The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Wolfgang Wintermeyer - One of the best experts on this subject based on the ideXlab platform.
-
cotranslational protein targeting to the membrane nascent chain transfer in a quaternary complex formed at the Translocon
Scientific Reports, 2018Co-Authors: Albena Draycheva, Sejeong Lee, Wolfgang WintermeyerAbstract:Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG Translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the Translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG–FtsY binding to RNC–SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the Translocon and repositioning of SRP in the post-transfer complex.
-
electrostatics and intrinsic disorder drive Translocon binding of the srp receptor ftsy
Angewandte Chemie, 2016Co-Authors: Albena Draycheva, Thomas Bornemann, Nilsalexander Lakomek, Wolfgang WintermeyerAbstract:Integral membrane proteins in bacteria are co-translationally targeted to the SecYEG Translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N-terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C-terminal membrane targeting sequence. The central A domain binds to the Translocon non-specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the Translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome-bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.
-
the bacterial srp receptor ftsy is activated on binding to the Translocon
Molecular Microbiology, 2016Co-Authors: Albena Draycheva, Thomas Bornemann, Sergey Ryazanov, Nilsalexander Lakomek, Wolfgang WintermeyerAbstract:Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the Translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the Translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid-embedded Translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the Translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the Translocon. We propose that FtsY activation at the Translocon ensures that ribosome-SRP complexes are directed to available Translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the Translocon achieved.
-
ribosome binding induces repositioning of the signal recognition particle receptor on the Translocon
Journal of Cell Biology, 2015Co-Authors: Patrick Kuhn, Andreas Vogt, Narcis-adrian Petriman, Albena Draycheva, Wolfgang Wintermeyer, Lukas Sturm, Friedel Drepper, Bettina Warscheid, Hans-georg KochAbstract:Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG Translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the Translocon is guided by the interaction between SRP and Translocon-bound FtsY in a quaternary targeting complex.
-
lateral opening of the bacterial Translocon on ribosome binding and signal peptide insertion
Nature Communications, 2014Co-Authors: Albena Draycheva, Thomas Bornemann, Marina V Rodnina, Wolfgang WintermeyerAbstract:Proteins are co-translationally inserted into the bacterial plasma membrane via the SecYEG Translocon by lateral release of hydrophobic transmembrane segments into the phospholipid bilayer. The trigger for lateral opening of the Translocon is not known. Here we monitor lateral opening by photo-induced electron transfer (PET) between two fluorophores attached to the two SecY helices at the rim of the gate. In the resting Translocon, the fluorescence is quenched, consistent with a closed conformation. Ribosome binding to the Translocon diminishes PET quenching, indicating opening of the gate. The effect is larger with ribosomes exposing hydrophobic transmembrane segments and vanishes at low temperature. We propose a temperature-dependent dynamic equilibrium between closed and open conformations of the Translocon that is shifted towards partially and fully open by ribosome binding and insertion of a hydrophobic peptide, respectively. The combined effects of ribosome and peptide binding allow for co-translational membrane insertion of successive transmembrane segments.
Arthur E Johnson - One of the best experts on this subject based on the ideXlab platform.
-
sequence specific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum sec61 Translocon
Molecular Biology of the Cell, 2008Co-Authors: David Pitonzo, Arthur E Johnson, Zhongying Yang, Yoshihiro Matsumura, William R. SkachAbstract:A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 Translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of Translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the Translocon in proximity to Sec61alpha. This interaction is initially weak, and TM8 spontaneously dissociates from the Translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61alpha photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the Translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the Translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61alpha can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.
-
the molecular mechanisms underlying bip mediated gating of the sec61 Translocon of the endoplasmic reticulum
Journal of Cell Biology, 2005Co-Authors: Nathan N Alder, Linda M Hendershot, Arthur E Johnson, Ying Shen, Jeffrey L BrodskyAbstract:The Sec61 Translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain–binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the Translocon, and that the reopening of the pore requires an ATP binding–induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain–binding region of BiP and membrane proteins. The mechanism by which BiP mediates Translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.
-
cotranslational integration and initial sorting at the endoplasmic reticulum Translocon of proteins destined for the inner nuclear membrane
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Suraj Saksena, Yuanlong Shao, Sharon C Braunagel, Max D Summers, Arthur E JohnsonAbstract:The current diffusion-retention model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding to nuclear proteins or DNA. Because some data indicate that the sorting of baculovirus envelope proteins to the INM is protein-mediated, we have examined the early stages of INM protein integration and sorting by using photocrosslinking. Both viral and host INM-directed proteins were integrated cotranslationally through the endoplasmic reticulum Translocon, and their nonrandom photocrosslinking to two Translocon proteins, Sec61α and translocating chain-associated membrane protein (TRAM), revealed that the first transmembrane sequence (TMS) of each viral and host INM-directed protein occupied a very similar location within the Translocon. Because few TMSs of non-INM-directed membrane proteins photocrosslink to TRAM, it seems that the INM-directed TMSs occupy different sites within the Translocon than do non-INM-directed TMSs. The distinct proximities of Translocon components to INM-directed TMSs strongly suggest that such TMSs are recognized and initially sorted within the Translocon. Taken together, these data indicate that membrane protein sorting to the INM is an active process involving specific nonnuclear proteins.
-
cotranslational protein integration into the er membrane is mediated by the binding of nascent chains to Translocon proteins
Molecular Cell, 2003Co-Authors: Peter J Mccormick, Arthur E Johnson, Yiwei Miao, Yuanlong Shao, Jialing LinAbstract:During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the Translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM α helix and signal-anchor sequence always faces Sec61α in the Translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61α and is retained at a fixed location within the Translocon, observations that are best explained by the binding of each TM sequence to a Translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the Translocon, nascent chain movement through the Translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.
-
the Translocon a dynamic gateway at the er membrane
Annual Review of Cell and Developmental Biology, 1999Co-Authors: Arthur E Johnson, Michael A Van WaesAbstract:▪ Abstract Cotranslational protein translocation across and integration into the membrane of the endoplasmic reticulum (ER) occur at sites termed Translocons. Translocons are composed of several ER membrane proteins that associate to form an aqueous pore through which secretory proteins and lumenal domains of membrane proteins pass from the cytoplasm to the ER lumen. These sites are not passive holes in the bilayer, but instead are quite dynamic both structurally and functionally. Translocons cycle between ribosome-bound and ribosome-free states, and convert between translocation and integration modes of operation. These changes in functional state are accompanied by structural rearrangements that alter Translocon conformation, composition, and interactions with ligands such as the ribosome and BiP. Recent studies have revealed that the Translocon is a complex and sophisticated molecular machine that regulates the movement of polypeptides through the bilayer, apparently in both directions as well as later...
Albena Draycheva - One of the best experts on this subject based on the ideXlab platform.
-
cotranslational protein targeting to the membrane nascent chain transfer in a quaternary complex formed at the Translocon
Scientific Reports, 2018Co-Authors: Albena Draycheva, Sejeong Lee, Wolfgang WintermeyerAbstract:Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG Translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the Translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG–FtsY binding to RNC–SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the Translocon and repositioning of SRP in the post-transfer complex.
-
electrostatics and intrinsic disorder drive Translocon binding of the srp receptor ftsy
Angewandte Chemie, 2016Co-Authors: Albena Draycheva, Thomas Bornemann, Nilsalexander Lakomek, Wolfgang WintermeyerAbstract:Integral membrane proteins in bacteria are co-translationally targeted to the SecYEG Translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N-terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C-terminal membrane targeting sequence. The central A domain binds to the Translocon non-specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the Translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome-bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.
-
the bacterial srp receptor ftsy is activated on binding to the Translocon
Molecular Microbiology, 2016Co-Authors: Albena Draycheva, Thomas Bornemann, Sergey Ryazanov, Nilsalexander Lakomek, Wolfgang WintermeyerAbstract:Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the Translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the Translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid-embedded Translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the Translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the Translocon. We propose that FtsY activation at the Translocon ensures that ribosome-SRP complexes are directed to available Translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the Translocon achieved.
-
ribosome binding induces repositioning of the signal recognition particle receptor on the Translocon
Journal of Cell Biology, 2015Co-Authors: Patrick Kuhn, Andreas Vogt, Narcis-adrian Petriman, Albena Draycheva, Wolfgang Wintermeyer, Lukas Sturm, Friedel Drepper, Bettina Warscheid, Hans-georg KochAbstract:Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG Translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the Translocon is guided by the interaction between SRP and Translocon-bound FtsY in a quaternary targeting complex.
-
lateral opening of the bacterial Translocon on ribosome binding and signal peptide insertion
Nature Communications, 2014Co-Authors: Albena Draycheva, Thomas Bornemann, Marina V Rodnina, Wolfgang WintermeyerAbstract:Proteins are co-translationally inserted into the bacterial plasma membrane via the SecYEG Translocon by lateral release of hydrophobic transmembrane segments into the phospholipid bilayer. The trigger for lateral opening of the Translocon is not known. Here we monitor lateral opening by photo-induced electron transfer (PET) between two fluorophores attached to the two SecY helices at the rim of the gate. In the resting Translocon, the fluorescence is quenched, consistent with a closed conformation. Ribosome binding to the Translocon diminishes PET quenching, indicating opening of the gate. The effect is larger with ribosomes exposing hydrophobic transmembrane segments and vanishes at low temperature. We propose a temperature-dependent dynamic equilibrium between closed and open conformations of the Translocon that is shifted towards partially and fully open by ribosome binding and insertion of a hydrophobic peptide, respectively. The combined effects of ribosome and peptide binding allow for co-translational membrane insertion of successive transmembrane segments.
Gunnar Von Heijne - One of the best experts on this subject based on the ideXlab platform.
-
forces on nascent polypeptides during membrane insertion and translocation via the sec Translocon
Biophysical Journal, 2018Co-Authors: Michiel J M Niesen, Gunnar Von Heijne, Rickard Hedman, Annika Mullerlucks, Thomas F MillerAbstract:Abstract During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide experiments and coarse-grained molecular dynamics to measure and elucidate the molecular origins of forces that are exerted on NCs during cotranslational membrane insertion and translocation via the Sec Translocon. The approach enables deconvolution of force contributions from NC-Translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a readout of conformational changes in the Sec Translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the Translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis.
-
mechanisms of integral membrane protein insertion and folding
Journal of Molecular Biology, 2015Co-Authors: Florian Cymer, Gunnar Von Heijne, Stephen H WhiteAbstract:The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require Translocons--often referred to as protein-conducting channels--for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of Translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded Translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-Translocon system of assembly. We conclude the review with a perspective suggesting a new view of Translocon-guided MP insertion.
-
spontaneous transmembrane helix insertion thermodynamically mimics Translocon guided insertion
Nature Communications, 2014Co-Authors: Martin B Ulmschneider, Gunnar Von Heijne, Nina Schiller, Jakob P Ulmschneider, B A Wallace, Stephen H WhiteAbstract:The favourable transfer free energy for a transmembrane (TM) α-helix between the aqueous phase and lipid bilayer underlies the stability of membrane proteins. However, the connection between the energetics and process of membrane protein assembly by the Sec61/SecY Translocon complex in vivo is not clear. Here, we directly determine the partitioning free energies of a family of designed peptides using three independent approaches: an experimental microsomal Sec61 Translocon assay, a biophysical (spectroscopic) characterization of peptide insertion into hydrated planar lipid bilayer arrays, and an unbiased atomic-detail equilibrium folding-partitioning molecular dynamics simulation. Remarkably, the measured free energies of insertion are quantitatively similar for all three approaches. The molecular dynamics simulations show that TM helix insertion involves equilibrium with the membrane interface, suggesting that the interface may play a role in Translocon-guided insertion.
-
a biphasic pulling force acts on transmembrane helices during Translocon mediated membrane integration
Nature Structural & Molecular Biology, 2012Co-Authors: Nurzian Ismail, Gunnar Von Heijne, Rickard Hedman, Nina SchillerAbstract:Most membrane proteins are co-translationally inserted into the membrane with the aid of Sec-type Translocons. Using so-called translation-arrest peptides derived from bacterial and mammalian proteins as natural force sensors, a new study now demonstrates how force is exerted on a nascent chain at two distinct points in a transmembrane helix during its transit through the Translocon channel into the membrane.
-
apolar surface area determines the efficiency of Translocon mediated membrane protein integration into the endoplasmic reticulum
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Karin Ojemalm, Stephen H White, Takashi Higuchi, Yang Jiang, Ulo Langel, Ingmarie Nilsson, Hiroaki Suga, Gunnar Von HeijneAbstract:Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 Translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the Translocon channel [van den Berg B, et al. (2004) Nature 427:36-44; Zimmer J, et al. (2008) Nature 455:936-943; Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107:17182-17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid's contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal/(mol · A(2)), the solvation parameter for transfer from Translocon to bilayer is 6-10 cal/(mol · A(2)), pointing to important differences between Translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a thermodynamic partitioning model and insights into the physical properties of the Translocon.
William R. Skach - One of the best experts on this subject based on the ideXlab platform.
-
the ribosome sec61 Translocon complex forms a cytosolically restricted environment for early polytopic membrane protein folding
Journal of Biological Chemistry, 2015Co-Authors: Melissa A Patterson, Prasanna K. Devaraneni, Zhongying Yang, William R. Skach, Anannya Bandyopadhyay, Josha Woodward, Leeann RooneyAbstract:Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-Translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to Translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome Translocon junction, the Sec61-Translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome Translocon complex.
-
cotranslational stabilization of sec62 63 within the er sec61 Translocon is controlled by distinct substrate driven translocation events
Molecular Cell, 2015Co-Authors: Brian J. Conti, Prasanna K. Devaraneni, Zhongying Yang, Larry L. David, William R. SkachAbstract:The ER Sec61 Translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates Translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62, and Sec63. Moreover, Sec62/63 stabilization within the Translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. These data show that cotranslational Translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.
-
sequence specific retention and regulated integration of a nascent membrane protein by the endoplasmic reticulum sec61 Translocon
Molecular Biology of the Cell, 2008Co-Authors: David Pitonzo, Arthur E Johnson, Zhongying Yang, Yoshihiro Matsumura, William R. SkachAbstract:A defining feature of eukaryotic polytopic protein biogenesis involves integration, folding, and packing of hydrophobic transmembrane (TM) segments into the apolar environment of the lipid bilayer. In the endoplasmic reticulum, this process is facilitated by the Sec61 Translocon. Here, we use a photocross-linking approach to examine integration intermediates derived from the ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR) and show that the timing of Translocon-mediated integration can be regulated at specific stages of synthesis. During CFTR biogenesis, the eighth TM segment exits the ribosome and enters the Translocon in proximity to Sec61alpha. This interaction is initially weak, and TM8 spontaneously dissociates from the Translocon when the nascent chain is released from the ribosome. Polypeptide extension by only a few residues, however, results in stable TM8-Sec61alpha photocross-links that persist after peptidyl-tRNA bond cleavage. Retention of these untethered polypeptides within the Translocon requires ribosome binding and is mediated by an acidic residue, Asp924, near the center of the putative TM8 helix. Remarkably, at this stage of synthesis, nascent chain release from the Translocon is also strongly inhibited by ATP depletion. These findings contrast with passive partitioning models and indicate that Sec61alpha can retain TMs and actively inhibit membrane integration in a sequence-specific and ATP-dependent manner.
