week4_key - MCB130 Week 4 Key 1 You have discovered a novel...

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MCB130 Week 4 – Key 1 1) You have discovered a novel integral membrane protein, and you suspect it’s a receptor of some sort involved in signaling between cells. The sequence of the protein suggests that it’s a polytopic membrane protein with 5 anchor domains and it’s N- terminus is cytoplasmic. (a) Draw a (linear) diagram of the protein, including all key domains, and an illustration of its topology in the plasma membrane. Make sure you can draw the linear diagram. It should include the 5 anchor domains and, after reading part (b), you might add a possible receptor domain in the C-terminus (because for cell-cell signaling you would expect the receptor to be outside the cell). You know there’s no N-terminal signal peptide because the N-terminus of the protein is in the cytoplasm. (b) You delete one of the anchor domains and find that the protein no longer binds its (extracellular) ligand. Suggest possible explanations of this finding? The ligand-binding (receptor) domain may be located in the C-teminus, which is no longer cytoplasmic. If the receptor domain is not in the C-terminus, deleting a portion of the protein my have caused other structural changes that have rendered the domain non-functional. (c) You delete the N-terminus and find that the protein also no longer binds the ligand. Explain this result. This is where I was a little confused. Note the correction. The correct answer would be that the first anchor domain has probably been removed. Just like in part (b), the topology of the protein would be disrupted and the C-terminus would now be located in the cytoplasm (as would the N-terminus). N-term C-term Extracellular Cytoplasm N-term C-term Cytoplasm Lumen Plasma Membrane ER Membrane
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MCB130 Week 4 – Key 2 (d) Suggest an experiment you might do to demonstrate your answer to (c) Fuse GFP to the C-terminus of the protein, and observe if it is extracellular. Use a protease protection assay to establish the topology of the protein and the truncated protein. A comparison of the two should demonstrate the change. Assuming you know what the receptor binds, attach the C-terminus of the protein to an affinitiy chromatography column and show that it is indeed the portion that binds the ligand. Others? 2) What’s the difference in targeting to and processing in the ER between type I and type
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week4_key - MCB130 Week 4 Key 1 You have discovered a novel...

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