Chapter 13.3

13.3 Protein Modifications, Folding, and Quality Control in the ER

Introduction

Membrane and soluble secretory proteins synthesized on the rough endoplasmic reticulum (ER) undergo four principal modifications prior to reaching their final destinations:

  1. Covalent addition and processing of carbohydrates (glycosylation) in the ER and Golgi complex.

  2. Formation of disulfide bonds in the ER.

  3. Proper folding of polypeptide chains and assembly of multisubunit proteins in the ER.

  4. Specific proteolytic cleavages in the ER, Golgi complex, and secretory vesicles.

These modifications enhance the native structure of secretory proteins and contribute to their stability within the extracellular environment, allowing for complex interactions necessary for cell communication.

Glycosylation

Types of Oligosaccharides

  • Glycoproteins: Proteins with carbohydrate chains.

  • O-linked oligosaccharides: Attached to hydroxyl groups in serine and threonine residues.

    • Example: Mucin-type O-linked chains in mucus and proteoglycans.

  • N-linked oligosaccharides: Attached to the amide nitrogen of asparagine residues.

    • Structure: Branched oligosaccharides predominantly added in the ER.

Synthesis of N-Linked Oligosaccharides

Biosynthesis begins in the rough ER with a preformed oligosaccharide precursor containing 14 residues:

  • Three glucose (Glc), nine mannose (Man), two N-acetylglucosamine (GlcNAc).

  • Modifications include addition or removal of monosaccharides in the ER and Golgi complex.

  • Core structure of 5 conserved residues remains intact across organisms.

Carbohydrate Modification Process

  • Dolichol phosphate is a lipid embedded in the ER membrane, initiating N-linked oligosaccharide assembly.

  • Tunicamycin inhibits the first enzyme, blocking N-linked oligosaccharide synthesis in cells.

  • The precursor oligosaccharide flips to the luminal side of the ER, where further residues are added sequentially.

    Disulfide Bonds Formation

Mechanism of Disulfide Bond Formation

  • Disulfide bonds stabilize protein structures by linking cysteine thiol groups (–SH).

  • Generally occur in the ER lumen via the enzyme protein disulfide isomerase (PDI) which facilitates:

    • Formation of new disulfide bonds.

    • Rearrangement of incorrectly formed disulfide bonds through thiol-disulfide transfer reactions,

  • Ero1 regenerates oxidized PDI, necessary for the continuous formation of disulfide bonds.

Importance in Protein Structure

  • Essential for the tertiary and quaternary structure of proteins, particularly soluble secretory proteins and exoplasmic domains of membrane proteins.

  • PDI operates efficiently in secretory cells (e.g., liver, pancreas).

Protein Folding and Assembly

Role of Molecular Chaperones

  • Rapid folding occurs as proteins are synthesized, facilitated by chaperones like BiP, which prevent aggregation and misfolding during synthesis.

  • Two other lectins:

    • Calnexin and calreticulin, recognize N-linked oligosaccharides and assist in quality control of protein folding by binding to unfolded or misfolded chains.

Quality Control Mechanisms

  • Misfolded proteins are retained, marking their condition, and avoiding transportation until properly folded.

  • Properly folded proteins can exit ER, whereas misfolded ones can trigger the unfolded protein response (UPR), enhancing expression of folding catalysts.

Unfolded Protein Response (UPR)

  • In response to misfolded proteins, mammalian cells upregulate genes encoding for chaperones and folding enzymes via pathways such as:

    • Ire1: Binding of BiP to Ire1 prevents dimerization. Misfolded proteins release BiP leading to increased Ire1 activity and subsequent production of chaperones.

    • Accumulated unfolded proteins lead to active conversion of Hac1, a transcription factor that increases chaperone levels.

    • ATF6: Another pathway where unfolded proteins lead to ATF6 proteolytic cleavage, activating transcription of ER chaperone genes.

Dislocation and Degradation of Misfolded Proteins

  • Misfolded proteins are dislocated back to the cytosol via a complex of membrane proteins known as the ERAD complex (ER-associated degradation).

  • Interaction with p97, an AAA ATPase, is crucial as it provides the energy necessary to pull misfolded proteins into the cytosol for degradation.

  • Proteins tagged with ubiquitin are targeted for proteasomal degradation, ensuring the removal of improperly folded proteins.

Conclusion

All N-linked oligosaccharides contain a core of two N-acetylglucosamine and three mannose residues with additional branches. O-linked oligosaccharides are generally shorter and bound to serine or threonine. The assembly of N-linked oligosaccharides starts on dolichol in the rough ER. Disulfide bonds, facilitated by PDI, stabilize protein structures, while molecular chaperones and quality control mechanisms ensure correct folding and assembly of proteins. Misfolding triggers UPR, enhancing cellular mechanisms for addressing improperly folded proteins.