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

Multi-spanning membrane protein loops are directed alternately into the cytosol or ER lumen during cotranslational integration. Nascent chain exposure is switched after a newly synthesized transmembrane segment (TMS) enters the ribosomal tunnel. FRET measurements revealed that each TMS is initially extended, but folds into a compact conformation after moving 6–7 residues from the peptidyltransferase center, irrespective of loop size. The ribosome-induced folding of each TMS coincided with its photocrosslinking to ribosomal protein L17 and an inversion of compartmental exposure. This correlation indicates that successive TMSs fold and bind at a specific ribosomal tunnel site that includes L17, thereby triggering structural rearrangements of multiple components in and on both sides of the ER membrane, most likely via TMS-dependent L17 and/or rRNA conformational changes transmitted to the surface. Thus, cyclical changes at the membrane during integration are initiated by TMS folding, even though nascent chain conformation and location vary dynamically in the ribosome tunnel. Nascent chains therefore control their own trafficking.

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Early encounters of a nascent membrane protein

The Journal of Cell Biology ◽

10.1083/jcb.200503035 ◽

2005 ◽

Vol 170 (1) ◽

pp. 27-35 ◽

Cited By ~ 41

Author(s):

Edith N.G. Houben ◽

Raz Zarivach ◽

Bauke Oudega ◽

Joen Luirink

Keyword(s):

Membrane Protein ◽

Ribosomal Proteins ◽

Transmembrane Domain ◽

Signal Recognition Particle ◽

Chain Complex ◽

Signal Recognition ◽

Inner Membrane ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Inner Membrane Protein

An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

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Functional ramifications of FRET-detected nascent chain folding far inside the membrane-bound ribosome

Biochemical Society Transactions ◽

10.1042/bst0320668 ◽

2004 ◽

Vol 32 (5) ◽

pp. 668-672 ◽

Cited By ~ 14

Author(s):

A.E. Johnson

Keyword(s):

Protein Trafficking ◽

Protein Biosynthesis ◽

Ribosomal Subunit ◽

Regulatory Control ◽

Secretory Proteins ◽

Induced Folding ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Membrane Bound ◽

Transmembrane Sequence

During protein biosynthesis, nascent protein chains are directed along a long narrow tunnel that spans the large ribosomal subunit. It has recently become clear that this structural feature has evolved to effect regulatory control over aspects of protein synthesis and protein trafficking. Since this control is nascent chain-specific, ribosomal components that form the tunnel must be involved in recognizing selected nascent proteins as they pass by. The present study focuses on one such situation in which nascent secretory proteins and membrane proteins are distinguished by the ribosome-induced folding of the latter's hydrophobic transmembrane sequence far inside the ribosomal tunnel and close to the peptidyltransferase centre.

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The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding

Journal of Biological Chemistry ◽

10.1074/jbc.m115.672261 ◽

2015 ◽

Vol 290 (48) ◽

pp. 28944-28952 ◽

Cited By ~ 5

Author(s):

Melissa A. Patterson ◽

Anannya Bandyopadhyay ◽

Prasanna K. Devaraneni ◽

Josha Woodward ◽

LeeAnn Rooney ◽

...

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Membrane Protein Folding ◽

Restricted Environment ◽

Complex Forms ◽

Polytopic Membrane Protein

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The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling

eLife ◽

10.7554/elife.25642 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 38

Author(s):

Ting Su ◽

Jingdong Cheng ◽

Daniel Sohmen ◽

Rickard Hedman ◽

Otto Berninghausen ◽

...

Keyword(s):

Secondary Structure ◽

Structure Formation ◽

Secondary Structure Formation ◽

Helix Formation ◽

Ribosomal Tunnel ◽

Nascent Chain ◽

Translational Arrest ◽

Exit Tunnel ◽

Α Helix ◽

Peptidyltransferase Center

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50–55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.

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Polytopic membrane protein folding and assemblyin vitroandin vivo(Review)

Molecular Membrane Biology ◽

10.1080/09687680410001697215 ◽

2004 ◽

Vol 21 (3) ◽

pp. 163-170 ◽

Cited By ~ 16

Author(s):

Paula J Booth ◽

Stephen High

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Membrane Protein Folding ◽

Polytopic Membrane Protein

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Faculty Opinions recommendation of Dissecting the ER-associated degradation of a misfolded polytopic membrane protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1097804.553924 ◽

2008 ◽

Author(s):

Davis Ng

Keyword(s):

Membrane Protein ◽

Polytopic Membrane Protein

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Correlation of membrane protein conformational and functional dynamics

Nature Communications ◽

10.1038/s41467-021-24660-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Raghavendar Reddy Sanganna Gari ◽

Joel José Montalvo‐Acosta ◽

George R. Heath ◽

Yining Jiang ◽

Xiaolong Gao ◽

...

Keyword(s):

Membrane Protein ◽

Conformational Changes ◽

Single Channel ◽

Conformational Dynamics ◽

Md Simulations ◽

High Temporal Resolution ◽

Dependent Manner ◽

Functional Dynamics ◽

Ph Dependent ◽

Open States

AbstractConformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.

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