ER Quality Control, Cystic Fibrosis Explanation, and Golgi Apparatus

ER Quality Control

  • Quality Control in the ER: Mechanism to determine if proteins are misfolded, preventing them from proceeding through the secretory pathway.

  • Properly Folded vs. Misfolded Proteins: Properly folded proteins bud off and are transported to the Golgi, while misfolded proteins are recognized, retained, and discarded.

  • Chaperones/Chaperonins: Proteins that bind to unfolded or misfolded proteins, assisting in their proper folding.

    • Example: BIP involved in transport into the ER.

Chaperone Mechanism in the ER Lumen

  • Sugar Asparagine End-Linked Sugar Chains: Proteins entering the ER lumen have sugar chains with glucose residues attached.

  • Glucosidases: Enzymes that cleave off glucose residues.

  • Calnexin: Binds to oligosaccharide chains with a single glucose residue.

  • Glucosyltransferase (GT): Adds glucoses back onto the chains.

  • Process:

    • Initial trimming of glucose residues.
    • Calnexin binds to the single glucose residue.
    • Glucosidase II clips off the glucose, allowing the protein to move on if properly folded.
    • If not fully folded, glucosyltransferase adds a new glucose residue, causing calnexin to bind again and retain the protein.

  • Repeated Cycle: If the protein remains incorrectly folded, glucosyltransferase continues to add glucose, and calnexin holds it in the ER.

  • Proteasome Degradation: If held too long, the misfolded protein is exported and degraded by the proteasome.

  • Glucosyltransferase Recognition:

    • Recognizes misfolded proteins through exposed nonpolar residues.
    • Binds to nonpolar stretches, adding a glucose residue to the oligosaccharide.
    • Calnexin then binds, retaining the protein in the ER.

  • Enzymes Involved: Glucosidases and glucosyltransferase.

  • ER Exit: To leave the ER lumen, all three glucoses must be removed.

Cystic Fibrosis and Quality Control

  • Cystic Fibrosis Overview: Genetic disease where mucus in the lungs becomes thick, leading to bacterial infections.

  • CFTR Protein: Cystic Fibrosis Transmembrane Regulator, a chloride channel that controls the level of water in lung mucus.

  • CFTR Mutation Effects: Mutations cause thicker mucus, resulting in lung infections.

  • F508 Mutation:

    • Most common mutation, involves a mutation or deletion of phenylalanine at position 508.
    • Protein doesn't act normally, triggering quality control.
    • Prevents CFTR from reaching the plasma membrane.

  • Quality Control Oversurveillance:

    • The F508 mutation doesn't severely impair CFTR function.
    • Quality control eliminates CFTR, even though it could function at the plasma membrane.
    • If CFTR reached the plasma membrane, cystic fibrosis might be avoided.

  • Research Focus: Efforts to allow mutant CFTR versions to evade quality control.

Protein Topology and the Secretory Pathway

  • Transmembrane Protein Orientation:

    • Tail in the cytosol and another tail with polysaccharides in the ER lumen.
    • Cytosolic side remains cytosolic throughout the secretory system.
    • ER lumen side ends up on the exterior of the cell.

  • Topological Equivalence:

    • Interior of the ER, vesicles, and Golgi are topologically equivalent to the exterior of the cell.
    • Cytosolic side of a transmembrane protein remains in the cytosol.

  • Orientation Maintenance: Secretory pathway maintains the cytosolic and external sides of proteins.

The Golgi Apparatus

  • Microscopy Images: TEM images show rough ER with ribosomes and vesicles budding off to the Golgi.

  • Cisternae: Sac-like structures in the Golgi (cis, medial, trans).

  • Cis and Trans Sides:

    • Cis side is closest to the ER and receives vesicles.
    • Trans side is closest to the plasma membrane.

  • Golgi Networks: Cis Golgi Network (CGN) and Trans Golgi Network (TGN).

  • Vesicle Budding: Vesicles bud off from the edges of the cisternae.

  • Cisternal Functions: Different cisternae have different functions.

  • TEM Staining Patterns:

    • Osmine tetroxide binds to lipids, more abundant in the cis region.
    • Mannosidase is predominantly in the medial region.
    • Nucleoside diphosphatase uses UDP and is involved in modifying sugars.

  • Maturation Process: Proteins mature as they move from one cisterna to another.

Glycosylation in the Golgi

  • Sugar Residue Modification: The Golgi primarily modifies sugar residues.

  • Sequential Modifications: Different cisternae perform different modifications.

  • Process Overview:

    • Mannose residues are trimmed and new sugar residues are added.
    • Loss of glucose residues (used for quality control).
    • Further trimming with mannosidases.
    • Transfer of new sugar residues.
    • Continued trimming and additions.

Clathrin Coat and Vesicle Formation

  • Clathrin Coat: Helps shape vesicles as they form around cargo. Triskelions naturally form rounded structures, facilitating vesicle formation.