Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

Lin, Pen Jen; Jongsma, C.; Pool, Martin; Johnson, Arthur E.

doi:10.1083/jcb.201103118

Cited by 31 publications

(36 citation statements)

References 79 publications

(172 reference statements)

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“…The efficiency of transfer, E, depends on several variables, but most importantly on the distance between donor and acceptor. Most FRET experiments measure and/or monitor the separation between a single donor dye and a single acceptor dye at specific sites in the same molecule (e.g., Woolhead et al, 2004; Lin et al, 2011b) or molecular complex (e.g., Johnson et al, 1982). However, protein interactions and topography at the two-dimensional membrane surface are sometimes characterized using a variation of the FRET technique that introduces acceptor-labeled PL molecules into liposomes.…”

Section: Resultsmentioning

confidence: 99%

“…We have not attempted to estimate the BOP-PE σ in KRMs because the amount and disposition of surface area occupied by proteins is unknown, as is the extent and distribution of BOP-PE association with ER membrane proteins. But the R 0 for NBD-BOP FRET is 47 Å (Lin et al, 2011b) and the translocon diameter is ~100 Å in native ER membranes (Hanein et al, 1996), so E between an NBD and a BOP on opposite sides of a translocon is essentially 0.…”

Section: Resultsmentioning

confidence: 99%

“…Since this process is co-translational, the cellular machinery must recognize each transmembrane segment (TMS) in the nascent chain, insert the TMS into the hydrophobic core of the phospholipid (PL) bilayer in the correct orientation, and direct the hydrophilic PMP loops that flank a TMS to opposite sides of the membrane. These complex operations are accomplished by the ribosome and translocon, acting as a coupled functional unit, along with other proteins in and on both sides of the membrane (Johnson, 2009; Lin et al, 2011a; Lin et al, 2011b). Yet many mechanisms involved in PMP integration are largely unknown or controversial.…”

Section: Introductionmentioning

confidence: 99%

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Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid

2012

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SUMMARY Most membrane proteins are integrated cotranslationally into the ER membrane at the translocon where nonpolar nascent protein transmembrane segments (TMSs) are widely believed to partition directly into the nonpolar membrane interior. However, a FRET approach that monitors the separation between a fluorescent-labeled TMS and fluorescent phospholipids diffusing in the bulk lipid reveals that TMSs do not immediately enter the lipid phase of the membrane. Instead, TMSs are retained at the translocon by protein-protein interactions until their release into bulk lipid is triggered by translation termination or, in some cases, by the arrival of another nascent chain TMS at a translocon. Nascent chain status and structural elements therefore dictate the timing of TMS release into the lipid phase by altering TMS and flanking sequence interactions with translocons, ribosomes, and associated proteins, thereby controlling when successive TMSs assemble in the bilayer and TMS-delineated loops fold.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid

2012

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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).…”

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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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“…The endoplasmic reticulum and the bacterial transport systems address these problems by co-translational integration. This presumably avoids aggregations of the multiple hydrophobic segments in the aqueous compartment and also may choreograph stepwise insertion of TMDs [63–65]. In E. coli the signal recognition particle (SRP), which consists of an SRP54 homologous protein and a 4.5 S RNA (Figure 4-B), binds the signal peptide or amino proximal TMD emerging from the ribosomal tunnel and escorts the nascent chain-ribosome complex to SecYEG, to YidC, or to a complex of both for co-translational integration [5,66].…”

Section: Membrane Protein Integrationmentioning

confidence: 99%

Intra-plastid protein trafficking: How plant cells adapted prokaryotic mechanisms to the eukaryotic condition

Celedon

Cline

2013

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Protein trafficking and localization in plastids involves a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called ‘conservative sorting’. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation.

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Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

Abstract: Interaction between L17 in the ribosome tunnel and folded nascent chain transmembrane segments during multi-spanning membrane protein synthesis triggers structural rearrangements in the ribosome that cause switching between cytosolic and ER lumenal targeting of the growing polypeptide.

Cited by 31 publications

References 79 publications

Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid

Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid

Functional asymmetry within the Sec61p translocon

Intra-plastid protein trafficking: How plant cells adapted prokaryotic mechanisms to the eukaryotic condition

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