Section 4: Protein synthesis and transport

Question 1

Describe the life cycle of an mRNA, from where it is transcribed to where it is translated

Answer 1

• mRNA is transcribed in the nucleus
• mRNA is translated in the cytoplasm
  
  • Once the signal sequence is translated, the ribosome either:
    
    1. Moves to the rough ER (ER signal sequence)
       
       • Completes translation here
       • Spits out the finished product into the lumen
    2. Completes synthesis in the cytoplasm
    • If no signal sequence, the product remains in the cytoplasm
    • If there is a non-ER signalling sequence (ex., mitochondria), the product moves to the specified location once synthesized

Question 2

How do we know that secretory proteins are in the ER?

Answer 2

• Proteins that go to the ER are secretory; destined to be secreted from the cell
• Ex., collagen, which is made in cells and secreted outside of the cell
  
  • Experiment: we isolate the ER and blend them into microsomes (functional pieces of ER)
    
    • Observe that protease do not degrade collagen because it inside of the microsomes
    • Only when we destroy the microsomes with detergent do we observe collagen on the SDS page

Question 3

How do proteins enter the ER?

Answer 3

• Cell free experiments demonstrate that translocation of secretory proteins into microsomes is coupled to translation
  
  • i.e., proteins CANNOT enter the microsome without the presence of the ribosomes, indicating translation and entry are coupled (co-translation translocation)

Question 4

Describe the steps of co-translation translocation

Answer 4

1. In the cytoplasm, the ribosome translating the protein with the ER signal sequence binds to the SRP
   
   • Translation pauses
2. The ribosome/peptide/SRP complex binds to the SRP receptor in the ER membrane
3. The binding triggers the opening of the translocon (protein channel)
   
   • Hydrolyses GTP to open
   • The SRP releases
   • The peptide is transferred into the channel
   • Translation resumes
4. The peptide is elongated by the ribosome, and during this process the signal peptidase cleaves the signal sequence
5. Elongation/translation is completed and the ribosome releases the protein into the ER and the translocon closes

Question 5

What sorts of protein modifications occur in the ER?

Answer 5

1. Removal of the signal sequence (as in step 4)
2. Glycosylation (adding sugars) of the protein
   
   • Forms disulfide bonds needed for the tertiary structure
3. Folding of the polypeptide gain

Question 6

Describe glycosylation

Answer 6

• It is the enzymatic transfer of a 14-reside oligosaccharide precursor from a dolichol (a membrane anchored phospholipid in the ER membrane) to an asparagine (Asn) reside
  
  • This transfer occurs as the peptide is being translocated and translated in the ER lumen
  • This transfer is done by oligosaccharyl transferase
• After, glycosidases modify the N-glycan to remove some sugars (depending on protein function)

Glycosylation affects: protein folding, protein stability, cell adhesion

Question 7

Describe protein folding in the ER

Answer 7

• 4 proteins: chaperone BiP, the lectins (sugar-binding) calnexin and calreticulin, and PDI
  
  • BiP, calnexin, and calreticulin prevents misfolding during elongation
    
    • BiP binds amino acids
    • Calnexin and calreticulin binds the sugars from the oligosaccharide
  • PDI catalyses disulfide bonds
• Once properly folded, the proteins are transported from the RER to the Golgi

Question 8

Describe signalling to the mitochondria

Answer 8

• Mitochondrial matrix targeting sequences are 20-50 amino acid N-terminus rich amphipathic sequences
• Cytosolic chaperones keep the proteins unfolded during transportation (folded = difficult to import)
• TOM protein receptors are on the outer membrane, TIM protein receptors are on the inner membrane

1. The TOM receptor binds to the matrix targeting sequence and shuffles
2. The TOM receptor binds to the TOM general import pore
   
   • The peptide is transported through the outer membrane
3. The TIM import pore receives the peptide
   
   • The TIM must contact the TOM to receive the peptide
   • ATP hydrolysis is needed to pull the peptide through
4. Once the peptide is in the matrix, the signal sequence is cleaved off