and TOC33. In response to repetitive heat stress, plants without TOC33 accumulate Hsp70 proteins and plants without TOC64 have a higher content of proteins involved in thylakoid structure determination when compared to wild-type plants.

High-resolution view of the type III secretion export apparatus in situ reveals membrane remodeling and a secretion pathway

Type III protein secretion systems are essential virulence factors for many important pathogenic bacteria. The entire protein secretion machine is composed of several substructures that organize into a holostructure or injectisome. The core component of the injectisome is the needle complex, which houses the export apparatus that serves as a gate for the passage of the secreted proteins through the bacterial inner membrane. Here, we describe a high-resolution structure of the export apparatus of the Salmonella type III secretion system in association with the needle complex and the underlying bacterial membrane, both in isolation and in situ. We show the precise location of the core export apparatus components within the injectisome and bacterial envelope and demonstrate that their deployment results in major membrane remodeling and thinning, which may be central for the protein translocation process. We also show that InvA, a critical export apparatus component, forms a multiring cytoplasmic conduit that provides a pathway for the type III secretion substrates to reach the entrance of the export gate. Combined with structure-guided mutagenesis, our studies provide major insight into potential mechanisms of protein translocation and injectisome assembly.

Polymer translocation across an oscillating nanopore: study of several distribution functions of relevant Brownian functionals

In this paper, we study polymer translocation dynamics across an oscillating nanopore by proposing and inspecting several probability distribution functions (PDFs) of relevant Brownian functionals which specify the translocation across the nanopore. We model such translocation process by an overdamped Langevin equation of collective variable x. We introduce several probability distribution functions (PDFs) to identify the translocation process. We consider elegant backward Fokker-Planck method to derive analytically closed form expressions of several PDFs associated with such stochastic process. For instance, an important quantity for translocation processes is the first passage time, i.e. the time the molecule takes to cross the nanopore with initial collective value of the molecule x0. We derive analytical expressions for: (i) the PDF P(tf|x0) of the first passage time tf which specify the lifetime of protein translocation process, (ii) the PDF P(A|x0) of the area A till the first passage time and it provides us numerous valuable information about the average size and reactivity of the process, and (iii) the PDF P(M) associated with the maximum value of collective mode, M, of the translocation process before the first passage time. Our analysis is limited to a regime where both drift and diffusion have the same periodic time dependence with a constant ratio between them. We further confirm our analytical predictions by computing the same PDFs with direct numerical simulations of the corresponding Langevin equation. We obtain a very good agreement of our theoretical predictions with the numerically simulated results. Finally, several nontrivial scaling behaviour in the asymptotic limits for the above mentioned PDFs are predicted, which can be verified further from experimental observation.

The Twin-Arginine Pathway for Protein Secretion.

The Tat pathway for protein translocation across bacterial membranes stands out for its selective handling of fully folded cargo proteins. In this review, we provide a comprehensive summary of our current understanding of the different known Tat components, their assembly into different complexes, and their specific roles in the protein translocation process. In particular, this overview focuses on the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus subtilis. Using these organisms as examples, we discuss structural features of Tat complexes alongside mechanistic models that allow for the Tat pathway's unique protein proofreading and transport capabilities. Finally, we highlight recent advances in exploiting the Tat pathway for biotechnological benefit, the production of high-value pharmaceutical proteins.

Simulation of single-protein nanopore sensing shows feasibility for whole-proteome identification

Single-molecule techniques for protein sequencing are making headway towards single-cell proteomics and are projected to propel our understanding of cellular biology and disease. Yet, single cell proteomics presents a substantial unmet challenge due to the unavailability of protein amplification techniques, and the vast dynamic-range of protein expression in cells. Here, we describe and computationally investigate the feasibility of a novel approach for single-protein identification using tri-color fluorescence and plasmonic-nanopore devices. Comprehensive computer simulations of denatured protein translocation processes through the nanopores show that the tri-color fluorescence time-traces retain sufficient information to permit pattern-recognition algorithms to correctly identify the vast majority of proteins in the human proteome. Importantly, even when taking into account realistic experimental conditions, which restrict the spatial and temporal resolutions as well as the labeling efficiency, and add substantial noise, a deep-learning protein classifier achieves 97% whole-proteome accuracies. Applying our approach for protein datasets of clinical relevancy, such as the plasma proteome or cytokine panels, we obtain ~98% correct protein identification. This study suggests the feasibility of a method for accurate and high-throughput protein identification, which is highly versatile and applicable.

Investigation of the Relative Activity of Monomer and Dimer SecYEG Translocons

The general secretory (Sec) pathway is the principal protein transport pathway in bacteria. Two key components of this system are the protein complex SecYEG, which forms a protein‐conducting channel within the plasma membrane, and the ATPase motor protein SecA, which drives the protein translocation process. Whether the SecYEG complex functions as a monomer or requires dimerization with a neighboring SecYEG complex for activity remains unclear. To address this important question, we have made use of both photobleaching and total internal reflection fluorescence (TIRF) microscopy to assess the fluorescently labeled SecY oligomeric state, as well as an in vitro protein translocation assay to assess Sec‐dependent protein transport activity. The key component of our system is the SecYEG proteoliposome, which is a lipid vesicle reconstituted solely with SecYEG protein. In this study, we reconstituted proteoliposomes at different lipid to SecYEG protein ratios to produce distinct vesicle populations containing either mostly SecYEG monomers or containing about half SecYEG monomers and half SecYEG dimers. Next, we utilized TIRF microscopy to determine the percent of singly or doubly reconstituted proteoliposomes existing within a given population based on the ratio of single or double photobleaching events. Finally, we optimized and performed an in vitro protein transport assay to assess the overall protein translocation activity of a given population. Initial findings from theses assays suggest that the monomer SecYEG proteoliposome population is more efficient at translocating protein compared to the population with substantial dimer content. Further studies will be required to fully confirm this finding. Ultimately, we hope to elucidate any differences in the protein translocation activity between these distinct populations in order to better understand the functionality of monomer and dimer SecYEG translocons in this universally conserved system.Support or Funding InformationFunding generously provided by the NIH through the National Institute of General Medical Sciences (GM110552)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assays for protein retrotranslocation in ERAD.

Elimination of misfolded proteins by endoplasmic reticulum (ER)-associated protein degradation (ERAD) ensures that proteins proceeding through the secretory pathway are correctly folded and processed, which is critical to minimize ER stress. All ERAD pathways include a protein translocation process termed retrotranslocation, in which ubiquitinated misfolded substrates are extracted from the ER and degraded by the cytosolic 26S proteasome. Despite being integral to ERAD, the retrotranslocation process has been largely obscure. Recently, an explosion of discoveries has provided key mechanistic insights into this novel route of protein transport. These advances were facilitated by the development of in vitro and in vivo assays that utilize components from the yeast Saccharomyces cerevisiae. The assays permit detailed study of the distinct steps in ERAD-linked retrotranslocation, including ubiquitination of selected ERAD substrates, substrate removal from the ER, maintenance of cytosolic substrate solubility in the cytosol, and substrate degradation. Here we provide detailed protocols for these assays that pertain to work on retrotranslocation of integral membrane proteins (ERAD-M substrates), with the expectation that these approaches can be adapted for many related biochemical processes.

Structure-based working model of SecDF, a proton-driven bacterial protein translocation factor.

The bacterial membrane protein SecDF enhances protein translocation across the membrane driven by the complex of SecA ATPase and SecYEG. Many newly synthesized proteins in the cytoplasm are programmed to be translocated to the periplasm via the narrow channel that is formed in the center of SecYEG. During the protein-translocation process, SecDF is proposed to undergo repeated conformational transitions to pull out the precursor protein from the SecYEG channel into the periplasm. Once SecDF captures the precursor protein on the periplasmic surface, SecDF can complete protein translocation even if SecA function is inactivated by ATP depletion, implying that SecDF is a protein-translocation motor that works independent of SecA. Structural and functional analyses of SecDF in 2011 suggested that SecDF utilizes the proton gradient and interacts with precursor protein in the flexible periplasmic region. The crystal structures of SecDF in different states at more than 3Å resolution were reported in 2017 and 2018, which further improved our understanding of the dynamic molecular mechanisms of SecDF. This review summarizes recent structural studies of SecDF.

Real-Time Nanopore-Based Recognition of Protein Translocation Success

A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules. Nanopore-based single-molecule DNA sequencing is the premier example. This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to pass through the nanopore and across the separating membrane. For disordered peptides, folded proteins, or block copolymers with heterogeneous charge densities, by contrast, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here, we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two “selectivity tags”—regions of different but uniform charge density—at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate that the selectivity tag technique will be broadly applicable to nanopore-based protein detection, analysis, and separation technologies, and to the elucidation of protein translocation processes in normal cellular function and in disease.

Energetics of Sheath Contraction in Contractile Injection Systems

The process of protein and DNA translocation across lipid membranes is central to the function of any organism. A large class of large multicomponent organelles, such as bacteriophage tails, Type VI Secretion System, R-type pyocins, Serratia antifeeding prophage, and others, translocate their substrates using a rigid tube/contractile sheath mechanism. Functionally and structurally, these ‘contractile injection systems’ resemble a stretched spring (or sheath) wound around a non-contractile tube. The system is locked in a high-energy metastable state by a baseplate structure that plays an important role in sheath assembly and contraction triggering. Upon interaction of the baseplate with a target cell membrane, the sheath contracts and drives the tube through the cell envelope. A full atomic model of the sheath and tube in the extended and contracted state is available for R-type pyocin, one of the simplest representatives of contractile injection systems. The structure suggests that the contraction is accomplished by rigid body rearrangement of sheath subunits and that the transformation is driven by the energy stored in the extended conformation of the sheath during assembly. Other data show that the contraction starts at the baseplate and propagates through the sheath as a wave. The individual subunits’ trajectories are however unknown. We will discuss how we can derive these trajectories from energetics considerations. These finding are important for understanding the substrates that can be translocated by contractile injection systems.

Functional Characterization of EscK (Orf4), a Sorting Platform Component of the Enteropathogenic Escherichia coli Injectisome.

The type III secretion system (T3SS) is an essential virulence determinant for enteropathogenic Escherichia coli (EPEC) colonization of intestinal epithelial cells. Multiple EPEC effector proteins are injected via the T3SS into enterocyte cells, leading to diarrheal disease. The T3SS is encoded within a genomic pathogenicity island termed the locus of enterocyte effacement (LEE). Here we unravel the function of EscK, a previously uncharacterized LEE-encoded protein. We show that EscK is central for T3SS biogenesis and function. EscK forms a protein complex with EscQ, the main component of the cytoplasmic sorting platform, serving as a docking site for T3S substrates. Our results provide a comprehensive functional analysis of an understudied component of T3SSs.

Biphasic actions of SecA inhibitors on Prl/Sec suppressors: Possible physiological roles of SecA-only channels

SecA is an essential component in the bacterial Sec-dependent protein translocation process. We previously showed that in addition to the ubiquitous, high-affinity SecYEG-SecDF·YajC protein translocation channel, there is a low-affinity SecA-only channel that elicits ion channel activity and promotes protein translocation. The SecA-only channels are less efficient, and like Prl suppressors, lack signal peptide specificity; they function in the absence of signal peptides. The presence of SecYEG-SecDF·YajC alters the sensitivity of ATPase inhibitor Rose Bengal. In this study, we found that the suppressor membranes are much more resistant to inhibition by Rose Bengal. Similar results have been found for a SecA-specific inhibitor. Moreover, biphasic responses of inhibition of ion current and protein translocation activities were observed for many PrlA/SecY and PrlG/SecE suppressor membranes, with a low IC50 value similar to that of the SecA-only channels and a very high IC50. However, the suppressor strains are as sensitive to the inhibitor as the parental strain, suggesting that SecA-only channels have some essential physiological function(s) in the cells that are inhibited by the specific SecA inhibitor.

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

Abstract SY44-03: Targeting the unfolded protein response in cancer

Abstract SY44-03: Targeting the unfolded protein response in cancer

Solution structure of the TatB component of the twin-arginine translocation system

The twin-arginine protein transport (Tat) system translocates fully folded proteins across lipid membranes. In Escherichia coli, the Tat system comprises three essential components: TatA, TatB and TatC. The protein translocation process is proposed to initiate by signal peptide recognition and substrate binding to the TatBC complex. Upon formation of the TatBC–substrate protein complex, the TatA subunits are recruited and form the protein translocation pore. Experimental evidences suggest that TatB forms a tight complex with TatC at 1:1 molar ratio and the TatBC complex contains multiple copies of both proteins. Cross-linking experiments demonstrate that TatB functions in tetrameric units and interacts with both TatC and substrate proteins. However, structural information of the TatB protein is still lacking, and its functional mechanism remains elusive. Herein, we report the solution structure of TatB in DPC micelles determined by Nuclear Magnetic Resonance (NMR) spectroscopy. Overall, the structure shows an extended ‘L-shape’ conformation comprising four helices: a transmembrane helix (TMH) α1, an amphipathic helix (APH) α2, and two solvent exposed helices α3 and α4. The packing of TMH and APH is relatively rigid, whereas helices α3 and α4 display notably higher mobility. The observed floppiness of helices α3 and α4 allows TatB to sample a large conformational space, thus providing high structural plasticity to interact with substrate proteins of different sizes and shapes.

Molecular Insights Revealing Interaction of Tim23 and Channel Subunits of Presequence Translocase

Tim23 is an essential channel-forming subunit of the presequence translocase recruiting multiple components for assembly of the core complex, thereby regulating the protein translocation process. However, understanding of the precise interaction of subunits associating with Tim23 remains largely elusive. Our findings highlight that transmembrane helix 1 (TM1) is required for homodimerization of Tim23, while, together with TM2, it is involved in preprotein binding within the channel. Based on our evidence, we predict that the TM1 and TM2 from each dimer are involved in the formation of the central translocation pore, aided by Tim17. Furthermore, TM2 is also involved in the recruitment of Tim21 and the presequence-associated motor (PAM) subcomplex to the Tim23 channel, while the matrix-exposed loop L1 generates specificity in their association with the core complex. Strikingly, our findings indicate that the C-terminal sequence of Tim23 is dispensable for growth and functions as an inhibitor for binding of Tim21. Our model conceptually explains the cooperative function between Tam41 and Pam17 subunits, while the antagonistic activity of Tim21 predominantly determines the bound and free forms of the PAM subcomplex during import.

Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Escherichia coli-the powerhouse for recombinant protein production-is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.