Protein Sorting, Translocation, and Trafficking Notes

Protein Sorting/Translocation/Trafficking

Protein ‘address labels’

  • Amino acid sequences within a polypeptide specify its destination: signal sequences.
  • These sequences are typically at the amino terminal end of the protein – the first part to be synthesised.
  • Enzyme systems within the cell recognise the signal sequence and transport the protein to the correct destination.
  • Signal sequences are usually removed when or before the protein arrives at its final destination.

Protein Synthesis and Sorting Decisions

  • A major sorting decision is made early in protein synthesis when specific proteins are synthesised either on free or membrane bound polyribosomes.

Ribosomes and Protein Synthesis

  • Proteins are made on ribosomes.
  • Ribosome pool is used to make cytosolic proteins.
  • An ER signal sequence on a growing protein directs the ribosome to continue protein synthesis on the rough ER.
  • The ribosome becomes attached to the cytosolic side of the membrane, and the protein is translocated into the ER while being synthesised.
  • Two fates of ER protein:
    • Membrane bound.
    • Lumen.

Cotranslational Import

  • Utilized by ribosomes synthesizing polypeptides destined for export from the cell
  • Ribosomes attach to ER membranes early in translation, and polypeptide chains are transferred across the ER membrane as synthesis takes place
  • This is called cotranslational import

Signal Recognition Particle (SRP)

  • Signal sequence on protein binds to signal recognition particle while protein is being synthesised.
  • SRP blocks further translation until it binds to the SRP-receptor.

Mechanism of Cotranslational Import

  • Signal recognition particle binds to receptor on ER membrane.
  • Translocon (a protein conducting channel) opens.
  • Protein synthesis resumes.
  • Protein passes through a pore into the ER lumen.
  • Signal peptidase (attached to membrane) removes the signal sequence.
  • If transmembrane signals are present within the sequence, stop-start signals are used.
  • GTP unblocks translation.
  • Translation continues (cotranslational insertion).
  • When the protein is complete, it is released into the ER.

Comparison of Mechanisms

  • Lumen protein (water soluble):
    • Synthesis passes the whole protein through, and the signal sequence is cleaved.
  • ER membrane protein (transmembrane):
    • Synthesis passes part of the protein through but is then stopped, and the signal sequence is cleaved.

Protein Folding and Quality Control in the ER

  • After polypeptides are released in the ER lumen, they fold into their final shape.
  • This process is helped by Hsp70, a chaperone protein.
  • Proteins that repeatedly fail to fold properly activate various quality control mechanisms.
  • One mechanism is the unfolded protein response (UPR), in which sensor molecules in the ER lumen detect the misfolded proteins.
  • The ER-associated degradation (ERAD) mechanism recognises misfolded or unassembled proteins and exports them to the cytosol.
  • Here, they are degraded by proteasomes.

Protein Glycosylation

  • Protein glycosylation can serve several different biological purposes.
  • Glycosylation is important for sorting secreted proteins.
  • For example, the phosphorylation of Man on N-glycan creates a recognition signal for sorting lysosomal proteins to lysosomes.
  • Sugars are added to proteins as they enter the ER.
  • Finally, proteins must be sorted and transported to their final destination
  • Original oligosaccharide is (Glc)3(Man)9(GlcNAc)2 This is usually trimmed by removal of (Glc)3Man

Pathways of Vesicular Transport

  • Vesicles transport proteins:
    • From the Golgi apparatus to lysosomes and the plasma membrane.
    • From the plasma membrane to lysosomes.
    • From endosomes to the plasma membrane.
    • Secretory pathway in red.
    • Endocytic pathway in green.

Vesicular Transport Stages

  • Soluble proteins move from the Golgi complex to secretory vesicles for secretion from the cell.
  • Transport vesicles from the ER carry their cargo to the Golgi complex.
  • Stages: budding – movement
  • Vesicles fuse to the Golgi and deposit their cargo inside the complex.
  • Cargo proteins are modified in the Golgi
  • Stages: budding – movement – fusion

Golgi Apparatus

  • Transport vesicles bud from one cisterna to fuse to the next.
  • Further oligosaccharide modifications on proteins occur.
  • Proteins are sorted based on signal sequences.
    • ER retention signal – returned to ER.
    • To endosomes – for lysosome degradation.
    • For secretion from cell.

Transport to the Cell Surface

  • Regulated exocytosis pathway:
    • Only operates in specialised secretory cells.
    • These cells produce many specialised secretory vesicles containing hormones, mucus, digestive enzymes, etc.
  • Constitutive exocytosis pathway:
    • There is a continuous stream of vesicles budding off the Golgi.
    • Function is to allow the cell to grow and expand.
    • This pathway delivers lipids and proteins to the cell membrane and proteins that diffuse into the matrix between cells.
    • It is the default pathway.

Vesicle Formation and Coating

  • Vesicles bud by forming a temporary coat.
  • Golgi budding has been very well studied.
  • EM shows bud associated with outer thickening.
  • Coat of the protein clathrin.
  • This coating occurs on the cytosolic side of the membrane.
  • Protein must bind to a receptor in the ER region associated with the proteins destination.
  • Various characteristics of the cargo protein are recognised i.e. aa sequence or added CHO.
  • Bud formation is facilitated by binding of Coat proteins (COPs).

SNARE Proteins and Vesicle Fusion

  • Once transport vesicle is formed and released, COPs are removed, revealing v-SNARE (vesicle), an integral protein.
  • v-SNARE binds to t-SNARE (target) in the target membrane.
  • This binding leads to fusion of the transport vesicle to the target membrane.
  • Cargo delivered.

Posttranslational Import

  • An alternative mechanism is employed for polypeptides destined for the cytosol or mitochondria, chloroplasts, peroxisomes, or nuclear interior.
  • After translation is complete, the polypeptides are released from ribosomes and remain in the cytosol or are taken up by the appropriate organelle.
  • Special targeting signals are required for this posttranslational import.

Organelle Import

  • Proteins destined for the nuclear interior, mitochondrion, chloroplast, or peroxisome are imported into these organelles after completion of translation.
  • These are synthesized on free ribosomes and released into the cytosol.

Nuclear Import

  • Each protein released to the cytosol has localization signals specific to the destination.
  • E.g., import into the nucleus requires nuclear localisation signals that target proteins for transport through nuclear pores.

Mitochondrial and Chloroplast Import

  • Nearly all polypeptides encoded by mitochondrial or chloroplast genes are subunits of multimeric proteins with one or more subunits encoded by nuclear genes.
  • Most mitochondrial and chloroplast polypeptides are synthesized on cytoplasmic ribosomes, released into the cytosol, and taken up by the organelle within a few minutes.
  • The targeting signal for mitochondrial and chloroplast polypeptides is a transit sequence located at the N-terminus of the polypeptide.
  • The mechanism is similar to cotranslational with the transit sequence transit peptidase.

Mitochondrial Protein Import

  • Most mitochondrial proteins are imported from the cytosol.
  • They are transported across both membranes in one step at sites where the two membranes come close.
  • The mitochondrial signal sequence is detected by an outer membrane import receptor protein, which is linked to an outer translocator protein.
  • The complex diffuses laterally in the membrane.
  • It finds an inner translocator protein and lines up.
  • These move cargo across with the help of chaperone proteins in the matrix, and the protein unfolds as it moves across.
  • The signal sequence is cleaved by signal peptidase inside, and the protein folds again into its mature form.

Summary

  • Signal sequences direct proteins to the ER.
  • In the ER, signal peptides are removed, and proteins are glycosylated.
  • Proteins move to Golgi apparatus in vesicles.
  • As proteins move through the Golgi apparatus, glycosylation is modified.
  • From the Golgi apparatus, proteins are directed in vesicles to the lysosomes or to the cell surface.