2. Your friend shows you her data. She has been analyzing a protein (CB353) whic

Question

2. Your friend shows you her data. She has been analyzing a protein (CB353) which is 576 amino acids long to determine which parts of the protein are responsible for its localization. She has created various deletion mutations in her protein and examined its localization, under normal conditions and also in the presence of leptomycin B, an inhibitor of the nuclear export machinery. Her data are depicted below. 9 marks total localization in the presence of eptomycin amino r acids nucleus cytoplasm nucleus cytoplasm 1-576 115-576 240-576 - 358-576- 240-357 15-357 1-114 15-239 From her data, answer the following questions: A. Which amino acids contain the nuclear localization signal (NLS)? Briefly explain. (3 marks) B. Which amino acids contain the nuclear export signal (NES)? Briefly explain. (3 marks) C. Describe a microscopic technique and procedure that your friend used to generate this data? (3 marks)

Explanation / Answer

A) 240-357 is NLS

Nuclear Localization signal (NLS) helps the protein to localize inside the nucleus. If that signal is lost then the protein will no longer remain inside the nucleus. In the above experiment the protein sequences are deleted from one end. The principle involves the loss in amino acid will cause a loss in NLS of protein.

In 358-576aa the protein is no more located in the nucleus which means that NLS is lost. But the signal was present in 240- 576. Which means that NLS is in between 240- 357. To confirm it further one more test is done. The segment 240-357 aa is found inside the nucleus which confirms that it has NLS which helps it and the protein to localize in the nucleus.

B) 115-357 is NES

Nuclear Export signal ( NES) is the amino acid sequence in a protein which act as a signal for the protein to export it from the nucleus to cytoplasm. similarly as we did for the NLS we will look for the amino acid which is related to the loss in nuclear export of protein.

1-576 is present in cytoplasm in normal conditions. But in presence of leptomycin B, a nuclear export inhibitory drug the same peptide is absent in cytoplasm. Means its movement is blocked. Similar is the case with 115-576.

But 240-576 is absent in cytoplasm even in normal condition ( when leptomycin Bis absent). That means the NES is absent in it. So the NES is present in 1-239. To further proof this whole peptide its three different segements are studied

1-114 and 115-239 : Both of them are absent in nucleus but present in cytoplasm in normal condition. Even in presence of leptomycin B when export machinary is not working they are present in cytoplasm. means they have nothing specific for NES

115-357 shows its localization in nucleus and in cytoplasm under normal condition. But in presence of inhibitor the protein remains in nucleus and does not transfer to cytoplasm that means it is being inhibited by the leptomycin B.

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