Protein Trafficking Methods

Protein Trafficking Ways

• There are four fundamentally different ways a protein is moved from one compartment to another:
  • Protein translocation: direct transport of specific proteins from the cytosol into a topologically distinct space (e.g., ER lumen or mitochondrial membrane)
  • Gated transport: movement of proteins and RNA molecules between the cytosol and the nucleus through nuclear pore complexes
  • Vesicular transport: transport of proteins and lipids between compartments using membrane-enclosed transport intermediates
  • Engulfment: formation of autophagosomes or other membrane-bound structures that engulf portions of the cytoplasm

Sorting Signals and Receptors

• Sorting signals are usually composed of amino acid side chains in a protein and come in two general varieties:
  • Signal patch: a specific three-dimensional arrangement of amino acids
  • Signal sequence: a linear sequence of about 5-10 predominantly hydrophobic amino acids
• Examples of signal sequences that direct proteins to different intracellular locations:
  • Proteins destined for mitochondria have signal sequences that include positively charged amino acids
  • Proteins destined for the nucleus have signal sequences composed primarily of positively charged amino acids
  • Proteins destined for peroxisomes have a signal sequence of three characteristic amino acids at their C-terminus

The Endoplasmic Reticulum (ER)

• The ER membrane typically constitutes more than half of the total membrane of an average animal cell
• The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extend throughout the cytosol
• The ER is specialized in regions that make intimate contacts with other organelles, such as mitochondria, plastids, endosomes, and the plasma membrane
• The ER can be specialized in regions that make intimate contacts with the plasma membrane, modulating levels of plasma membrane phosphoinositides

Signal Sequence Discovery

• The signal hypothesis was formulated to explain the observations of secreted water-soluble proteins that are first translocated across the ER membrane
• The signal sequence is a linear sequence of about 5-10 predominantly hydrophobic amino acids at the N-terminus of the polypeptide chain
• The signal sequence is cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed

Signal-Recognition Particle (SRP) Directs the ER Signal Sequence to a Specific Receptor at the ER

• SRP is a large complex that binds to the signal sequence and guides it to the ER membrane
• SRP is a hinged rod-like structure that can wrap along the large ribosomal subunit
• The signal-recognition particle (SRP) exposes a binding site for an SRP receptor, which brings the SRP-ribosome complex to an unoccupied protein translocator in the ER membrane

Post-Translational Translocation

• Post-translational translocation: proteins are completely synthesized in the cytosol as precursors before they are imported into the ER
• In yeast and bacteria, post-translational translocation is more common across the ER membrane and the evolutionarily related bacterial plasma membrane