Related Experiment Video

Updated: Aug 8, 2026

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
09:42

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Published on: June 19, 2012

Cotranslational folding and calnexin binding during glycoprotein synthesis

W Chen1, J Helenius, I Braakman

  • 1Department of Cell Biology, Yale School of Medicine, New Haven, CT 06510-8002, USA.

Proceedings of the National Academy of Sciences of the United States of America
|July 3, 1995
PubMed

Insights

Influenza hemagglutinin undergoes cotranslational folding in the endoplasmic reticulum, involving disulfide bond formation and N-linked glycosylation. The chaperone calnexin plays a role in this process, influenced by oligosaccharide modifications.

Area of Science:

  • Molecular Biology
  • Protein Folding
  • Virology

Background:

  • Influenza hemagglutinin (HA) is a crucial viral glycoprotein involved in host cell entry.
  • Understanding the cotranslational folding of HA is essential for comprehending viral assembly and infectivity.
  • The endoplasmic reticulum (ER) is the primary site for glycoprotein folding and maturation.

Purpose of the Study:

  • To investigate the cotranslational folding pathway of influenza hemagglutinin within live cells.
  • To elucidate the roles of disulfide bond formation, N-linked glycosylation, and chaperone interactions during HA synthesis.

Main Methods:

  • Utilized short pulses of radiolabeling followed by immunoprecipitation.
  • Employed a two-dimensional SDS-PAGE system (non-reducing then reducing) to separate nascent polypeptides.
  • Analyzed polypeptide length, oxidation state, and interactions with calnexin.

Main Results:

  • Provided evidence for cotranslational disulfide bond formation and the generation of conformational epitopes.
  • Demonstrated N-linked glycosylation occurring during translation.
  • Showed oligosaccharide-dependent binding of calnexin to incompletely folded HA via trimmed oligosaccharides.

Conclusions:

  • N-linked oligosaccharides and calnexin are integral to the cotranslational folding of hemagglutinin.
  • Inhibition of glycosylation or oligosaccharide trimming perturbs the HA folding pathway.
  • These findings highlight the interplay between nascent polypeptide modifications and chaperone-assisted folding.

More Related Videos

Quantitative Immunofluorescence to Measure Global Localized Translation09:13

Quantitative Immunofluorescence to Measure Global Localized Translation

Published on: August 22, 2017

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase08:59

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Published on: February 12, 2019

Related Concept Videos

Nuclear Protein Sorting01:34

Nuclear Protein Sorting

Nuclear protein sorting is the selective trafficking of histones, polymerases, gene regulatory proteins into the nucleus and exporting RNAs and ribosomes to the cytosol. It is a tightly controlled process that regulates gene expression within a cell.
Proteins targeted to the nucleus carry nuclear localization signals or NLS recognized by import receptors in the cytosol. Similarly, proteins with nuclear export signals are recognized by export receptors. Import and export receptors are...
Directing Proteins to the Rough Endoplasmic Reticulum01:34

Directing Proteins to the Rough Endoplasmic Reticulum

The organelle-specific signaling sequences direct proteins synthesized in the cytosol to their final destination like ER, mitochondria, peroxisomes, etc. Some of the proteins directed to ER are then trafficked via vesicles to other organelles within the cell or the extracellular environment through the Golgi complex. For example, the rough ER synthesizes soluble proteins for transportation to the lysosomes or secretion out of the cell. It can also synthesize transmembrane proteins that can...
Cotranslational Protein Translocation01:20

Cotranslational Protein Translocation

Translocation of proteins across membranes is an ancient process that occurs even in bacteria and archaebacteria. In fact, the components of the translocation machinery are still conserved between prokaryotes and eukaryotes.
Sec61 channel partners for cotranslational translocation
During cotranslational translocation, the Sec61 channel partners with the signal recognition particle (SRP), the signal recognition particle receptor (SR), and the ribosomes to transport the nascent polypeptide chain...
Protein Folding Quality Check in the RER01:29

Protein Folding Quality Check in the RER

ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
Post-translational Translocation of Proteins to the RER01:27

Post-translational Translocation of Proteins to the RER

A sizable fraction of proteins destined for ER are first synthesized in the cell cytosol and then transported across the ER membrane–a process called post-translational translocation. Similar to cotranslationally translocated proteins, these proteins also use the Sec translocon complex to enter the ER lumen.
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
Protein Translocation Machinery on the ER Membrane01:28

Protein Translocation Machinery on the ER Membrane

The translocon complex situated on the ER membrane is the main gateway for the protein secretory pathway. It facilitates the transport of nascent peptides into the ER lumen and their insertion into the ER membrane.
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the translocon complex.