Vesicular Transport Notes

Vesicular Transport

Vesicular Transport

  • Vesicular transport is essential for moving materials within the cell.
  • Mutations can block transport at different stages (A, B, C; D, E, F; G, H, I).
  • Yeast secretory (sec) mutants are temperature-sensitive and denser than normal.

Isolation of Yeast Sec Mutants

  • Novick & Schekman isolated yeast sec mutants. They generated temperature-sensitive mutants (ts) that have a higher density than normal.
  • Screen by EM Vacuole Nucleus Golgi Mother Cell

Golgi Transport Assay

  • Involves donor and acceptor Golgi stacks, cytosol, ATP, and 3H-GlcNAc.
  • Measures the incorporation of 3H-GlcNAc into transported glycoprotein over time.
  • Requires both ATP and cytosol.

VSV G protein ts045

  • Temperature-sensitive mutant that accumulates at 40°C in the ER.

Clathrin-mediated Endocytosis

  • Example: Endocytosis of LDL (cholesterol).
  • Involves LDL receptors, clathrin-coated pits, adaptor proteins.
  • Lower pH in endosomes leads to dissociation of the ligand from its receptor.

AP2 Adaptor Protein Complex

  • The AP2 complex has a Hinge, Trunk, a-appendage. It contains ẞ2, μ2 σ2 α subunits.

Shibire Mutation

  • The Shibire mutation in Drosophila affects synaptic vesicle recycling.

Synaptic Vesicle Cycle

  • Involves docking, priming, fusion, and recycling.
  • Impairment of the dynamin

Clathrin

  • Clathrin is important the formation of a clathrin-coated vesicle.

Tethering Factors

  • EEA1 is a tethering factor with Rab5 binding sites, reaching 80nm in length.

SNARE-mediated Membrane Apposition

  • Model for SNARE mediated membrane apposition

Ca2+-dependent Synaptic Vesicle Exocytosis

  • Depends on SNARE proteins and synaptotagmin (Ca2+ sensor).

Role of SNAREs in Membrane Fusion

  • Neurotoxins (BoNT/A, BoNT/B, etc.) target SNAREs.

Toxin Resistance

  • Rats and chickens are resistant to certain toxins due to amino acid substitutions in the cleavage site of VAMP1/VAMP2.

Viral Infection

  • Infection of cells by influenza virus.
  • Early endosome (pH ~6) to Late endosome (pH ~5)

Viral Fusion Proteins and SNAREs

  • Share a similar hairpin-like structure.