Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

Lin, Pen-Jen; Jongsma, C.; Liao, Shuren; Johnson, Arthur E.

doi:10.1083/jcb.201103117

Cited by 29 publications

(56 citation statements)

References 43 publications

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“…For in vitro transcription, the tatA or tatA-NTstrep regions in the corresponding pGEM constructs were amplified using the pGEMrecognizing primers pGEM-5-F (5'-CCC AGT CAC GAC GTT GTA AAA CG-3') and pGEM-5-R (5'-CTT CCG GCT CGT ATG TTG TG-3'), and the purified PCR-product was used as template for SP6 RNA-polymerase according to the suppliers protocol (NEB). Translation was done according to standard protocols (62).…”

Section: Methodsmentioning

confidence: 99%

“…TatA variants were inserted into inverted membrane vesicles (IMVs) prepared from E. coli strain DADE/pBW-tatBC (52) by employing wheat-germ extract-based in vitro translation according to the protocol of Lin et al (62), but in the presence of IMVs (4 OD280) and with 600 pmol/ml tetramethylrhodamine (TMR)-cysteinetRNA Cys .…”

Section: In Vitro Reconstitution Of Cysteine-labeled Tata Into Invertmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: In Vitro Reconstitution Of Cysteine-labeled Tata Into Invertmentioning

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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“…Time-resolved Spectral Measurements-Fluorescence lifetimes were measured as detailed in Lin et al (25). The background phase and modulation data from a sample lacking NBD were subtracted from an equivalent NBD-containing sample that was prepared in parallel (26).…”

Section: Methodsmentioning

confidence: 99%

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support β-Barrel Pore Insertion

Farrand

Hotze

Sato

et al. 2015

Journal of Biological Chemistry

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Background:The cholesterol-dependent cytolysins (CDCs) have an identical cholesterol-binding motif but exhibit different binding parameters. Results: Binding affinity is altered by the loop three (L3) structure, which impacts pore-forming efficiency. Conclusion: The L3 structure affects its equilibrium between stabilizing (inserted) and destabilizing (uninserted) membrane interactions. Significance: The L3 structure provides CDCs with cellular selectivity by discriminating lipid environments surrounding membrane cholesterol.

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“…In addition, the ribosome was implicated in influencing the translocon with respect to TM integration and pore sealing (e.g., refs. [16][17][18]. Molecular dynamics (MD) analysis suggested that modulation of the channel's gating kinetics is controlled by the TM's hydrophobicity (19)(20)(21).…”

mentioning

confidence: 99%

Functional asymmetry within the Sec61p translocon

Demirci

Junne

Baday

et al. 2013

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

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The Sec61 translocon forms a pore to translocate polypeptide sequences across the membrane and offers a lateral gate for membrane integration of hydrophobic (H) segments. A central constriction of six apolar residues has been shown to form a seal, but also to determine the hydrophobicity threshold for membrane integration: Mutation of these residues in yeast Sec61p to glycines, serines, aspartates, or lysines lowered the hydrophobicity required for integration; mutation to alanines increased it. Whereas four leucines distributed in an oligo-alanine H segment were sufficient for 50% integration, we now find four leucines in the N-terminal half of the H segment to produce significantly more integration than in the C-terminal half, suggesting functional asymmetry within the translocon. Scanning a cluster of three leucines through an oligo-alanine H segment showed high integration levels, except around the position matching that of the hydrophobic constriction in the pore where integration was strongly reduced. Both asymmetry and the position effect of H-segment integration disappeared upon mutation of the constriction residues to glycines or serines, demonstrating that hydrophobicity at this position within the translocon is responsible for the phenomenon. Asymmetry was largely retained, however, when constriction residues were replaced by alanines. These results reflect on the integration mechanism of transmembrane domains and show that membrane insertion of H segments strongly depends not only on their intrinsic hydrophobicity but also on the local conditions in the translocon interior. Thus, the contribution of hydrophobic residues in the H segment is not simply additive and displays cooperativeness depending on their relative position.protein translocation | transmembrane helix T he conserved Sec61/SecY translocon provides a passage for hydrophilic polypeptide sequences across the membrane of the endoplasmic reticulum (ER) or the bacterial plasma membrane (1). It consists of Sec61α (Sec61p in yeast) with 10 transmembrane domains and the single spanning proteins Sec61β (Sbh1p) and Sec61γ (Sss1p), corresponding in bacteria to SecY/ SecG/SecE. The translocon forms a compact helix bundle that can open a pore across the membrane with a lateral gate toward the lipid membrane. In the idle state, the central pore is closed by a constriction of six, almost invariably hydrophobic side chains and a luminal plug helix. The plug closes and stabilizes the inactive translocon and is displaced by translocating peptides, while the constriction residues form a gasket around them, keeping the translocon sealed to ions and small molecules (2-4).The lateral gate allows transmembrane helices (TMs) to insert into the lipid phase. Systematic quantitative analyses of membrane integration of mildly hydrophobic sequences (H segments) defined the contribution of each amino acid to the probability of insertion into or transfer across the bilayer (5-9). These studies yielded "biological hydrophobicity scales," similar to scales determined b...

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Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

Abstract: Vastly different folded transmembrane segments of nascent multispanning membrane proteins each induce structural changes in the ribosome tunnel and translocon that target the loops of the growing polypeptide alternately into the cytosol or ER lumen.

Cited by 29 publications

References 43 publications

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

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

The Cholesterol-dependent Cytolysin Membrane-binding Interface Discriminates Lipid Environments of Cholesterol to Support β-Barrel Pore Insertion

Functional asymmetry within the Sec61p translocon

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