In vitro import experiments with semi-intact cells suggest a role of the Sec61 paralog Ssh1 in mitochondrial biogenesis.

Mitochondrial biogenesis relies on the synthesis of hundreds of different precursor proteins in the cytosol and their subsequent import into the organelle. Recent studies suggest that the surface of the endoplasmic reticulum (ER) actively contributes to the targeting of some mitochondrial precursors. In the past, in vitro import experiments with isolated mitochondria proved to be extremely powerful to elucidate the individual reactions of the mitochondrial import machinery. However, this in vitro approach is not well suited to study the influence of non-mitochondrial membranes. In this study, we describe an in vitro system using semi-intact yeast cells to test a potential import relevance of the ER proteins Erg3, Lcb5 and Ssh1, all being required for efficient mitochondrial respiration. We optimized the conditions of this experimental test system and found that cells lacking Ssh1, a paralog of the Sec61 translocation pore, show a reduced import efficiency of mitochondrial precursor proteins. Our results suggest that Ssh1, directly or indirectly, increases the efficiency of the biogenesis of mitochondrial proteins. Our findings are compatible with a functional interdependence of the mitochondrial and the ER protein translocation systems.

The Sec61/SecY complex is inherently deficient in translocating intrinsically disordered proteins.

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or ß-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or ß-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and ß-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon.

SecA functions in vivo as a discrete anti-parallel dimer to promote protein transport.

SecA ATPase motor protein plays a central role in bacterial protein transport by binding substrate proteins and the SecY channel complex and utilizing its ATPase activity to drive protein translocation across the plasma membrane. SecA has been shown to exist in a dynamic monomer-dimer equilibrium modulated by translocation ligands, and multiple structural forms of the dimer have been crystallized. Since the structural form of the dimer remains a controversial and unresolved question, we addressed this matter by engineering ρ-benzoylphenylalanine along dimer interfaces corresponding to the five different SecA X-ray structures and assessing their in vivo photo-crosslinking pattern. A discrete anti-parallel 1M6N-like dimer was the dominant if not exclusive dimer found in vivo, whether SecA was cytosolic or in lipid or SecYEG-bound states. SecA bound to a stable translocation intermediate was crosslinked in vivo to a second SecA protomer at its 1M6N interface, suggesting that this specific dimer likely promotes active protein translocation. Taken together, our studies strengthen models that posit, at least in part, a SecA dimer-driven translocation mechanism.

The Canonical and Accessory Sec System of Gram-positive Bacteria.

The Sec system is present in all bacteria and responsible for the translocation of the majority of proteins across the cytoplasmic membrane. The system consists of two principal components: the ATPase motor protein, SecA, and the protein-conducting channel, SecYEG. In addition to this canonical Sec system, several Gram-positive bacteria also possess a so-called accessory Sec system. This is a specialized translocation system that is responsible for the export of a subset of secretory proteins, including virulence factors. The accessory Sec system consists of a second SecA paralog, termed SecA2, with or without a second SecY paralog, termed SecY2. In some bacteria, the accessory Sec system is dependent on the canonical Sec system for functionality, while in other bacteria, they can function independently. In this review, we provide an overview of the current knowledge of the canonical and accessory Sec system of Gram-positive bacteria with a focus on the primary component of the Sec translocase, SecA and SecYEG.

Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins.

The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W ) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins.IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.

Voltage Sensing in Bacterial Protein Translocation.

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY's half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b's dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b's dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

Sec translocon has an insertase-like function in addition to polypeptide conduction through the channel.

The Sec translocon provides a polypeptide-conducting channel, which is insulated from the hydrophobic lipidic environment of the membrane, for translocation of hydrophilic passenger polypeptides. Its lateral gate allows a downstream hydrophobic segment (stop-transfer sequence) to exit the channel laterally for integration into the lipid phase. We note that this channel model only partly accounts for the translocon function. The other essential role of translocon is to facilitate de novo insertion of the N-terminal topogenic segment of a substrate polypeptide into the membrane. Recent structural studies suggest that de novo insertion does not use the polypeptide-conducting channel; instead, it takes place directly at the lateral gate, which is prone to opening. We propose that the de novo insertion process, in concept, is similar to that of insertases (such as YidC in bacteria and EMC3 in eukaryotes), in which an intramembrane surface of the machinery provides the halfway point of insertion.

The Conserved Role of YidC in Membrane Protein Biogenesis.

YidC insertase plays a pivotal role in the membrane integration, folding, and assembly of a number of proteins, including energy-transducing respiratory complexes, both autonomously and in concert with the SecYEG channel in bacteria. The YidC family of proteins is widely conserved in all domains of life, with new members recently identified in the eukaryotic endoplasmic reticulum membrane. Bacterial and organellar members share the conserved 5-transmembrane core, which forms a unique hydrophilic cavity in the inner leaflet of the bilayer accessible from the cytoplasm and the lipid phase. In this chapter, we discuss the YidC family of proteins, focusing on its mechanism of substrate insertion independently and in association with the Sec translocon.

LncRNA LINC02418 regulates proliferation and apoptosis of non-small cell lung cancer cells by regulating miR-4677-3p/SEC61G.

OBJECTIVE: Increasing studies indicated that long non-coding RNA (lncRNA) has crucial roles in cancer development, including non-small cell lung cancer (NSCLC). LINC02418 was reported to promote colorectal cancer development. However, whether LINC02418 has a role in NSCLC remains to be explored. MATERIALS AND METHODS: First, expression level of LINC02418 in NSCLC tissues and normal tissues was analyzed at ENCORI. Moreover, expression level of LINC02418 in NSCLC cells and normal cell was analyzed with quantitative real-time PCR. Cell counting kit-8 assay, transwell invasion assay, and flow cytometry assay were used to analyze cell proliferation, cell invasion, and cell apoptosis. RESULTS: LINC02418 was found as upregulated expression in both NSCLC tissues and cells. Functional assays showed that LINC02418 knockdown suppressed NSCLC cell proliferation and invasion but promoted cell apoptosis, while the overexpression of LINC02418 exerts opposite effects. Mechanistically, we showed LINC02418 could interact with microRNA-4677-3p (miR-4677-3p) to regulate Sec61 gamma subunit (SEC61G) expression. CONCLUSIONS: These results indicated that LINC02418 functions as an oncogene, and regulated miR-4677-3p/SEC61G axis to accelerate NSCLC progression.

Transport of Folded Proteins by the Tat System.

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.