5. Consider cargo proteins that are destined for secretion to the extracellular
ID: 163360 • Letter: 5
Question
5. Consider cargo proteins that are destined for secretion to the extracellular space. For each of the following pairs of mutations or conditions, predict where the cargo will end up (ex. in the cytoplasm, a specific organelle, transport vesicles, multiple places, extracellular space) for: (i) & (ii) Each individual condition; & (iii) The combination of both.
A. (i) Normal Sar1 is mutated so that it hydrolyzes GTP immediately after binding GTP.
(ii) The signal recognition particle is mutated to be unable to bind to the SRP receptor.
(iii) Both
B. (i) A mitochondrial signal sequence is added to the C-terminus of the cargo protein.
(ii) v-SNAREs on COPII coated vesicles are mutated to be unable to bind t-SNAREs
(iii) Both
C. (i) COPII coat proteins are mutated so they don’t bind to the ER membrane.
(ii) COPI coat proteins are mutated so they don’t bind to the Golgi membrane.
(iii) Both
D. (i) Normal Sar1 is mutated so it binds tightly to GDP and cannot exchange it for GTP. (This leads to Sar1*-GDP being the only form of the protein in the cell.)
(ii) A transmembrane domain is added to the cargo protein.
(iii) Both
Explanation / Answer
Consider cargo proteins that are destined for secretion to the extracellular space. For each of the following pairs of mutations or conditions, predict where the cargo will end up (ex. in the cytoplasm, a specific organelle, transport vesicles, multiple places, extracellular space) for: (i) & (ii) Each individual condition; & (iii) The combination of both.
A. Sar1-GTP locates itself to the membrane and it serves as the binding site for the Sec23/Sec24 protein coat complex. The Sar1 GTP is disassembled only after the vesicle coat is completely assembled. The vesicle is released from the donor membrane, after the activity of the SarGTPase activity.
(i) Normal Sar1 is mutated so that it hydrolyzes GTP immediately after binding GTP.
If the GTP is hydrolysed IMMEDIATELY after binding to the Sar1 protein, then the Sec23/Sec24 protein coat complex would not bind to the membrane.
(ii) The signal recognition particle is mutated to be unable to bind to the SRP receptor.
The signal recognition particle (SRP) is an abundant conserved ribonucleoprotein, it specifies proteins onto the palms membrane and the endoplasmic reticulum, if it is unable to bind the SRP, it will not bind the signal proteins such as Sar proteins onto the membrane.
(iii) Both
With a mutation in both, neither would the Sar protein bind onto the membrane, and even if it binds somehow , the Sar protein would not be able to bind to the Sec23/Sec24 proteins, as it will get hydrolysed as soon as the GTP binds onto it.
C. (i) COPII coat proteins are mutated so they don’t bind to the ER membrane.
CopII proteins transport the proteins from the RER to the Golgi apparatus. If they are not able to bind to the ER, then they will not be able to transport the protein onto the Golgi apparatus.
(ii) COPI coat proteins are mutated so they don’t bind to the Golgi membrane.
If COPI does not bind onto the Golgi apparatus , it will not be able to transport the protein onto the ER.
(iii) Both
No proteins would be transported between RER or Golgi apparatus, either by retrograde transport, or anterograde transport
D. (i) Normal Sar1 is mutated so it binds tightly to GDP and cannot exchange it for GTP. (This leads to Sar1*-GDP being the only form of the protein in the cell.)
Sar-GDP complex interacts with the membrane to exchange it with GTP, in order to bind to coat vesicles. If GTP is not exchanged for , then it will not bind to the membrane coat vesicles.
(ii) A transmembrane domain is added to the cargo protein.
(iii) Both
No attachment of the membrane vesicles as no GDP would be exchanged for coat vesicles.
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