The cryo-EM structure of an ERAD protein channel formed by tetrameric human Derlin-1

Endoplasmic reticulum–associated degradation (ERAD) is a process directing misfolded proteins from the ER lumen and membrane to the degradation machinery in the cytosol. A key step in ERAD is the translocation of ER proteins to the cytosol. Derlins are essential for protein translocation in ERAD, but the mechanism remains unclear. Here, we solved the structure of human Derlin-1 by cryo–electron microscopy. The structure shows that Derlin-1 forms a homotetramer that encircles a large tunnel traversing the ER membrane. The tunnel has a diameter of about 12 to 15 angstroms, large enough to allow an α helix to pass through. The structure also shows a lateral gate within the membrane, providing access of transmembrane proteins to the tunnel, and thus, human Derlin-1 forms a protein channel for translocation of misfolded proteins. Our structure is different from the monomeric yeast Derlin structure previously reported, which forms a semichannel with another protein.

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

Science ◽

10.1126/science.aav6740 ◽

2018 ◽

Vol 363 (6422) ◽

pp. 84-87 ◽

Cited By ~ 32

Author(s):

Samuel Itskanov ◽

Eunyong Park

Keyword(s):

Electron Microscopy ◽

Saccharomyces Cerevisiae ◽

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Binding Sites ◽

Protein Translocation ◽

Secretory Proteins ◽

Conducting Channel ◽

Cryo Electron Microscopy ◽

Lateral Gate

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.

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The structure of the bacterial protein translocation complex SecYEG

Biochemical Society Transactions ◽

10.1042/bst0331225 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1225-1230 ◽

Cited By ~ 5

Author(s):

I. Collinson

Keyword(s):

Protein Transport ◽

Protein Translocation ◽

Membrane Insertion ◽

Bacterial Protein ◽

X Ray ◽

Substrate Protein ◽

Cryo Electron Microscopy ◽

Membrane Bound ◽

Pass Through ◽

Translational Mechanisms

Proteins destined for secretion, membrane insertion or organellar import contain signal sequences that direct them to the membrane. Once there, transport machines receive and translocate them appropriately across or into the membrane. The related SecY and Sec61 protein translocation complexes are ubiquitous components of machines that are essential for protein transport. They co-operate with various partners such that the substrate polypeptide is pulled or pushed through the membrane by post- or co-translational mechanisms. In bacteria and archaea, the SecY complex (SecYEG/SecYEβ) is a heterotrimer, which associates with ribosomes so that the polypeptide is threaded through the channel during its synthesis. Bacteria possess an additional pathway, whereby the newly synthesized substrate protein is maintained in an unfolded conformation and is engaged by the ATPase SecA and delivered to the translocon. Recent medium- (cryo-electron microscopy) and high-resolution (X-ray) structures of the Sec complex have dramatically increased our understanding about how proteins pass through membranes, but have posed a number of new questions. The Sec complex is active as an oligomer, but the structure indicates that the protein-conducting channel is formed by a monomer of SecYEG. Structures of the membrane-bound dimer of Escherichia coli SecYEG and the detergent-solubilized monomer of Methanococcus jannaschii SecYEβ will be described and discussed in the context of the mechanism that underlies protein secretion and membrane insertion.

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Positive Charges Determine the Topology and Functionality of the Transmembrane Domain in the Chloroplastic Outer Envelope Protein Toc34

The Journal of Cell Biology ◽

10.1083/jcb.141.4.895 ◽

1998 ◽

Vol 141 (4) ◽

pp. 895-904 ◽

Cited By ~ 21

Author(s):

Timo May ◽

Jürgen Soll

Keyword(s):

Envelope Protein ◽

Transmembrane Domain ◽

Protein Translocation ◽

Small Subunit ◽

Lipid Phase ◽

Outer Envelope ◽

Double Positive ◽

Membrane Anchor ◽

Bisphosphate Carboxylase ◽

Α Helix

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation Ncyto-Cin. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid residues NH2- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.

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Translocation channel gating kinetics balances protein translocation efficiency with signal sequence recognition fidelity

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0070 ◽

2011 ◽

Vol 22 (17) ◽

pp. 2983-2993 ◽

Cited By ~ 21

Author(s):

Steven F. Trueman ◽

Elisabet C. Mandon ◽

Reid Gilmore

Keyword(s):

Structural Transition ◽

Protein Translocation ◽

Signal Sequence ◽

Channel Gating ◽

Critical Event ◽

Protein Insertion ◽

Sequence Recognition ◽

Translocation Efficiency ◽

Lateral Gate ◽

Insight Into

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence–binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in channel gating. The translocation defect caused by L7 mutations could be suppressed by the prl class of sec61 alleles, which reduce the fidelity of signal sequence recognition. The prl mutants are proposed to act by destabilizing the closed conformation of the translocation channel. Our results indicate that the equilibrium between the open and closed conformations of the protein translocation channel maintains a balance between translocation activity and signal sequence recognition fidelity.

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How does Sec63 affect the conformation of Sec61 in yeast?

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008855 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1008855

Author(s):

Pratiti Bhadra ◽

Lalitha Yadhanapudi ◽

Karin Römisch ◽

Volkhard Helms

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Pathway ◽

Endoplasmic Reticulum Membrane ◽

Intermolecular Contact ◽

Cryo Electron Microscopy ◽

Ring Diameter ◽

Lateral Gate ◽

Co Precipitation ◽

Dynamics Simulations ◽

Sec61 Channel

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the accessory membrane protein Sec62 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate.

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Regulation of ER-associated degradation via p97/VCP-interacting motif

Biochemical Society Transactions ◽

10.1042/bst0360818 ◽

2008 ◽

Vol 36 (5) ◽

pp. 818-822 ◽

Cited By ~ 14

Author(s):

Petek Ballar ◽

Shengyun Fang

Keyword(s):

Endoplasmic Reticulum ◽

Ubiquitin Ligase ◽

Terminal Region ◽

Misfolded Proteins ◽

Interacting Protein ◽

Aaa Atpase ◽

Endoplasmic Reticulum Associated Degradation ◽

Er Membrane

p97/VCP (valosin-containing protein) is a cytosolic AAA (ATPase associated with various cellular activities) essential for retrotranslocation of misfolded proteins during ERAD [ER (endoplasmic reticulum)-associated degradation]. gp78, an ERAD ubiquitin ligase, is one of the p97/VCP recruitment proteins localized to the ER membrane. A newly identified VIM (p97/VCP-interacting motif) in gp78 has brought about novel insights into mechanisms of ERAD, such as the presence of a p97/VCP-dependent but Ufd1-independent retrotranslocation during gp78-mediated ERAD. Additionally, SVIP (small p97/VCP-interacting protein), which contains a VIM in its N-terminal region, negatively regulates ERAD by uncoupling p97/VCP and Derlin1 from gp78. Thus SVIP may protect cells from damage by extravagant ERAD.

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Mobility of the SecA 2-helix-finger is not essential for polypeptide translocation via the SecYEG complex

The Journal of Cell Biology ◽

10.1083/jcb.201205191 ◽

2012 ◽

Vol 199 (6) ◽

pp. 919-929 ◽

Cited By ~ 37

Author(s):

Sarah Whitehouse ◽

Vicki A.M. Gold ◽

Alice Robson ◽

William J. Allen ◽

Richard B. Sessions ◽

...

Keyword(s):

Conformational Changes ◽

Protein Translocation ◽

Energy Transduction ◽

Power Stroke ◽

Active Complex ◽

Essential Protein ◽

The Core ◽

Substrate Protein ◽

Ideal Position ◽

Protein Channel

The bacterial ATPase SecA and protein channel complex SecYEG form the core of an essential protein translocation machinery. The nature of the conformational changes induced by each stage of the hydrolytic cycle of ATP and how they are coupled to protein translocation are not well understood. The structure of the SecA–SecYEG complex revealed a 2-helix-finger (2HF) of SecA in an ideal position to contact the substrate protein and push it through the membrane. Surprisingly, immobilization of this finger at the edge of the protein channel had no effect on translocation, whereas its imposition inside the channel blocked transport. This analysis resolves the stoichiometry of the active complex, demonstrating that after the initiation process translocation requires only one copy each of SecA and SecYEG. The results also have important implications on the mechanism of energy transduction and the power stroke driving transport. Evidently, the 2HF is not a highly mobile transducing element of polypeptide translocation.

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Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

The Journal of Cell Biology ◽

10.1083/jcb.202103199 ◽

2021 ◽

Vol 221 (1) ◽

Author(s):

Jason C. Casler ◽

Natalie Johnson ◽

Adam H. Krahn ◽

Areti Pantazopoulou ◽

Kasey J. Day ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Transmembrane Proteins ◽

Membrane Traffic ◽

Yeast Saccharomyces Cerevisiae ◽

Clathrin Adaptor ◽

Pass Through ◽

Kinetics Of

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5–dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5–dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

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Molecular Modeling of Signal Peptide Recognition by Eukaryotic Sec Complexes

International Journal of Molecular Sciences ◽

10.3390/ijms221910705 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10705

Author(s):

Pratiti Bhadra ◽

Volkhard Helms

Keyword(s):

Protein Translocation ◽

Conformational Dynamics ◽

Primary Component ◽

Signal Peptides ◽

Accessory Proteins ◽

Simulation Studies ◽

Peptide Recognition ◽

Substrate Peptide ◽

Modelling Studies ◽

Lateral Gate

Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways.

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Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters

Journal of Bacteriology ◽

10.1128/jb.00432-10 ◽

2010 ◽

Vol 192 (21) ◽

pp. 5588-5602 ◽

Cited By ~ 22

Author(s):

Elvira Marín ◽

Gustavo Bodelón ◽

Luis Ángel Fernández

Keyword(s):

Disulfide Bonds ◽

Protein Translocation ◽

Human Pathogens ◽

Passenger Domain ◽

Extracellular Milieu ◽

Functional Features ◽

Α Helix ◽

Terminal Domains

ABSTRACT Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.

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