mcb130-fa07-mt1-Weis-soln - MCB130 Midterm Key Name: SID:...

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MCB130 Midterm Name: Key SID: GSI’s Name: 1 1. Peroxisomes are small, membrane-enclosed organelles that function in the degradation of fatty acids and in the degradation of H 2 O 2 . Peroxisomes are not part of the secretory pathway and peroxisomal enzymes are targeted directly from the cytoplasm to the lumen of peroxisomes via a peroxisomal targeting signal (PTS). a. Design a general experimental strategy to identify the PTS in your f avorite p eroxisomal p rotein 1 (FPP1). [15 pts] The question is asking to describe the experiments to find a peroxisomal targeting signal sequence without assuming the signal is one that has already been described. You cannot assume that the signal sequence is cleaved, this is not a valid assumption and experiments based solely on this assumption received only minimal credit for experimental design. Need to specifically explain how to first find the PTS in the FPP1 protein, need to explain an experiment that would narrow down to a specific region or find it based on mutagenesis. It is important to not only explain how to test a potential PTS but also explain how the PTS was found in the first place. After the PTS sequence is found, need to show specifically that the PTS is both necessary and sufficient for localization to the peroxisome. [ 2 pts for clearly finding the PTS sequence from the FPP1 protein and recognizing that it needs to be necessary and sufficient] To show that the PTS sequence is necessary for localization of the FPP1 protein to the peroxisome, need to either make a point mutation or small deletion in the PTS of FPP1. Then it is important to have an experiment to show that the FPP1 protein with a defective PTS is not able to localize to the peroxisome. [ 5 pts . for clearly defining the PTS as necessary.] Examples of possible experiments include 1) tagging FPP1 with GFP and using microscopy to visualize the cellular localization (need to mention control that FPP1-GFP is able to localize to peroxisome and FPP1-PTS* mutant-GFP was not able to localize to
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mcb130-fa07-mt1-Weis-soln - MCB130 Midterm Key Name: SID:...

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