Protein Processing and Quality Control in the Endoplasmic Reticulum

The Endoplasmic Reticulum and Protein Processing

Protein Targeting and Translocation in vitro

• In vitro translation: Proteins translated in a test tube using:
  • Cell extract (ribosomes, cytosolic factors).
  • mRNA.
  • Radioactive amino acids.
  • Organelle membranes (ER, mitochondria).
• Outcomes:
  • Not targeted or imported: Signal remains attached.
  • Targeted & imported: Signal is cleaved.
• Cell-free assay: Allows for easy manipulation by:
  • Using different mRNA.
  • Adding or removing soluble components.

Studying Protein Targeting

• Use SDS-PAGE to separate radioactive proteins by size to analyze protein targeting and translocation.
• Molecular Weight Analysis:
  • With ER membranes:
    • Protein with signal: -12 kDa.
    • Protein without signal: -9.5 kDa.
  • No ER membranes: Larger protein.
• Radioactive label used to detect protein products.
• Reduced protein size confirms signal cleavage inside the ER.

Protein Translocation into the ER Lumen

• ER signal sequence binds to Sec61 translocator, opening a channel.
• Polypeptide threaded through the channel as a loop during translation (co-translational translocation).
• Signal sequence cleaved by signal peptidase, releasing the protein into the ER lumen.

Membrane Protein Insertion

• Additional hydrophobic sequences anchor transmembrane proteins in the lipid bilayer.
• Orientation is fixed with the N-terminus in the lumen.
• Signal sequence binds Sec61, opening the channel.
• Hydrophobic stop-transfer sequence halts polypeptide movement through the channel.
• Stop-transfer sequence released into the bilayer, forming a transmembrane domain.

Multi-Pass Transmembrane Proteins

• Possess an internal ER signal sequence (start-transfer sequence).
• Internal or start-transfer sequence acts as ER targeting signal.
• Binds Sec61 and opens channel to initiate translocation.
• Stop-transfer sequence enters channel, halting translocation.
• Alternating start- and stop-transfer sequences create complex multi-pass membrane proteins.

Ways Membrane Proteins Associate with Lipid Bilayer

• (A) Transmembrane: Integral membrane proteins spanning the lipid bilayer. COOH group in either Extracellular Space or Cytosol with NH₂ group in the opposite location respectively
• (B) Monolayer-Associated: Integral membrane proteins associated with one leaflet of the lipid bilayer.
• (C) Lipid-Linked: Peripheral membrane proteins linked to the lipid bilayer via a lipid anchor.
• (D) Protein-Attached: Peripheral membrane proteins attached to other membrane proteins.

Protein Folding in the ER

• After translocation, polypeptide chain folds into correct 3D conformation in the ER lumen.
• Folding is assisted by molecular chaperones in the ER lumen.
  • BiP (an ATPase) binds to exposed hydrophobic residues.
  • Calnexin binds to N-glycosylated proteins.
• Chaperone proteins guide the folding of newly synthesized polypeptide chains.

Protein Modifications in the ER

• Signal sequence cleavage.
• Disulphide bond formation (oxidation).
• Glycosylation (covalent attachment of carbohydrate).

Disulphide Bond Formation

• Formed by oxidation of cysteine side chains and stabilizes folded protein structure.
• Catalyzed by protein disulphide isomerase inside the ER lumen.
• The ER lumen provides an oxidizing environment.

N-Linked Glycosylation

• Occurs on Asparagine (Asn) residues.
• Oligosaccharide transferred to protein from dolichol (a special lipid donor).
• Catalyzed by oligosaccharyl transferase (OST).
• OST glycosylates Asn residues in a specific consensus sequence: Asn-X-Ser or Asn-X-Thr (X cannot be Proline).

Functions of N-Glycosylation

• Assists in protein folding.
• Modified to create mannose-6-phosphate tags, which act as a lysosome sorting signal.
• Acts as a ligand for specific cell-cell recognition events.

Glycocalyx

• Eukaryotic cells coated in carbohydrates attached to proteins and lipids, forming the glycocalyx.
• Forms a protective layer outside the cell.
• Made at the ER and Golgi before delivery to the plasma membrane.

Protein Destination After ER

• Some proteins function in the ER.
• Most are destined for secretion or other subcellular locations.
• Packaged into membrane-bound transport vesicles, transporting them along the secretory pathway to their final destination.

Quality Control in the ER

• ER is the entry point to the secretory pathway.
• Proteins (and lipids) destined for the Golgi, lysosomes, & plasma membrane are all made at the ER.
• Exit from the ER is controlled to ensure protein quality.
• Misfolded proteins are potentially harmful, chaperones bind to them preventing them from leaving the ER.

Unfolded Protein Response (UPR)

• Build-up of misfolded proteins in the ER lumen triggers the UPR.
• ER size and function is controlled by demand.

Summary Points

• Hydrophobic signal sequences (internal or start transfer, stop transfer sequences) determine the arrangement of transmembrane proteins in the lipid bilayer.
• Proteins are folded and may undergo disulphide bond formation and N-glycosylation in the ER.
• ER acts as the entry point for the secretory pathway.
• Proteins that cannot fold correctly are retained by ER quality control.
• Build-up of unfolded/misfolded proteins in the ER activates the unfolded protein response (UPR).