what are the exceptions to cotranslational translocation in eukaryotes
yeast, mitochondria and the nucleus
what translocation does yeast, mitochondria and the nucleus undergo
post translational translocation
why does yeast undergo post translational translocation
some yeast proteins are fully translated in the cytosol before going to the ER, so a ribosome can’t push it into the ER
describe the steps of post translational translocation in yeast
1) after translation, the signal sequence interacts with the Sec61 translocon
2) the N-terminal enters the ER lumen
3) BiP, an ER chaperone, binds the polypeptide
4) BiP is bound to ATP, which hydrolyses to ADP and makes BiP clamp tightly onto the protein
5) this pulls the protein and prevents it from sliding back out through the pore
6) BiP then releases the protein as it binds new ATP
7) another BiP molecule binds a new segment of the polypeptide and each cycle pulls in more of the protein
8) a signal peptidase cleaves off the signal sequence and the protein folds into its final structure
what is the mitochondrial targeting signal called
amphipathic alpha helix
describe the steps of post translational translocation in mitochondria
1) the “pre-protein” in the cytosol contains the amphipathic alpha helix
2) the protein moves towards the mitochondria, with the help of chaperones using ATP to keep the protein unfolded
3) the protein binds the TOM complex on the mitochondria’s outer membrane
4) TOM passes the protein to the TIM complex in the inner mitochondrial membrane
5) both complexes line up to allow the protein to enter the mitochondrial matrix
6) a matrix chaperone, using ATP, binds the protein, preventing it from moving back out of the mitochondria
7) the inner mitochondrial membrane has an electrical gradient that helps to pull the protein through the TIM complex and into the matrix
8) in the matrix, the signal sequence is cleaved off and the protein folds into its final structure
what factors help to pull the protein into the mitochondrial matrix
- matrix chaperones that use ATP
- the electrical gradient across the inner mitochondrial membrane and matrix
what type of eukaryotic post translational translocation requires the protein to be unfolded after translation
mitchondria
describe the steps of post translational translocation in the nucleus
1) nuclear pore complexes (NPCs) allow large proteins to enter via active transport.
2) proteins directed for the nucleus contain a nuclear localisation signal (NLS), which contains positively charged amino acids
3) in the cytosol, the NLS is recognised then bound by importins, which take the protein through the NPCs
4) in the nucleus, a protein called Ran-GTP then binds importin, causing it to release the protein
how were the genes involved in protein trafficking discovered
Randy Schekman
- randomly mutated yeast cells to identify genes involved in vesicle trafficking
- studied the effects of these mutations
- by determining which gene was mutated in each case, he was able to map which genes controlled specific steps of the secretory pathway
describe how proteins leave the ER lumen
1) Sar1 in the cytosol, bound to GDP, activates when it binds to GTP
2) Sar1 inserts into the ER membrane
3) Sar1-GTP then recruits COPII proteins to the ER membrane
4) COPII recognises and binds to membrane proteins that have “tails” sticking out of the cytosol
5) COPII begins to aggregate, forming a cage structure that induces curvature, eventually forming the vesicle bud
6) the cargo proteins will be carried to the golgi
what happens after a vesicle buds off the golgi
1) once the vesicles leave the ER, the COPII coat is removed, exposing the vesicle’s membrane proteins
2) the vesicles travel through the cytosol via motor proteins
3) the membrane proteins, known as v-SNARES, bind to proteins on the target membrane, t-SNARES.
4) once they bind to their pair, the lipid bilayers fuse, and the cargo proteins are released into the golgi
