Translocation Of Secretory Proteins Research Articles

UFM1 E3 ligase promotes recycling of 60S ribosomal subunits from the ER.

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.

Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation

Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.

A unified evolutionary origin for the ubiquitous protein transporters SecY and YidC

BackgroundProtein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known.ResultsThe features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY’s halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC’s sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains.ConclusionsSecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells.

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

ABSTRACTN-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

Cancer associated mutations in Sec61γ alter the permeability of the ER translocase.

Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the Sec61 complex, a heterotrimeric protein complex possessing two essential sub-units, Sec61p/Sec61α and Sss1p/Sec61γ and the non-essential Sbh1p/Sec61β subunit. In addition to forming a protein conducting channel, the Sec61 complex maintains the ER permeability barrier, preventing flow of molecules and ions. Loss of Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C-terminus is juxtaposed to the key gating module of Sec61p/Sec61α and is important for gating the translocon. Inspection of the cancer genome database identifies six mutations in highly conserved amino acids of Sec61γ/Sss1p. We identify that five out of the six mutations identified affect gating of the ER translocon, albeit with varying strength. Together, we find that mutations in Sec61γ that arise in malignant cells result in altered translocon gating dynamics, this offers the potential for the translocon to represent a target in co-therapy for cancer treatment.

Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

The conserved C-terminus of Sss1p is required to maintain the endoplasmic reticulum permeability barrier

The endoplasmic reticulum (ER) is the entry point to the secretory pathway and major site of protein biogenesis. Translocation of secretory and integral membrane proteins across or into the ER membrane occurs via the evolutionarily conserved Sec61 complex, a heterotrimeric channel that comprises the Sec61p/Sec61α, Sss1p/Sec61γ, and Sbh1p/Sec61β subunits. In addition to forming a protein-conducting channel, the Sec61 complex also functions to maintain the ER permeability barrier, preventing the mass free flow of essential ER-enriched molecules and ions. Loss in Sec61 integrity is detrimental and implicated in the progression of disease. The Sss1p/Sec61γ C terminus is juxtaposed to the key gating module of Sec61p/Sec61α, and we hypothesize it is important for gating the ER translocon. The ER stress response was found to be constitutively induced in two temperature-sensitive sss1 mutants (sss1ts ) that are still proficient to conduct ER translocation. A screen to identify intergenic mutations that allow for sss1ts cells to grow at 37 °C suggests the ER permeability barrier to be compromised in these mutants. We propose the extreme C terminus of Sss1p/Sec61γ is an essential component of the gating module of the ER translocase and is required to maintain the ER permeability barrier.

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG–SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Нові мішені фармакологічної корекції когнітивних розладів за умов пренатальної дії гіпоксії

В огляді наведено сучасні концепції механізмів пошкоджень клітин головного мозку за умов пренатального впливу хронічної гіпоксії. На основі сучасних даних про механізми адаптації організму до стану гіпоксії пропонуються нові перспективні напрями пошуку ефективних лікарських засобів для фармакологічної корекції наслідків пренатальної гіпоксії. Пренатальна гіпоксія є найпоширенішою причиною смертності та інвалідності новонароджених дітей. Хронічна пренатальна гіпоксія призводить до значних змін у розвитку когнітивних функцій у постнатальному періоді життя, які базуються на морфологічних змінах у структурах мозку, що беруть участь у навчанні та пам'яті. Результати досліджень останніх років внесли великий вклад у розкриття механізмів формування стану гіпоксії різного генезу та індукованих нею порушень метаболічних і функціональних процесів на рівні клітини й субклітинних структур. Визначено ряд морфофункціональних детермінант (специфічні рецептори, регуляторні білки, внутрішньоклітинні ферменти, мітохондріальні іонні канали і т. д.), які беруть безпосередню участь у розвитку термінової та довготривалої адаптації клітини й усього організму до гіпоксії. Аналіз літератури з досліджень молекулярних механізмів дії пренатальної гіпоксії показав, що основою гіпоксичних уражень головного мозку новонароджених є каскад біохімічних і молекулярних процесів, що призводять до ексайтотоксичності, окиснювального та нітрозуючого стресів і запалення. Одночасно з цим за гіпоксії починають функціонувати й компенсаторно­пристосувальні механізми, що підвищують стійкість організму до дефіциту кисню. В огляді особливу увагу приділено обговоренню даних щодо ролі молекулярних детермінант ендогенної нейропротекції, таких як HSP70 і HIF­1a. Транскрипційна система HIF­1 є ключовим регулятором адаптивних механізмів відповідей на гіпоксію на локальному та системному рівнях. Зміни в експресії генів HIF­1 у пренатальному розвитку запускають епігенетичні механізми, що призводять до негативних змін у розвитку нервової системи. Важливим компонентом системи ендогенної нейропротекції є сімейство білків теплового шоку HSP70, які сприяють широкому спектру процесів фолдингу, рефолдингу білків, мембранній транслокації органелярних і секреторних білків, а також здійснюють контроль за активністю регуляторних білків. Вплив HSP на стабілізацію HIF­1 забезпечує активацію процесів проліферації, апоптозу, ангіогенезу за умов гіпоксії. Білки HSP70 / HIF­1a­ системи можуть бути специфічними мішенями для впливу фармакологічних агентів з метою регуляції процесів адаптації організму до гіпоксії. Тому пошук нових ефективних нейропротекторних засобів повинен бути спрямований на активацію HSP70/HIF­1a­системи та використання агентів, здатних забезпечувати модуляцію генів, що кодують синтез білків HSP70 і HIF­1a.

Posttranslational translocon architecture

Protein Translocation About a third of proteins are transported into endoplasmic reticulum by the universally conserved Sec61 protein-conducting channel. Itskanov and Park determined a cryo–electron microscopy structure of the Sec complex from yeast, which mediates posttranslational translocation of many secretory proteins across the endoplasmic reticulum membrane. The study reveals how Sec63 activates the Sec61 channel for substrate polypeptide insertion. The structure also explains the mutually exclusive binding of Sec63 and the ribosome to the channel. Science , this issue p. [84][1] [1]: /lookup/doi/10.1126/science.aav6740

Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress.

In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coliIMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

Positive zip coding in small protein translocation

Most newly synthesized proteins destined for the secretory pathway contain a signal peptide (SP) that triggers cotranslational translocation into the endoplasmic reticulum (ER). However, how small polypeptides undergo ER translocation is not fully understood. In this issue of JBC, Guo et al. describe a mechanism for posttranslational translocation of small secretory proteins featuring a positive charge within the SP N-terminal region. Defects in this element disrupt proper secretion and explain the effects of genetic mutations associated with one type of diabetes.

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 β-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 β-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 β-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.

The meta and bioinformatics analysis of GRP78 expression in gastric cancer.

GRP78 is a molecular chaperone located in endoplasmic reticulum, and induces folding and assembly of newly-synthesized proteins, proteasome degradation of aberrant proteins, and translocation of secretory proteins, autophagy, and epithelial-mesenchymal transition. We performed a systematic meta- and bioinformatics analysis through multiple online databases up to March 14, 2017. It was found that up-regulated GRP78 expression in gastric cancer, compared with normal mucosa at both protein and mRNA levels (p < 0.05). GRP78 expression was positively correlated with depth of invasion, TNM staging and dedifferentiation of gastric cancer (p < 0.05), while its mRNA expression was negatively correlated with depth of invasion, histological grading and dedifferentiation (p < 0.05). A positive association between GRP78 expression and unfavorable overall survival was found in patients with gastric cancer (p < 0.005). A higher GRP78 mRNA expression was positively correlated with overall and progression-free survival rates of all cancer patients, even stratified by aggressive parameters, or as an independent factor (p < 0.05). These findings indicated that GRP78 expression might be employed as a potential marker to indicate gastric carcinogenesis and subsequent progression, even prognosis.

The Endoplasmic Reticulum Unfolded Protein Response in Neurodegenerative Disorders and Its Potential Therapeutic Significance.

In eukaryotic cells, the endoplasmic reticulum (ER) is the cell compartment involved in secretory protein translocation and quality control of secretory protein folding. Different conditions can alter ER function, resulting in the accumulation of unfolded or misfolded proteins within the ER lumen. Such a condition, known as ER stress, elicits an integrated adaptive response known as the unfolded protein response (UPR) that aims to restore proteostasis within the secretory pathway. Conversely, in prolonged cell stress or insufficient adaptive response, UPR signaling causes cell death. ER dysfunctions are involved and contribute to neuronal degeneration in several human diseases, including Alzheimer, Parkinson and Huntington disease and amyotrophic lateral sclerosis. The correlations between ER stress and its signal transduction pathway known as the UPR with neuropathological changes are well established. In addition, much evidence suggests that genetic or pharmacological modulation of UPR could represent an effective strategy for minimizing the progressive neuronal loss in neurodegenerative diseases. Here, we review recent results describing the main cellular mechanisms linking ER stress and UPR to neurodegeneration. Furthermore, we provide an up-to-date panoramic view of the currently pursued strategies for ameliorating the toxic effects of protein unfolding in disease by targeting the ER UPR pathway.

Positive Charge in the n‐region of the Signal Peptide Contributes to Efficient Post‐translational Translocation of Small Secretory Proteins

A majority of secretory proteins in mammalian cells undergo signal recognition particle (SRP) dependent co‐translationally translocation across the endoplasmic reticulum (ER). However, due to minimal time for SRP recognition, many small secretory proteins can be post‐translationally tanslocated into the ER. Several chaperones and cellular machinery have been identified to be involved in post‐translational translocation of these small secretory proteins. However, the intrinsic signals of small secretory proteins that contribute to efficient post‐translational translocation remain unknown. Here, we analyzed signal sequences of eukaryotic secretory proteins and found that the percentage of the proteins with positive charge in the n‐region of their signal peptide (SP) increases substantially as the polypeptides become shorter. Eliminating the n‐region positive charge selectively impaired translocation of small secretory proteins. This translocation impairment appeared to be mainly caused by the defect of post‐translational translocation. Slowing down protein translational rate of small secretory proteins could alleviate their translocation defects caused by loss of SP N‐terminal positive charge. Together, these data revealed a previously unrecognized role of n‐region positive charge in ensuring efficiency of post‐translational translocation of small secretory proteins.Support or Funding InformationThis work was supported by NIH RO1‐DK088856, and by the research grants from The National Natural Science Foundation of China 81070629, 81370895, 81570699.

Signal-3L 2.0: A Hierarchical Mixture Model for Enhancing Protein Signal Peptide Prediction by Incorporating Residue-Domain Cross-Level Features.

Signal peptides play key roles in targeting and translocation of integral membrane proteins and secretory proteins. However, signal peptides present several challenges for automatic prediction methods. One challenge is that it is difficult to discriminate signal peptides from transmembrane helices, as both the H-region of the peptides and the transmembrane helices are hydrophobic. Another is that it is difficult to identify the cleavage site between signal peptides and mature proteins, as cleavage motifs or patterns are still unclear for most proteins. To solve these problems and further enhance automatic signal peptide recognition, we report a new Signal-3L 2.0 predictor. Our new model is constructed with a hierarchical protocol, where it first determines the existence of a signal peptide. For this, we propose a new residue-domain cross-level feature-driven approach, and we demonstrate that protein functional domain information is particularly useful for discriminating between the transmembrane helices and signal peptides as they perform different functions. Next, in order to accurately identify the unique signal peptide cleavage sites along the sequence, we designed a top-down approach where a subset of potential cleavage sites are screened using statistical learning rules, and then a final unique site is selected according to its evolution conservation score. Because this mixed approach utilizes both statistical learning and evolution analysis, it shows a strong capacity for recognizing cleavage sites. Signal-3L 2.0 has been benchmarked on multiple data sets, and the experimental results have demonstrated its accuracy. The online server is available at www.csbio.sjtu.edu.cn/bioinf/Signal-3L/ .

Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis.

ABSTRACTMycolactone is the exotoxin virulence factor produced by Mycobacterium ulcerans, the pathogen responsible for Buruli ulcer. The skin lesions and immunosuppression that are characteristic of this disease result from the action of mycolactone, which targets the Sec61 complex and inhibits the co-translational translocation of secretory proteins into the endoplasmic reticulum. In this study, we investigate the effect of mycolactone on the Sec61-dependent biogenesis of different classes of transmembrane protein (TMP). Our data suggest that the effect of mycolactone on TMP biogenesis depends on how the nascent chain initially engages the Sec61 complex. For example, the translocation of TMP lumenal domains driven by an N-terminal cleavable signal sequence is efficiently inhibited by mycolactone. In contrast, the effect of mycolactone on protein translocation that is driven solely by a non-cleavable signal anchor/transmembrane domain depends on which flanking region is translocated. For example, while translocation of the region N-terminal to a signal anchor/transmembrane domain is refractive to mycolactone, C-terminal translocation is efficiently inhibited. Our findings highlight the diversity of Sec61-dependent translocation and provide a molecular basis for understanding the effect of mycolactone on the biogenesis of different TMPs.

Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease.

Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σE protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.