2005_pset6 - 7.06 Problem Set #6, Spring 2005 1. You work...

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Glyc mutant 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? 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? 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: WT cells ATF6 ~ 70 kDa ATF6 ~ 30 kDa
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the same antibody in these two different cell lines, she is surprised to see the following: WT cell – ATF6 on ER membrane Glycosylation mutant cell – ATF6 in nucleus What do the results of her Western blot and immunofluorescence experiments tell you about ATF6, and how do these results make sense given the function of ATF6? d. You track the production of your protein from part a) by performing a time course on live wild-type cells. You grow your cells in medium containing radioactive amino acids for a short time. (This is called a “pulse” of radioactivity.) These radioactive amino acids incorporate into all newly made proteins during the “pulse.” You then switch the cells to media containing only cold (non-radioactive) media. (This is called the “chase.”) Immediately after the pulse and during various times during the chase period, you solubilize the ER membrane, immunoprecipitate your protein of interest with a monoclonal antibody, and run it on an SDS-PAGE gel. (Note that the loading buffer you use does not contain any reducing agents.) You then take a picture using
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2005_pset6 - 7.06 Problem Set #6, Spring 2005 1. You work...

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