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

Fujita, Hideaki; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

doi:10.1242/jcs.086850

Cited by 29 publications

(31 citation statements)

References 31 publications

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“…Although such strong discrimination against positively charged amino acids was not observed in eukaryotic signal sequences, long strings of positively charged residues placed downstream of the signal sequence can block translocation of eukaryotic secretory proteins [50-52]. What is the nature of this inhibition of translocation?…”

Section: Resultsmentioning

confidence: 99%

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

Nilsson

Lara

Hessa

et al. 2015

Journal of Molecular Biology

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The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes (RNCs) to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP-signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP-ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP-signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP-signal sequence affinity and targeting to the translocon.

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Section: Resultsmentioning

confidence: 99%

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

Nilsson

Lara

Hessa

et al. 2015

Journal of Molecular Biology

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“…translocation at least in vitro (Fujita et al, 2012) and thus might neutralize an additional inhibitory effect on integration. Finally, a longer oligoproline peptide is likely to assume its sterically preferred conformation as a polyproline type II helix, which is very extended with 3.1-Å spacing per residue.…”

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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“…An electrostatic interaction between the negatively charged ribosomal RNA helix 59 and nascent IMPs suggests an attractive molecular mechanism for retaining positively charged loop regions on the cytoplasmic side of the membrane during integration into the lipid phase. Support for such a mechanism is provided by the observation that positively charged residues can indeed arrest the movement of nascent proteins through the PCC in a salt-sensitive manner 40 . Our findings thus provide a starting point for designing experiments that address this potential mechanism in more detail.…”

Section: Discussionmentioning

confidence: 99%

Visualization of a polytopic membrane protein during SecY-mediated membrane insertion

et al. 2014

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The biogenesis of polytopic membrane proteins occurs co-translationally on ribosomes that are tightly bound to a membrane-embedded protein-conducting channel: the Sec-complex. The path that is followed by nascent proteins inside the ribosome and the Sec-complex is relatively well established; however, it is not clear what the fate of the N-terminal transmembrane domains (TMDs) of polytopic membrane proteins is when the C-terminal TMDs domains are not yet synthesized. Here, we present the sub-nanometer cryo-electron microscopy structure of an in vivo generated ribosome-SecY complex that carries a membrane insertion intermediate of proteorhodopsin (PR). The structure reveals a pre-opened Sec-complex and the first two TMDs of PR already outside the SecY complex directly in front of its proposed lateral gate. Thus, our structure is in agreement with positioning of N-terminal TMDs at the periphery of SecY, and in addition, it provides clues for the molecular mechanism underlying membrane protein topogenesis.

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

Cited by 29 publications

References 31 publications

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

Integration of transmembrane domains is regulated by their downstream sequences

Visualization of a polytopic membrane protein during SecY-mediated membrane insertion

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