Question

You want to tag the extracellular part of a single-pass transmembrane receptor with GFP, which requires that you modify the DNA sequence encoding the receptor to also include the DNA sequence encoding GFP.   
a) Would you tag the receptor so that GFP is fused to that portion of the polypeptide that is translocated into the ER lumen during translation? Or instead, would GFP be fused to that portion of the polypeptide that remains in the cytosol during translation? (2 points)
b) Briefly explain your answer to a) by using a simple diagram that clearly illustrates the transport of the receptor from the ER to the Golgi and then plasma membrane. (4 points)

Answer 1

I would tag GFP to the portion of the protein that remains in the cytosol during translation because the portion of protein that translocated into the cytoplasm contains ER localisation signal, if we will tag them with GFP it would not be able to translocate into the ER for processing and finally targeting to the membrane will be affected. Hence the proteins if intend to check the localisation should always be tagged at the region which does not contain protein localisation signal.