Insertion of short transmembrane helices by the Sec61 translocon

Jaud, Simon; Fernández-Vidal, Mónica; Nilsson, IngMarie; Meindl-Beinker, Nadja M.; Hübner, Nadja C.; Tobias, Douglas J.; Heijne, Gunnar von; White, Stephen H.

doi:10.1073/pnas.0900638106

Cited by 82 publications

(140 citation statements)

References 28 publications

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“…In many cases, the TM topology of a signal sequence is likely to be determined within a short range. The positive charges flanking an H-segment enhance stop translocation (Jaud et al, 2009;Kuroiwa et al, 1990;Kuroiwa et al, 1991;Lerch-Bader et al, 2008). The TM topology is achieved as soon as the positive charges reach the translocon.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Accessibility of this segment in any orientation to the aqueous surface in the course of substrateinduced conformational changes should thus become detectable. Dyes at positions 22,23,26,27,28,32,33,34,35,42, and 45 cover the region from the hinge to the APH and an adjacent conserved negatively charged stretch. Position 51 served as positive control for quenching, as it is already positioned in an exposed region that is not anymore required for functionality (25).…”

Section: A Cell-growth Effect Of the Tata Membrane-anchor That Is Commentioning

confidence: 99%

“…Notably, a stretch of 12 hydrophobic residuesalthough very short -is sufficiently long for a complete membrane-integration of a peptide by the Sec61 translocon in eukaryotes (42).…”

Section: Substrate-inducedmentioning

confidence: 99%

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

Hou¹,

Heidrich²,

Mehner-Breitfeld

et al. 2018

Journal of Biological Chemistry

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The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as model organism, we now demonstrate in vivo that the N-terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N-terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membranedestabilizing effects of the short TatA membrane anchor are compensated by the membraneimmersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.The Tat system serves to transport folded proteins in bacteria, archaea, plant plastids, and possibly plant mitochondria (1-4). In Escherichia coli, the Tat system consists of TatA, TatB, and TatC components. TatA and TatB are similar, and two-component "minimal" Tat systems exist in which TatA exerts functions of TatA and TatB (5). TatA/B components are N-terminally membraneanchored by a very short hydrophobic transmembrane domain (TMD), which is followed by a short hinge region, an amphipathic helix (APH), and a variable C-terminal domain (6). TatC is a polytopic membrane protein with six TMDs (7,8). TatB tightly interacts with TatC (9, 10). TatA associates with these TatBC complexes and is thought to permeabilize the membrane for protein transport (11)(12)(13)(14). TatBC complexes recognize and tightly bind the signal peptides of the cargo proteins throughout the translocation process (15,16).The mechanism by which this translocation is achieved must be unusual and is not understood (17). A currently favored model suggests that the N-termini of multiple TatA molecules at the translocon site weaken the membrane upon substrate-binding, thereby permitting a TatCmediated pulling of the substrate through the Control of membrane-weakening by TatA 2 destabilized membrane ("membrane-weakening and pulling mechanism", 18). Molecular dynamics simulations support this view ...

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“…Although hydrophobicity is the predominant factor (16), it is insufficient per se to determine whether or not a given polypeptide segment will be integrated into the membrane (17,18). Both residues immediately flanking a transmembrane (TM) segment (19) and those in other TM segments upstream or downstream (18,20) can also contribute to the membrane insertion propensity of the segment. Experiments have demonstrated that the polypeptide chain can interact with lipids while still in the channel (21)(22)(23), leading to the proposal that insertion is a thermodynamic partitioning between channel and membrane (23)(24)(25).…”

mentioning

confidence: 99%

Free-energy cost for translocon-assisted insertion of membrane proteins

Gumbart

Chipot

Schulten

2011

Proc. Natl. Acad. Sci. U.S.A.

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Nascent membrane proteins typically insert in a sequential fashion into the membrane via a protein-conducting channel, the Sec translocon. How this process occurs is still unclear, although a thermodynamic partitioning between the channel and the membrane environment has been proposed. Experiment-and simulationbased scales for the insertion free energy of various amino acids are, however, at variance, the former appearing to lie in a narrower range than the latter. Membrane insertion of arginine, for instance, requires 14-17 kcal∕mol according to molecular dynamics simulations, but only 2-3 kcal∕mol according to experiment. We suggest that this disagreement is resolved by assuming a two-stage insertion process wherein the first step, the insertion into the translocon, is energized by protein synthesis and, therefore, has an effectively zero free-energy cost; the second step, the insertion into the membrane, invokes the translocon as an intermediary between the fully hydrated and the fully inserted locations. Using free-energy perturbation calculations, the effective transfer free energies from the translocon to the membrane have been determined for both arginine and leucine amino acids carried by a background polyleucine helix. Indeed, the insertion penalty for arginine as well as the insertion gain for leucine from the translocon to the membrane is found to be significantly reduced compared to direct insertion from water, resulting in the same compression as observed in the experiment-based scale.membrane-protein insertion | SecY | ribosome | hydrophobicity scale N early all membrane proteins found in the inner membranes of bacterial cells and the membranes of eukaryotic cells are inserted concomitant with their synthesis by the ribosome, i.e., cotranslationally (1). Insertion into the bilayer does not happen directly, but rather occurs via a highly conserved protein-conducting channel in the membrane, the Sec translocon (2-4). At an early stage of synthesis, the ribosome docks to the channel, forming a tightly bound complex (5, 6). The polypeptide is then inserted into the translocon prior to entering the membrane, the former step requiring a driving force, such as nucleotide hydrolysis, a membrane potential gradient, or the pressure exerted by the growing nascent chain (2).In addition to aiding the insertion of membrane proteins, the translocon also allows certain nascent proteins to cross the membrane (2). Structures of the Sec translocon (7-9) reveal two apparent gates, one transverse for the passage of soluble proteins across the membrane and one lateral for the exit of membrane proteins (7,10,11). The lateral gate is formed at the interface of two halves of SecY (7, 12) (see Fig. 1B). This gate fluctuates during translocation of the nascent polypeptide (13,14), although what factors govern its opening and closing are unclear (8,9,15).Given the two pathways presented by the translocon, there must exist a way to discriminate between proteins destined for the lipid membrane environment and proteins destined ...

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Insertion of short transmembrane helices by the Sec61 translocon

Cited by 82 publications

References 28 publications

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

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

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding

Free-energy cost for translocon-assisted insertion of membrane proteins

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