You want to identify where a peroxisome-targeting signal sequence is in a yeast

Question

You want to identify where a peroxisome-targeting signal sequence is in a yeast protein called thiolase. To do so, you create a set of hybrid proteins that contain different parts of the thiolase protein sequence fused to another protein, histidinol dehydrogenase (HDH). You express these hybrid proteins (a-e) in different strains instead of the normal HDH (wt). If the hybrid proteins are imported into the peroxisome, the HDH portion of the protein cannot function, and the yeast cells do not grow on a medium lacking histidine. Your results are shown on the right.

A. Which of the proteins a-e localize to the cytoplasm?

B. Which of the proteins a-e localize to the peroxisome?

C. What range of amino acid positions in the thiolase protein contains the signal sequence that targets proteins to the peroxisome? 1-50 50-100 100-125 125-150 150-200 Please explain your conclusion for Part C.

growth on medium lacking histidine wt HDH 200 thiolase 50 200 100 200 125 200 150 200 The numbers above each protein indicate the amino acids from thiolase present in the hybrid protein. indicates growth on medium lacking histidine; indicates lack of growth on medium lacking histidine.

Explanation / Answer

A. Wt (wild type), c, d & e localized in the cytoplasm. Because they possess intact HDH gene and can grow in lacking Histidine. They retain in cytoplasm, because they don't have functional peroxysomal gene.

B. a & b are localized in peroxysomes. It is due to they have functional peroxysomal signaling, that span in between 1-100 amino acid length.

C. Thiolase, a peroxysomal target span in bebetween 50-100 aminoacids. Because in b construct, 1-49 amino acids not there. Still it enter into peroxysomes. So this is acctual portion for perosysomal localization.

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