16.1 Protein targeting to organelles

Protein Targeting and Translocation

Role of signal sequences

Signal sequences are short stretches of amino acids that act as address labels, directing newly made proteins to the correct organelle. Without them, proteins would have no way to reach their functional destination, and the cell's compartmentalized organization would fall apart.

• Signal sequences are typically located at the N-terminus of the polypeptide chain
• Different organelles recognize different signal sequences:
  • ER signal sequence: a stretch of hydrophobic amino acids that routes proteins to the endoplasmic reticulum
  • Mitochondrial targeting sequence: usually positively charged and amphipathic, directing proteins to mitochondria
  • Nuclear localization signal (NLS): rich in basic amino acids like lysine and arginine, enabling import through nuclear pores
• Once a protein reaches its destination, signal peptidases typically cleave the signal sequence off. One notable exception: the NLS is not cleaved, because nuclear proteins must re-enter the nucleus after every cell division when the nuclear envelope reforms.

Mechanisms of protein translocation

Proteins cross organelle membranes through specialized protein complexes called translocons, which form aqueous channels spanning the lipid bilayer. The specific translocon and energy source differ depending on the target organelle.

• Co-translational translocation: the protein is threaded into the organelle while it's still being synthesized on the ribosome. This is the primary route for ER-targeted proteins.
• Post-translational translocation: the protein is fully synthesized in the cytosol before it crosses the membrane. This is how proteins reach mitochondria, chloroplasts, and peroxisomes.
• Energy to drive translocation comes from ATP hydrolysis (e.g., chaperones like BiP in the ER lumen or mtHsp70 in the mitochondrial matrix) and, in the case of mitochondria, the electrochemical gradient (membrane potential) across the inner membrane.

Co-translational vs. post-translational targeting

The core distinction is when translocation happens relative to protein synthesis.

Co-translational targeting:

• Translation and translocation happen at the same time
• Depends on the signal recognition particle (SRP) and the SRP receptor on the ER membrane
• Because the protein enters the ER as it's being made, there's little risk of misfolding in the cytosol

Post-translational targeting:

• The protein is fully synthesized in the cytosol first
• Cytosolic chaperone proteins (such as Hsp70 family members) bind the completed polypeptide to keep it in an unfolded, translocation-competent state
• Used for import into mitochondria, chloroplasts, and peroxisomes

Function of the signal recognition particle

The SRP is a ribonucleoprotein complex made up of a small RNA molecule (7SL RNA in mammals) and six protein subunits. It's the critical link between a ribosome synthesizing an ER-bound protein and the ER membrane itself.

Here's how SRP-mediated targeting works, step by step:

1. As the ribosome translates an ER-destined mRNA, the hydrophobic signal sequence emerges from the ribosomal exit tunnel.
2. SRP binds the signal sequence and simultaneously contacts the ribosome, causing a pause in translation. This pause prevents the growing polypeptide from folding prematurely in the cytosol.
3. The SRP-ribosome-nascent chain complex diffuses to the ER membrane and binds the SRP receptor (also called the docking protein).
4. Both SRP and its receptor hydrolyze GTP, which triggers the release of SRP and the transfer of the ribosome-nascent chain complex to the Sec61 translocon.
5. Translation resumes, and the polypeptide is threaded co-translationally through the translocon channel into the ER lumen.
6. The signal sequence is cleaved by signal peptidase on the lumenal side of the ER membrane.

SRP is recycled back to the cytosol to target another ribosome, making the process catalytic rather than stoichiometric. Without functional SRP, ER-destined proteins accumulate in the cytosol and misfold, which can trigger cellular stress responses.