Section 4: Protein synthesis and transport Flashcards
1
Q
Describe the life cycle of an mRNA, from where it is transcribed to where it is translated
A
- mRNA is transcribed in the nucleus
- mRNA is translated in the cytoplasm
- Once the signal sequence is translated, the ribosome either:
- Moves to the rough ER (ER signal sequence)
- Completes translation here
- Spits out the finished product into the lumen
- 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
- Moves to the rough ER (ER signal sequence)
- Once the signal sequence is translated, the ribosome either:
2
Q
How do we know that secretory proteins are in the ER?
A
- 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
- Experiment: we isolate the ER and blend them into microsomes (functional pieces of ER)
3
Q
How do proteins enter the ER?
A
- 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)
4
Q
Describe the steps of co-translation translocation
A
- In the cytoplasm, the ribosome translating the protein with the ER signal sequence binds to the SRP
- Translation pauses
- The ribosome/peptide/SRP complex binds to the SRP receptor in the ER membrane
- 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
- The peptide is elongated by the ribosome, and during this process the signal peptidase cleaves the signal sequence
- Elongation/translation is completed and the ribosome releases the protein into the ER and the translocon closes
5
Q
What sorts of protein modifications occur in the ER?
A
- Removal of the signal sequence (as in step 4)
- Glycosylation (adding sugars) of the protein
- Forms disulfide bonds needed for the tertiary structure
- Folding of the polypeptide gain
6
Q
Describe glycosylation
A
- 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
7
Q
Describe protein folding in the ER
A
-
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
- BiP, calnexin, and calreticulin prevents misfolding during elongation
- Once properly folded, the proteins are transported from the RER to the Golgi
8
Q
Describe signalling to the mitochondria
A
- 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
- The TOM receptor binds to the matrix targeting sequence and shuffles
- The TOM receptor binds to the TOM general import pore
- The peptide is transported through the outer membrane
- 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
- Once the peptide is in the matrix, the signal sequence is cleaved off