Cell Biology: Protein Glycosylation and ER Functions

N-linked Glycosylation

• The amino acid sequence Asn-X-Ser/Thr on a protein is required for N-linked glycosylation.
• Each oligosaccharide chain is transferred as a whole unit to the Asn from a lipid called dolichol.
• Oligosaccharides are further modified in the ER and Golgi apparatus.

ER Exit

• The exit from the ER is controlled to ensure protein quality.
• Proteins do not leave the ER if they:
  • have an ER retention signal (KDEL sequence at the C-terminus)
  • are misfolded
    • retained by chaperones
  • are incorrectly assembled into multimeric proteins
    • retained by chaperones

Unfolded Protein Response (UPR)

• The UPR is triggered if protein folding takes too long in the ER.
• The UPR increases ER and folding machinery (chaperones) to help fold proteins quicker.
• The UPR slows down ER protein synthesis to give folding machinery time to catch up.

Golgi Network

• cis Golgi network: faces the ER
  • buds vesicles that return to the ER or continue through the Golgi.
• trans Golgi network: faces the plasma membrane
  • buds vesicles headed to the plasma membrane or other compartments.
• Vesicles from the ER do not fuse with the trans Golgi network.

Golgi Modifications

• Different stacks of the Golgi contain different enzymes, allowing for progressive processing of proteins as they move from the cis to the trans face.
• Proteins exiting the trans Golgi network are sorted based on their next destination.

Exocytosis

• Constitutive secretion:
  • the default pathway, does not require a specific signal sequence
  • continuous process
• Regulated secretion:
  • occurs in some cells
  • proteins aggregate and are stored in secretory vesicles until a signal is received.
• An example of regulated secretion:
  • Increased glucose levels in the blood trigger the release of insulin from pancreatic β cells.
  • This process involves glucose metabolism, ATP-sensitive K+ channels, voltage-gated Ca2+ channels, and SNARE proteins.
• In this process, increased ATP levels activate K+ channels and do not hyperpolarize the membrane.

Endocytosis

• Endocytosis is the uptake of material into a cell by membrane invagination and internalization of the resulting vesicle.
• Phagocytosis:
  • uptake of particles into large vesicles (phagosomes)
  • occurs in phagocytic cells (dendritic cells, macrophages, and neutrophils)
  • Phagosomes fuse with lysosomes.
• Pinocytosis:
  • uptake of fluids and molecules in small vesicles.
  • Occurs via clathrin-coated pits and vesicles.
  • Balanced with exocytosis.
  • Vesicles fuse with endosomes.
  • Pinocytosis is generally a non-specific process
• Receptor-mediated endocytosis:
  • selective endocytosis of specific macromolecules.
  • LDL, low-density lipoproteins (cholesterol + proteins)

Viruses and Endocytosis

• Viruses can enter cells through receptor-mediated endocytosis.
• These vesicles can fuse with lysosomes leading to the release of the viral genome into the cytoplasm and subsequent replication.

Endosomes

• Endosomes are sorting compartments.
• Receptors can be recycled to the same plasma membrane, directed to lysosomes for degradation, or transcytosed to a new plasma membrane area.

Clathrin Coats

• Clathrin-coated vesicles are a major component of the cell's transport system.
• The primary protein in clathrin-coated vesicles is clathrin.
• Each clathrin subunit is made of three large and three small polypeptide chains that form a three-legged structure called a triskelion.
• Triskelions readily assemble into polyhedral cages.

Vesicle Formation

• Clathrin-coated vesicle formation is driven by the assembly of a protein coat.
• Cargo receptors recognize transport signals on cargo molecules and bind to them.
• Adaptins connect coat proteins to cargo receptors.
• Clathrin proteins form basket-like cages that shape membranes into vesicles.
• Dynamin, a GTP-binding protein, pinches off the vesicle.
• The clathrin coat disassembles after the vesicle is pinched off.

Coated Vesicles

• COP (coat protein) coated vesicles mediate membrane transport.
• COPII mediates anterograde transport from the ER to the Golgi.
• COPI mediates retrograde transport from the Golgi to the ER.
• Clathrin participates in endocytosis at the plasma membrane and transport between the Golgi and endosomes.

Vesicle Movement

• Kinesins move cargo from the cell body towards the cell periphery (anterograde transport).
• Dynein moves cargo back toward the cell body (retrograde transport).

Vesicle Fusion

• Protein-protein interactions mediate the fusion of transport vesicles with appropriate membranes.

Receptor-mediated Endocytosis

• The fate of receptor proteins after endocytosis depends on the type of receptor.

Lysosomes

• Lysosomes are the sites of intracellular digestion.
• Lysosomes digest extracellular materials and old organelles.
• They are acidic due to the activity of an H+ pump.
• They contain transporters that export metabolites.
• Lysosomal membrane proteins are glycosylated for protection.
• The signal sequence for Lysosomal targeting is mannose-6-phosphate (M6P), recognized at the trans Golgi network.

Lysosomal Pathways

• Autophagy: enclosure of an old organelle in a double membrane, creating an autophagosome.

Phagocytosis

• Phagocytosis is the uptake of large particles by certain cells, primarily immune cells like macrophages and neutrophils.