Question

(Based on a true story) Dr. Schmephanie Schmoo is studying a secreted protein called Lefty. To see what happens to Lefty protein after it’s secreted from cells, she fuses a green fluorescent protein the N-terminus of Lefty (GFP-Lefty). However, when she expresses this fluorescent fusion protein in cells, she finds that green fluorescence is located in the cytoplasm of cells and not in the extracellular space as expected. How would you explain this result? (Note: GFP on its own is normally a cytoplasmic protein).

Answer 1

Question:

(Based on a true story) Dr. Schmephanie Schmoo is studying a secreted protein called Lefty. To see what happens to Lefty protein after it’s secreted from cells, she fuses a green fluorescent protein the N-terminus of Lefty (GFP-Lefty). However, when she expresses this fluorescent fusion protein in cells, she finds that green fluorescence is located in the cytoplasm of cells and not in the extracellular space as expected. How would you explain this result? (Note: GFP on its own is normally a cytoplasmic protein).

Answer:

Secretary proteins are translated by endoplasmic reticulum membrane bound ribosome and enter into endoplasmic reticulum from where it go to golgi complex and followed by vesicular transport it secreted from cells.

When the mRNA of a secretary protein is come into cytosol from nucleus through nuclear pore this mRNA is recognized by the small ribosomal subunit and then large ribosomal subunit come and bind to it and starts translation. After translation of few portion of the protein it contains a signal sequence at its N-terminus (amino-terminus) end (a short sequence of 15 to 35 amino acids that contain a sequence of at least six non-polar amino acids). Then signal recognize particle (SRP) come and recognized the signal sequence and bind to both signal sequence and large ribosomal subunit which cause a pause in protein synthesis. There is a SRP receptor in the membrane of endoplasmic reticulum. The signal sequence and ribosome bound SRP come to the membrane of endoplasmic reticulum and bind to the SRP receptor. After then the ribosomal complex bind to the translocator protein known as translocon in the membrane of endoplasmic reticulum and the newly synthesized secretary protein translocated into endoplasmic reticulum and follow the secretary pathway to finally secret from the cell.

In her study Dr. Schmephanie Schmoo fused a green fluorescent protein the N-terminus of the secreted protein Lefty (GFP-Lefty), so the SRP didn’t find the signal sequence in the N-terminus end and it can’t bind to it. As a result the ribosomal complex with the protein didn’t bind to the translocator protein or translocon in the membrane of endoplasmic reticulum and the GFP-Lefty protein can’t enter into the endoplasmic reticulum. So it didn’t follow the secretary pathway and didn’t secret from the cells. Thus the GFP-Lefty protein remains in the cytoplasm.

So, when she expresses this fluorescent fusion protein in cells, she finds that green fluorescence is located in the cytoplasm of cells and not in the extracellular space as expected (as GFP on its own is normally a cytoplasmic protein).