2005_pset6_ans_rev - 7.06 Problem Set#6 Spring 2005 1 You...

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7.06 Problem Set #6, Spring 2005 1. You work in a lab studying cell adhesion in mammalian cells. Many cell adhesion proteins, such as CAMs, are extensively glycosylated. The oligosaccharides attached to these molecules can interact with sugar-binding domains on other cells, which can assist in cell movement. You are studying gene that encodes a secreted protein that is predicted to be involved in cell adhesion. The predicted molecular weight of the polypeptide (without attached sugars) that would be produced from the gene you study is 40kDa. This predicted protein has several cysteine residues, and two potential N- linked glycosylation sites consisting of the Asn-X-Ser motif (where X can be any amino acid). a. To determine if this protein is indeed glycosylated, you decide to disable glycosylation and then see if this affects your protein. What are three ways that you can prevent glycosylation of your protein from occurring? 1. You can mutate oligosaccharyl transferase (enzyme 1), which transfers the 14- residue oligosaccharide precursor to the Asn residue as it enters the ER lumen. Doing this in a cell line would block glycosylation for all proteins in those cells. Note that a mutant in enzyme 1 should be a conditional mutant. 2. You can block the first step of precursor formation by treating the cells with the drug tunicamycin. Doing this in a cell line would block glycosylation for all proteins in those cells. 3. You can mutate the Asn residues on your protein in Asn – X – Ser/Thr sequences to other residues. This would block glycosylation of only your protein. b. You choose to block glycosylation for all proteins in your cell line by using a mutant cell line that you call the “Glyc” mutant cell line. How can you monitor whether disruption of glycosylation in these cells prevents the proper folding of your protein? If your protein is normally glycosylated and enzyme 1 is inactivated in your cells, when the protein enters the ER lumen, it could misfold due to the loss of the stabilizing effects of the sugar side chains. Misfolded proteins are retained in the ER due to the unfolded protein response. If you have an antibody for your protein, you can do immunofluorescence to determine if it is retained in the ER in the absence of glycosylation. Alternatively, after remaining unfolded for an extended period in the ER lumen, your protein will be transported back to the cytosol where it will be degraded. If this occurred, you could look for the absence of your protein on a Western blot.
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Glyc mutant c. Your colleague is studying protein translocation into the ER and decides to use your Glyc mutant cell line to study the glycosylation of her protein of interest -- the ATF6 protein, which is a trans-membrane protein localized to the ER membrane. She sees the following result on her Western blot when she probes wild-type and mutant cells with a monoclonal anti-ATF6 antibody: Furthermore, when she looks at the localization of ATF6 by immunofluorescence using
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2005_pset6_ans_rev - 7.06 Problem Set#6 Spring 2005 1 You...

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