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

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
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.

scholarly journals The yeast ERAD-C ubiquitin ligase Doa10 recognizes an intramembrane degron

2015 ◽  
Vol 209 (2) ◽  
pp. 261-273 ◽  
Author(s):  
Gregor Habeck ◽  
Felix A. Ebner ◽  
Hiroko Shimada-Kreft ◽  
Stefan G. Kreft
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Ubiquitin Ligase ◽  
Protein Translocation ◽  
Dependent Manner ◽  
Β Subunit ◽  
Human Orthologue ◽  
Protein Ligases ◽  
Prevailing View

Aberrant endoplasmic reticulum (ER) proteins are eliminated by ER-associated degradation (ERAD). This process involves protein retrotranslocation into the cytosol, ubiquitylation, and proteasomal degradation. ERAD substrates are classified into three categories based on the location of their degradation signal/degron: ERAD-L (lumen), ERAD-M (membrane), and ERAD-C (cytosol) substrates. In Saccharomyces cerevisiae, the membrane proteins Hrd1 and Doa10 are the predominant ERAD ubiquitin-protein ligases (E3s). The current notion is that ERAD-L and ERAD-M substrates are exclusively handled by Hrd1, whereas ERAD-C substrates are recognized by Doa10. In this paper, we identify the transmembrane (TM) protein Sec61 β-subunit homologue 2 (Sbh2) as a Doa10 substrate. Sbh2 is part of the trimeric Ssh1 complex involved in protein translocation. Unassembled Sbh2 is rapidly degraded in a Doa10-dependent manner. Intriguingly, the degron maps to the Sbh2 TM region. Thus, in contrast to the prevailing view, Doa10 (and presumably its human orthologue) has the capacity for recognizing intramembrane degrons, expanding its spectrum of substrates.

scholarly journals Interaction mapping of the Sec61 translocon identifies two Sec61α regions interacting with hydrophobic segments in translocating chains

2018 ◽  
Vol 293 (44) ◽  
pp. 17050-17060 ◽  
Author(s):  
Yuichiro Kida ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Bilayer ◽  
Secretory Pathway ◽  
Cross Linking ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Conducting Channel ◽  
Nascent Chain ◽  
Biochemical Analyses ◽  
Lateral Gate

Many proteins in organelles of the secretory pathway, as well as secretory proteins, are translocated across and inserted into the endoplasmic reticulum membrane by the Sec61 translocon, a protein-conducting channel. The channel consists of 10 transmembrane (TM) segments of the Sec61α subunit and possesses an opening between TM2b and TM7, termed the lateral gate. Structural and biochemical analyses of complexes of Sec61 and its ortholog SecY have revealed that the lateral gate is the exit for signal sequences and TM segments of translocating polypeptides to the lipid bilayer and also involved in the recognition of such hydrophobic sequences. Moreover, even marginally hydrophobic (mH) segments insufficient for membrane integration can be transiently stalled in surrounding Sec61α regions and cross-linked to them, but how the Sec61 translocon accommodates these mH segments remains unclear. Here, we used Cys-scanned variants of human Sec61α expressed in cultured 293-H cells to examine which channel regions associate with mH segments. A TM segment in a ribosome-associated polypeptide was mainly cross-linked to positions at the lateral gate, whereas an mH segment in a nascent chain was cross-linked to the Sec61α pore-interior positions at TM5 and TM10, as well as the lateral gate. Of note, cross-linking at position 180 in TM5 of Sec61α was reduced by an I179A substitution. We therefore conclude that at least two Sec61α regions, the lateral gate and the pore-interior site around TM5, interact with mH segments and are involved in accommodating them.

scholarly journals Cryo–electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1372-1375 ◽  
Author(s):  
Ana S. Ramírez ◽  
Julia Kowal ◽  
Kaspar P. Locher
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Secretory Proteins ◽  
Catalytic Subunits ◽  
Protein Substrates ◽  
Cryo Electron Microscopy ◽  
Structural Differences ◽  
High Mannose ◽  
Equivalent Region ◽  
High Mannose Glycan

Oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose glycan onto secretory proteins in the endoplasmic reticulum. Mammals express two distinct OST complexes that act in a cotranslational (OST-A) or posttranslocational (OST-B) manner. Here, we present high-resolution cryo–electron microscopy structures of human OST-A and OST-B. Although they have similar overall architectures, structural differences in the catalytic subunits STT3A and STT3B facilitate contacts to distinct OST subunits, DC2 in OST-A and MAGT1 in OST-B. In OST-A, interactions with TMEM258 and STT3A allow ribophorin-I to form a four-helix bundle that can bind to a translating ribosome, whereas the equivalent region is disordered in OST-B. We observed an acceptor peptide and dolichylphosphate bound to STT3B, but only dolichylphosphate in STT3A, suggesting distinct affinities of the two OST complexes for protein substrates.

scholarly journals Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62

2020 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Transmembrane Helices ◽  
Conducting Channel ◽  
Essential Step ◽  
Gating Mechanisms ◽  
Cryo Electron Microscopy ◽  
Gate Opening ◽  
Lateral Gate ◽  
Sec61 Channel ◽  
Hierarchical Manner

SummaryThe universally conserved Sec61/SecY channel mediates transport of many newly synthesized polypeptides across membranes, an essential step in protein secretion and membrane protein integration1-5. The channel has two gating mechanisms—a lipid-facing lateral gate, through which hydrophobic signal sequences or transmembrane helices (TMs) are released into the membrane, and a vertical gate, called the plug, which regulates the water-filled pore required for translocation of hydrophilic polypeptide segments6. Currently, how these gates are controlled and how they regulate the translocation process remain poorly understood. Here, by analyzing cryo-electron microscopy (cryo-EM) structures of several variants of the eukaryotic post-translational translocation complex Sec61-Sec62-Sec63, we reveal discrete gating steps of Sec61 and the mechanism by which Sec62 and Sec63 induce these gating events. We show that Sec62 forms a V-shaped structure in front of the lateral gate to fully open both gates of Sec61. Without Sec62, the lateral gate opening narrows, and the vertical pore becomes closed by the plug, rendering the channel inactive. We further show that the lateral gate is opened first by interactions between Sec61 and Sec63 in both cytosolic and luminal domains, a simultaneous disruption of which fully closes the channel. Our study defines the function of Sec62 and illuminates how Sec63 and Sec62 work together in a hierarchical manner to activate the Sec61 channel for post-translational translocation.

Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae

2000 ◽  
Vol 381 (9-10) ◽  
pp. 1025-1028 ◽  
Author(s):  
Anke Prinz ◽  
Enno Hartmann ◽  
Kai-Uwe Kalies
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Protein Translocation ◽  
Growth Conditions ◽  
Ribosome Binding ◽  
Ribosome Binding Sites ◽  
Tight Association ◽  
Biochemical Analyses

Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane

1999 ◽  
Vol 112 (23) ◽  
pp. 4185-4191 ◽  
Author(s):  
K. Romisch
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Transmembrane Proteins ◽  
Retrograde Transport ◽  
Conducting Channel ◽  
Peptide Cleavage ◽  
Sec61 Channel ◽  
Er Membrane

Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.

scholarly journals Cryo-electron Microscopy of Membrane Proteins

2013 ◽  
pp. 325-341 ◽  
Author(s):  
Kenneth N. Goldie ◽  
Priyanka Abeyrathne ◽  
Fabian Kebbel ◽  
Mohamed Chami ◽  
Philippe Ringler ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Cryo Electron Microscopy

scholarly journals Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

2016 ◽  
Vol 27 (6) ◽  
pp. 930-940 ◽  
Author(s):  
Yuichiro Kida ◽  
Yudai Ishihara ◽  
Hidenobu Fujita ◽  
Yukiko Onishi ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Lipid Phase ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Transmembrane Segment ◽  
Conducting Channel ◽  
Transmembrane Segments ◽  
Lipid Environment ◽  
Sec61 Channel

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.

scholarly journals Immunoisolaton of the Yeast Golgi Subcompartments and Characterization of a Novel Membrane Protein, Svp26, Discovered in the Sed5-Containing Compartments

2005 ◽  
Vol 25 (17) ◽  
pp. 7696-7710 ◽  
Author(s):  
Hironori Inadome ◽  
Yoichi Noda ◽  
Hiroyuki Adachi ◽  
Koji Yoda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Membrane Protein ◽  
Considerable Portion ◽  
Marker Proteins ◽  
Evolutionarily Conserved ◽  
Golgi Compartment

ABSTRACT The Golgi apparatus consists of a set of vesicular compartments which are distinguished by their marker proteins. These compartments are physically separated in the Saccharomyces cerevisiae cell. To characterize them extensively, we immunoisolated vesicles carrying either of the SNAREs Sed5 or Tlg2, the markers of the early and late Golgi compartments, respectively, and analyzed the membrane proteins. The composition of proteins was mostly consistent with the position of each compartment in the traffic. We found six uncharacterized but evolutionarily conserved proteins and named them Svp26 (Sed5 compartment vesicle protein of 26 kDa), Tvp38, Tvp23, Tvp18, Tvp15 (Tlg2 compartment vesicle proteins of 38, 23, 18, and 15 kDa), and Gvp36 (Golgi vesicle protein of 36 kDa). The localization of Svp26 in the early Golgi compartment was confirmed by microscopic and biochemical means. Immunoprecipitation indicated that Svp26 binds to itself and a Golgi mannosyltransferase, Ktr3. In the absence of Svp26, a considerable portion of Ktr3 was mislocalized in the endoplasmic reticulum. Our data suggest that Svp26 has a novel role in retention of a subset of membrane proteins in the early Golgi compartments.

scholarly journals How does Sec63 affect the conformation of Sec61 in yeast?

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.

Sign in / Sign up

Export Citation Format

Share Document