Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes (original) (raw)
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Structural insight into the protein translocation channel
Current Opinion in Structural Biology, 2004
A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time.
Bacterial protein translocation requires only one copy of the SecY complex in vivo
Journal of Cell Biology, 2012
The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between...
Cell, 2007
Many proteins are translocated across the bacterial plasma membrane by the interplay of the cytoplasmic ATPase SecA with a proteinconducting channel, formed from the evolutionarily conserved heterotrimeric SecY complex. Here, we have used purified E. coli components to address the mechanism of translocation. Disulfide bridge crosslinking demonstrates that SecA transfers both the signal sequence and the mature region of a secreted substrate into a single SecY molecule. However, protein translocation involves oligomers of the SecY complex, because a SecY molecule defective in translocation can be rescued by linking it covalently with a wild-type SecY copy. SecA interacts through one of its domains with a nontranslocating SecY copy and moves the polypeptide chain through a neighboring SecY copy. Oligomeric channels with only one active pore likely mediate protein translocation in all organisms.
Structure of the SecY channel during initiation of protein translocation
Nature, 2013
Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY-or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis 1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase 3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryoelectron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N-and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Journal of Molecular Biology, 1999
The SecYEG complex is a major component of the protein translocation apparatus in the cytoplasmic membrane of bacteria. We have puri®ed a translocationally active complex of the two subunits, SecY and SecE, from Bacillus subtilis. As demonstrated by electron microscopy, SecY/E forms ring structures in detergent solution and in intact lipid bilayers, often with a quasi-pentagonal appearance in projection. The particles represent oligomeric assemblies of the SecY/E complex and are similar to those formed by the eukaryotic Sec61p complex. We propose that these SecY/E rings represent protein-conducting channels and that the two essential membrane components SecY and SecE are suf®cient for their formation.
Translocation of proteins through the Sec61 and SecYEG channels
Current Opinion in Cell Biology, 2009
The Sec61 and SecYEG translocation channels mediate the selective transport of proteins across the endoplasmic reticulum and bacterial inner membrane, respectively. These channels are also responsible for the integration of membrane proteins. To accomplish these two critical events in protein expression, the transport channels undergo conformational changes to permit the export of lumenal domains and the integration of transmembrane spans. Novel insight into how these channels open during protein translocation has been provided by a combination of the analysis of new channel structures, biochemical characterization of translocation intermediates, molecular dynamics simulations, and in vivo and in vitro analysis of structure-based Sec61 and SecY mutants.
Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes
Nature, 2007
A decisive step in the biosynthesis of many proteins is their partial or complete translocation across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. Most of these proteins are translocated through a protein-conducting channel that is formed by a conserved, heterotrimeric membrane-protein complex, the Sec61 or SecY complex. Depending on channel binding partners, polypeptides are moved by different mechanisms: the polypeptide chain is transferred directly into the channel by the translating ribosome, a ratcheting mechanism is used by the endoplasmic reticulum chaperone BiP, and a pushing mechanism is used by the bacterial ATPase SecA. Structural, genetic and biochemical data show how the channel opens across the membrane, releases hydrophobic segments of membrane proteins laterally into lipid, and maintains the membrane barrier for small molecules.
The structure of the Sec complex and the problem of protein translocation
EMBO reports, 2006
Proteins synthesized in the cytosol either remain there or are localized to a specific membrane and subsequently translocated to another cellular compartment. These extracytosolic proteins have to cross, or be inserted into, a phospholipid bilayer-a process governed by membrane-bound protein transporters designed to recognize and receive appropriate polypeptides and thread them through the membrane. One such translocation complex, SecY/Sec61, is found in every cell, in either the plasma membrane of bacteria and archaea or the endoplasmic reticulum membrane of eukaryotes. Recent structural findings, combined with previous genetic and biochemical studies, have helped to describe how the passage of proteins through the membrane might occur, but several points of uncertainty remain.
Structure of the post-translational protein translocation machinery of the ER membrane
Nature, 2018
Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane 1,2. Proteins with hydrophobic signal sequences are first recognized by the signal recognition particle (SRP) 3,4 and then moved co-translationally through the Sec61/SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass SRP and are moved through the channel post-translationally 5,6. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62/Sec63 complex 7-9 , and substrates are moved through the channel by the luminal BiP ATPase 9. How the Sec62/63 complex activates the Sec61 channel for posttranslational translocation is unclear. Here, we report the electron cryo-microscopy (cryo-EM) structure of the Sec complex from S. cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71, and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting with both cytosolic loops in the C-terminal half of Sec61 and trans-membrane (TM) segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70 (ref. 10). Our structure shows how the Sec61 channel is activated for post-translational protein translocation. The Sec61 channel is formed from the multi-spanning Sec61 protein and two singlespanning proteins (called Sbh1 and Sss1 in S. cerevisiae) 7,8. Sec61 and its prokaryotic homolog SecY consist of two halves that form an hourglass-shaped pore with a constriction Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Preserving the membrane barrier for small molecules during bacterial protein translocation
Nature, 2011
Many proteins are translocated through the SecY channel in bacteria and archaea, and the related Sec61 channel in eukaryotes 1. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids 2. While the cytoplasmic cavity of the channel is empty, the extra-cellular cavity is filled with a short helix, the plug 2 , which moves out of the way during protein translocation 3,4. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial 2,5-8. Here, we have addressed this issuein intact E. coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. In the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation the pore ring forms a gasket-like seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms suggests a universal mechanism by which the membrane barrier is maintained during protein translocation. Bacteria offer a unique opportunity to test the permeation of small molecules through the protein translocation channel, as the channel is located in the plasma membrane and is therefore accessible in intact cells. To test the permeability of the resting channel, we compared E. coli wild-type SecY, expected to be sealed, with a plug-deletion mutant(ΔP), which should be constitutively open(Fig. S1); although a new plug may form from neighboring polypeptide segments 9 , it likely blocks the channel only transiently 8. Wild-type and ΔP mutant SecY channels were expressed under an inducible promoter at about the same level as the endogenous protein (Fig. S2). Expression of the ΔP mutant caused only a moderate growth defect (Fig. S2). Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: