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

Kida, Yuichiro; Ishihara, Yudai; Fujita, Hideaki; Onishi, Yukiko; Sakaguchi, Masao

doi:10.1091/mbc.e15-09-0672

Cited by 6 publications

(17 citation statements)

References 56 publications

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“…A study by von Heijne and colleagues reported that >30% of TMDs inefficiently integrate into the membrane because they contain marginally hydrophobic TMDs [77]. The integration of these TMDs requires the presence of other stabilizing TMDs [78–81]. To determine whether the αENaC TMDs poorly integrate, we performed free energy calculations for membrane insertion (http://dgpred.cbr.su.se/index.php?p=home).…”

Section: Discussionmentioning

confidence: 99%

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein

Buck

Jordahl

Yates

et al. 2017

Biochemical Journal

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In the kidney, the epithelial sodium channel (ENaC) regulates blood pressure through control of sodium and volume homeostasis, and in the lung, ENaC regulates the volume of airway and alveolar fluids. ENaC is a heterotrimer of homologous α-, β- and γ-subunits, and assembles in the endoplasmic reticulum (ER) before it traffics to and functions at the plasma membrane. Improperly folded or orphaned ENaC subunits are subject to ER quality control and targeted for ER-associated degradation (ERAD). We previously established that a conserved, ER lumenal, molecular chaperone, Lhs1/GRP170, selects αENaC, but not β- or γ-ENaC, for degradation when the ENaC subunits were individually expressed. We now find that when all three subunits are co-expressed, Lhs1-facilitated ERAD was blocked. To determine which domain–domain interactions between the ENaC subunits are critical for chaperone-dependent quality control, we employed a yeast model and expressed chimeric α/βENaC constructs in the context of the ENaC heterotrimer. We discovered that the βENaC transmembrane domain was sufficient to prevent the Lhs1-dependent degradation of the α-subunit in the context of the ENaC heterotrimer. Our work also found that Lhs1 delivers αENaC for proteasome-mediated degradation after the protein has become polyubiquitinated. These data indicate that the Lhs1 chaperone selectively recognizes an immature form of αENaC, one which has failed to correctly assemble with the other channel subunits via its transmembrane domain.

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Section: Discussionmentioning

confidence: 99%

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein

Buck

Jordahl

Yates

et al. 2017

Biochemical Journal

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“…It thus requires energy to pull the Hsegment out and the following more hydrophilic sequence into the translocon, most likely through Brownian motion of the already translocated polypeptide and associated chaperones. Indeed, it has been shown experimentally that marginally hydrophobic segments stall at the translocon and cause the subsequent chain to accumulate on the cytosolic side, and to eventually loop out into the cytosol (Kida et al, 2016;Onishi et al, 2013). Accordingly, the sequence C-terminal to the H-segment has time and opportunity to influence H-segment integration.…”

Section: Discussionmentioning

confidence: 99%

Integration of transmembrane domains is regulated by their downstream sequences

Junne

Spiess

2017

Journal of Cell Science

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The Sec61 translocon catalyzes translocation of proteins into the endoplasmic reticulum and the lateral integration of transmembrane segments into the lipid bilayer. Integration is mediated by the hydrophobicity of a polypeptide segment consistent with thermodynamic equilibration between the translocon and the lipid membrane. Integration efficiency of a generic series of increasingly hydrophobic sequences (H-segments) was found to diverge significantly in different reporter constructs as a function of the ∼100 residues that are C-terminal to the H-segments. The hydrophobicity threshold of integration was considerably lowered through insertion of generic ∼20-residue peptides either made of flexible glycine-serine repeats, containing multiple negative charges, or consisting of an oligoproline stretch. A highly flexible, 100-residue glycine-serine stretch maximally enhanced this effect. The apparent free energy of integration was found to be changed by more than 3 kcal/mol with the downstream sequences tested. The C-terminal sequences could also be shown to affect integration of natural mildly hydrophobic sequences. The results suggest that the conformation of the nascent polypeptide in the protected cavity between the ribosome and translocon considerably influences the release of the H-segment into the bilayer.

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“…We previously demonstrated that designed mH segments in ribosome-bound nascent chains are stalled in surrounding Sec61␣ regions and cross-linked to them (30). Therefore, we next examined which Cys positions in Sec61␣ are cross-linked to mH segments inserted in a secretory protein, rat serum albumin (RSA).…”

Section: Stalling Mh Segments Interact With the Pore-interior Site Atmentioning

confidence: 99%

“…Membrane insertion of such mH segments is supported by the following TM segments forming an N lumen /C cyto orientation (25)(26)(27)(28). Additionally, synthetic and natural mH segments can be stalled in the membrane (29,30), and the stalling is enhanced by the following positively charged residues (31,32). Site-specific cross-linking and chemical modification of stalling mH segments indicated that such segments are in an aqueous location and surrounded with Sec61␣ regions (30).…”

mentioning

confidence: 99%

“…Additionally, synthetic and natural mH segments can be stalled in the membrane (29,30), and the stalling is enhanced by the following positively charged residues (31,32). Site-specific cross-linking and chemical modification of stalling mH segments indicated that such segments are in an aqueous location and surrounded with Sec61␣ regions (30). How the Sec61 channel accommodates these mH segments, however, remained unclear.…”

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confidence: 99%

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

Kida

Sakaguchi

2018

Journal of Biological Chemistry

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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.

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

Cited by 6 publications

References 56 publications

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein

Integration of transmembrane domains is regulated by their downstream sequences

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

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