scholarly journals Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

2021 ◽  
Author(s):  
Pratiti Bhadra ◽  
Scott Dos Santos ◽  
Igor Gamayun ◽  
Tillman Pick ◽  
Clarissa Neumann ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Docking ◽  
Protein Translocation ◽  
Translation Termination ◽  
Potential Interaction ◽  
Mycobacterium Ulcerans ◽  
Resistance Mutations ◽  
Interaction Sites ◽  
Chain Complexes ◽  
Nascent Chain

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon “breathing” during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone’s stabilisation of the latter underpins the phenotype observed.

scholarly journals Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

2021 ◽  
Author(s):  
Pratiti Bhadra ◽  
Scott Dos Santos ◽  
Igor Gamayun ◽  
Tillman Pick ◽  
Joy Ogbechi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Docking ◽  
Protein Translocation ◽  
Translation Termination ◽  
Potential Interaction ◽  
Mycobacterium Ulcerans ◽  
Resistance Mutations ◽  
Interaction Sites ◽  
Chain Complexes ◽  
Nascent Chain

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon breathing during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactones stabilisation of the latter underpins the phenotype observed.

scholarly journals Characterization of Posttranslational Formylglycine Formation by Luminal Components of the Endoplasmic Reticulum

2001 ◽  
Vol 276 (50) ◽  
pp. 47021-47028 ◽  
Author(s):  
Jens Fey ◽  
Martina Balleininger ◽  
Ljudmila V. Borissenko ◽  
Bernhard Schmidt ◽  
Kurt von Figura ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Cysteine Residue ◽  
Ph Optimum ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Biochemical Identification

Cα-formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established anin vitroassay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprisingin vitrosynthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. Thein vitroassay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is ≈10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca2+. Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

scholarly journals NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

2012 ◽  
Vol 23 (16) ◽  
pp. 3027-3040 ◽  
Author(s):  
Ying Zhang ◽  
Uta Berndt ◽  
Hanna Gölz ◽  
Arlette Tais ◽  
Stefan Oellerer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Signal Sequences ◽  
Chain Complexes ◽  
Ribosomal Tunnel ◽  
Nascent Chain ◽  
Combined Data

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.

scholarly journals Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

1987 ◽  
Vol 104 (2) ◽  
pp. 201-208 ◽  
Author(s):  
M Wiedmann ◽  
T V Kurzchalia ◽  
H Bielka ◽  
T A Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Group 4 ◽  
Lysine Residues ◽  
Nascent Chain

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

scholarly journals Regulation of the Ribosome–Membrane Junction at Early Stages of Presecretory Protein Translocation in the Mammalian Endoplasmic Reticulum

1997 ◽  
Vol 139 (7) ◽  
pp. 1697-1708 ◽  
Author(s):  
Christopher V. Nicchitta ◽  
Tianli Zheng
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Membrane Topology ◽  
Hydrophobic Core ◽  
Conducting Channel ◽  
Nascent Chain ◽  
Potential Mode

A series of fusion protein constructs were designed to investigate the contribution of secretory nascent chains to regulation of the ribosome–membrane junction in the mammalian endoplasmic reticulum. As a component of these studies, the membrane topology of the signal sequence was determined at stages of protein translocation immediately after targeting and before signal sequence cleavage. Truncated translation products were used to delimit the analysis to defined stages of translocation. In a study of secretory protein precursors, formation of a protease-resistant ribosome–membrane junction, currently thought to define the pathway of the translocating nascent chain, was observed to be precursor- and stage-dependent. Analysis of the binding of early intermediates indicated that the nascent chain was bound to the membrane independent of the ribosome, and that the binding was predominately electrostatic. The membrane topology of the signal sequence was determined as a function of the stage of translocation, and was found to be identical for all assayed intermediates. Unexpectedly, the hydrophobic core of the signal sequence was observed to be accessible to the cytosolic face of the membrane at stages of translocation immediately after targeting as well as stages before signal sequence cleavage. Removal of the ribosome from bound intermediates did not disrupt subsequent translocation, suggesting that the active state of the protein-conducting channel is maintained in the absence of the bound ribosome. A model describing a potential mode of regulation of the ribosome–membrane junction by the nascent chain is presented.

scholarly journals Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

2018 ◽  
Author(s):  
Wenfei Li ◽  
Fred R. Ward ◽  
Kim F. McClure ◽  
Stacey Tsai-Lan Chang ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Structural Basis ◽  
Small Subset ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractSmall molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

scholarly journals Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

2006 ◽  
Vol 17 (9) ◽  
pp. 3860-3869 ◽  
Author(s):  
Julia Schaletzky ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Signal Recognition Particle ◽  
Structural Data ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Ribosome Binding ◽  
High Affinity ◽  
Chain Complexes ◽  
Nascent Chain

We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC–SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC–SRP complexes can overcome the competition by nontranslating ribosomes.

scholarly journals Signal Sequences Encode Information for Protein Folding in the Endoplasmic Reticulum

2020 ◽  
Author(s):  
Sha Sun ◽  
Xia Li ◽  
Malaiyalam Mariappan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Sequences ◽  
Sequence Dependent ◽  
Proteomic Approach ◽  
Multiple Protein ◽  
Nascent Chain ◽  
In Cells

SummaryRoughly one-third of newly synthesized proteins enter into the endoplasmic reticulum (ER) via the Sec61 translocon. It is unclear how nascent chains bind correct chaperones and properly fold upon entering the ER lumen. We find that signal sequences harbor information to recruit specific chaperones for protein folding in the ER. Using a substrate-trapping proteomic approach, we identify that marginally hydrophobic signal sequences are transiently clogged at the Sec61 translocon, which recruits BiP chaperone through Sec63 to bind onto nascent chains. Surprisingly, this privileged BiP binding not only releases clogged nascent chains into the ER lumen but also prevent inappropriate interactions and promotes folding and maturation. Signal sequence swapping bypasses BiP-dependent unclogging and translocation, but the translocated nascent chain is terminally misfolded after binding the wrong chaperone in the ER lumen. Thus, signal sequence-dependent chaperone recruitment explains why signal sequences are paradoxically diverse and use multiple protein translocation pathways in cells.

scholarly journals Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

1985 ◽  
Vol 100 (6) ◽  
pp. 1913-1921 ◽  
Author(s):  
V Siegel ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
7Sl Rna ◽  
Nascent Chain ◽  
Specific Proteins

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Sign in / Sign up

Export Citation Format

Share Document