Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

Kida, Yuichiro; Morimoto, Fumiko; Sakaguchi, Masao

doi:10.1083/jcb.200707050

Cited by 44 publications

(52 citation statements)

References 46 publications

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“…N-domain translocation starts with entry of the SA-I sequence into the translocon. In the case of longer N-domains, the hydrophobic segment (H-segment) of the SA-I sequence acquires the TM orientation before the N-domain is completely translocated (7,10). It is also possible that the stroke of the N terminus of the SA-I sequence across the membrane traps the last ␤-strand detached from the first strand in the translocon and leads to unfolding of the N-domain.…”

Section: Resultsmentioning

confidence: 99%

“…The SBP-tag of 38 residues binds SAv with high affinity and has been used to arrest N-domain translocation mediated by the SA-I sequence (7). The arrested translocation can be resumed by biotin (7). We demonstrate here that the motive force for translocation can be quantitatively estimated using these probes.…”

mentioning

confidence: 89%

“…On the other hand, a large aqueous environment of the translocon has been observed (6). Moreover, the translocon possesses such remarkable flexibility that two translocating hydrophilic polypeptide chains can be simultaneously accommodated (7). The dynamics of the Sec61/ SecY channel and cooperation of the channels within the oligomeric form, however, remain to be solved (8).…”

mentioning

confidence: 99%

“…These domains have been successfully used to analyze mitochondrial protein import (12,13). The SBP-tag of 38 residues binds SAv with high affinity and has been used to arrest N-domain translocation mediated by the SA-I sequence (7). The arrested translocation can be resumed by biotin (7).…”

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confidence: 99%

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Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Kida

Morimoto

Sakaguchi

2009

Journal of Biological Chemistry

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Many proteins are translocated across and integrated into the endoplasmic reticulum membrane. The type I signal anchor sequence mediates the translocation of its preceding region through the endoplasmic reticulum membrane, but the source of the motive force has been unclear. Here, we characterized the motive force for N-terminal domain translocation using two probes. First, an Ig-like domain of the muscle protein titin (I27 domain) or its mutants were fused to the N termini, and translocation was examined in a cell-free translation system supplemented with rough microsomal membrane. The N-terminal translocation efficiencies correlated with the mechanical instabilities of the I27 mutants. When the I27 domain was separated from the signal anchor sequence by inserting a spacer, even the most unstable mutant stalled on the cytoplasmic side, whereas its downstream portion spanned the membrane. Proline insertion into the signal anchor sequence also caused a large translocation defect. Second, a streptavidin-binding peptide tag was fused to the N terminus. Titration of streptavidin in the translation system allowed us to estimate the translocation motive force operative on the tag. The motive force was decreased by the proline insertion into the signal anchor sequence as well as by separation from the signal anchor sequence. When the streptavidin-binding peptide tag was separated from the signal anchor, the proline insertion did not induce further deficits in the motive force for the tag. On the basis of the findings obtained by using these two independent techniques, we conclude that the signal sequence itself provides the motive force for N-terminal domain translocation within a limited upstream region.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 89%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Kida

Morimoto

Sakaguchi

2009

Journal of Biological Chemistry

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“…The chemical crosslinking was performed essentially as previously described (Kida et al, 2007). The products were treated on ice for 60 minutes with 10 mM BMOE or its solvent dimethyl sulfoxide.…”

Section: In Vitro Synthesis and Membrane Translocationmentioning

confidence: 99%

Positive charges on the translocating polypeptide chain arrest movement through the translocon

Fujita

Yamagishi

Kida

et al. 2011

Journal of Cell Science

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SummaryPolypeptide chains synthesized by membrane-bound ribosomes are translocated through, and integrated into, the endoplasmic reticulum (ER) membrane by means of the protein translocation channel, the translocon. Positive charges on the nascent chain determine the orientation of the hydrophobic segment as it is inserted into the translocon and enhance the stop-translocation of translocating hydrophobic segments. Here we show that positive charges temporarily arrested ongoing polypeptide chain movement through the ER translocon by electrostatic interaction, even in the absence of a hydrophobic segment. The C-terminus of the polypeptide chain was elongated during the arrest, and then the full-length polypeptide chain moved through the translocon. The translocation-arrested polypeptide was not anchored to the membrane and the charges were on the cytoplasmic side of the membrane. The arrest effect was prevented by negatively charged residues inserted into the positive-charge cluster, and it was also suppressed by high salt conditions. We propose that positive charges are independent translocation regulators that are more active than previously believed.

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Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

Marothy

Elofsson

2015

Protein Science

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Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment.

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Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

Cited by 44 publications

References 46 publications

Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Positive charges on the translocating polypeptide chain arrest movement through the translocon

Marginally hydrophobic transmembrane α‐helices shaping membrane protein folding

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