Protein Translocation Channel Research Articles

Discovery and biological evaluation of an adamantyl-amide derivative with likely MmpL3 inhibitory activity

A series of molecules containing bulky lipophilic scaffolds was screened for activity against Mycobacterium tuberculosis and a number of compounds with antimycobacterial activity were identified. The most active compound, (2E)-N-(adamantan-1-yl)-3-phenylprop-2-enamide (C1), has a low micromolar minimum inhibitory concentration, low cytotoxicity (therapeutic index = 32.26), low mutation frequency and is active against intracellular Mycobacterium tuberculosis. Whole genome sequencing of mutants resistant to C1 showed a mutation in mmpL3 which may point to the involvement of MmpL3 in the antimycobacterial activity of the compound. In silico mutagenesis and molecular modelling studies were performed to better understand the binding of C1 within MmpL3 and the role that the specific mutation may play in the interaction at protein level. These analyses revealed that the mutation increases the energy required for binding of C1 within the protein translocation channel of MmpL3. The mutation also decreases the solvation energy of the protein, suggesting that the mutant protein might be more solvent-accessible, thereby restricting its interaction with other molecules. The results reported here describe a new molecule that may interact with the MmpL3 protein, providing insights into the effect of mutations on protein-ligand interactions and enhancing our understanding of this essential protein as a priority drug target.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Exploring voltage sensing in protein-translocating channel SecYEG.

The conserved protein translocation channel SecYEG facilitates protein transport across as well as integration into the membrane. To maintain ion gradients across the membrane, essential for many cellular processes, it is of utmost importance that the Sec channel remains impermeable to these ions despite its bulky substrates. It has been shown via electrophysiological experiments that physiological levels of transmembrane voltage allow the channel to stay impermeable. Yet, the substrate-bound SecYEG becomes leaky once the absolute value of the transmembrane voltage drops below a certain threshold [1,2]. We proposed that the dipole moment of helix 2b is responsible for voltage sensing and that other complex parts must also contribute [3]. Molecular dynamics simulations suggested the involvement of the 5th helix dipole. Replacing S188 on its cytoplasmatic terminus allowed reverting its electrostatic contribution. Here, we measured the kinetics of voltage-gated closings of the thus mutated bacterial SecYEG translocon, reconstituted into planar bilayers. The translocon openings were triggered by the binding of ribosome nascent chain complexes harboring the voltage sensor domain of the voltage-gated potassium channel KvAP. S188E exhibited an increased voltage sensitivity, whereas S188R partially de-sensitized the translocon. Our results confirm the earlier hypothesis, in which the dipole moment of multiple TM-helices serves as SecYEG's voltage sensor. Support of the Austrian Science Fund (FWF), grant P34584 to PP, is gratefully acknowledged.

Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61β/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol.

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Membrane protein biogenesis at the ER: the highways and byways.

The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle, which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.

Structure of the Inhibited State of the Sec Translocon

SummaryProtein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.

Observing the nonvectorial yet cotranslational folding of a multidomain protein, LDL receptor, in the ER of mammalian cells

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.

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.

Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum inSaccharomyces cerevisiae

Mammalian cells express two oligosaccharyltransferase complexes, STT3A and STT3B, that have distinct roles in N-linked glycosylation. The STT3A complex interacts directly with the protein translocation channel to mediate glycosylation of proteins using an N-terminal–to–C-terminal scanning mechanism. N-linked glycosylation of proteins in budding yeast has been assumed to be a cotranslational reaction. We have compared glycosylation of several glycoproteins in yeast and mammalian cells. Prosaposin, a cysteine-rich protein that contains STT3A-dependent glycosylation sites, is poorly glycosylated in yeast cells and STT3A-deficient human cells. In contrast, a protein with extreme C-terminal glycosylation sites was efficiently glycosylated in yeast by a posttranslocational mechanism. Posttranslocational glycosylation was also observed for carboxypeptidase Y–derived reporter proteins that contain closely spaced acceptor sites. A comparison of two recent protein structures indicates that the yeast OST is unable to interact with the yeast heptameric Sec complex via an evolutionarily conserved interface due to occupation of the OST binding site by the Sec63 protein. The efficiency of glycosylation in yeast is not enhanced for proteins that are translocated by the Sec61 or Ssh1 translocation channels instead of the Sec complex. We conclude that N-linked glycosylation and protein translocation are not directly coupled in yeast cells.

Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites.

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

Structure of the posttranslational Sec protein-translocation channel complex from yeast.

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo-electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61's lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.

Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.

Conserved motifs on the cytoplasmic face of the protein translocation channel are critical for the transition between resting and active conformations

The Sec61 complex is the primary cotranslational protein translocation channel in yeast (Saccharomyces cerevisiae). The structural transition between the closed inactive conformation of the Sec61 complex and its open and active conformation is thought to be promoted by binding of the ribosome nascent-chain complex to the cytoplasmic surface of the Sec61 complex. Here, we have analyzed new yeast Sec61 mutants that selectively interfere with cotranslational translocation across the endoplasmic reticulum. We found that a single substitution at the junction between transmembrane segment TM7 and the L6/7 loop interferes with cotranslational translocation by uncoupling ribosome binding to the L6/7 loop from the separation of the lateral gate transmembrane spans. Substitutions replacing basic residues with acidic residues in the C-terminal tail of Sec61 had an unanticipated impact upon binding of ribosomes to the Sec61 complex. We found that similar charge-reversal mutations in the N-terminal tail and in cytoplasmic loop L2/3 did not alter ribosome binding but interfered with translocation channel gating. These findings indicated that these segments are important for the structural transition between the inactive and active conformations of the Sec61 complex. In summary our results have identified additional cytosolic segments of the Sec61 complex important for promoting the structural transition between the closed and open conformations of the complex. We conclude that positively charged residues in multiple cytosolic segments, as well as bulky hydrophobic residues in the L6/7-TM7 junction, are required for cotranslational translocation or integration of membrane proteins by the Sec61 complex.

SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels.

Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.

Structural determinants for protein unfolding and translocation by the Hsp104 protein disaggregase.

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr257 and Tyr662) involved in substrate binding. However, the role of conserved aliphatic residues (Lys256, Lys258, and Val663) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr257 and Tyr662 are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.

DC2 and KCP2 mediate the interaction between the oligosaccharyltransferase and the ER translocon.

In metazoan organisms, the STT3A isoform of the oligosaccharyltransferase is localized adjacent to the protein translocation channel to catalyze co-translational N-linked glycosylation of proteins in the endoplasmic reticulum. The mechanism responsible for the interaction between the STT3A complex and the translocation channel has not been addressed. Using genetically modified human cells that are deficient in DC2 or KCP2 proteins, we show that loss of DC2 causes a defect in co-translational N-glycosylation of proteins that mimics an STT3A-/- phenotype. Biochemical analysis showed that DC2 and KCP2 are responsible for mediating the interaction between the protein translocation channel and the STT3A complex. Importantly, DC2- and KCP2-deficient STT3A complexes are stable and enzymatically active. Deletion mutagenesis revealed that a conserved motif in the C-terminal tail of DC2 is critical for assembly into the STT3A complex, whereas the lumenal loop and the N-terminal cytoplasmic segment are necessary for the functional interaction between the STT3A and Sec61 complexes.

Robust Ligand Binding to the Protein Translocation Complex (Secyeg) Requires a Lipid Environment

Many secretory proteins pass the membrane barrier via the bacterial SecY complex. SecY is assisted by the motor protein SecA, which is thought to push the polypeptides through the SecY pore with its two-helix finger. This model requires ATP molecules to exchange continuously during translocation. Whether the resulting change in SecA-SecY binding affinity leads to SecA release is unknown. Here we have tested the hypothesis by monitoring SecA binding to the SecY complex using lanthanide resonance energy transfer between a fluorescent label on the tip of its two-helix finger and Tb3+ in a genetically engineered binding pocket on SecY. In the absence of ATP, binding only occurred to SecY molecules that were reconstituted into lipid bilayers, but not to SecY molecules in detergent. In a fraction of these binding events we observed a deep insertion of SecA's two-helix finger into the SecY-channel. These penetrations resulted in relocation of SecY's plug as indicated by measurements of the distance between fingertip and plug. The ‘‘plug’’ is a short helix that is (i) normally located in the center of the channel adjacent to its hydrophobic constriction zone (pore ring) and (ii) blocks the passage of small molecules through the idle SecY complex (1). Observation of ion channel activity in reconstituted planar lipid bilayers confirmed that the plug had moved out of the pore. As previously observed in the case of ribosome nascent chain complexes (2), physiological values of the membrane potential were required to close the channel. We conclude that (i) SecA binding is sufficient to open the protein translocation channel in its lipid environment and (ii) the current transport model needs to be revised to account for SecY's voltage sensitivity.The project was supported by the Austrian Science Fund (P25844, P28213).1. Saparov SM,..,Pohl P (2007) Mol Cell 26:501-509.2. Knyazev DG, ⋯, Pohl P (2014) J Biol Chem 289:24611-24616.

Tuning membrane protein mobility by confinement into nanodomains.

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.