Protein Trafficking Methods

Study Notes

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 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 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

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

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: 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