Question

Q 3: You are trying to identify the peroxisome-targeting sequence in the thiolase enzyme in yeast. The thiolase enzyme normally resides in the peroxisome and therefore must contain amino acid sequences that are used to target the enzyme for import into the peroxisome. To identify the targeting sequences, you create a set of hybrid genes that encode fusion proteins containing part of the thiolase protein fused to another protein, histidinol dehydrogenase (HDH). HDH is a cytosolic enzyme required for the synthesis of the amino acid histidine and cannot function if it is localized in the peroxisome. You genetically engineer a series of yeast cells to express these fusion proteins instead of their own versions of these enzymes. If the fusion proteins are imported into the peroxisome, the HDH portion of the protein cannot function and the yeast cells cannot grow on a medium lacking histidine. You obtain the results growth on medium lacking histidine HDH 200 thiolase 50 200 200 200 150 200 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. What region of the thiolase protein contains the peroxisomal targeting sequence? Explain your answer. (10)

Answer 1

Region from 1 to 125 contains peroxisomal targeting sequence.

As question says -

HDH enzyme can function only in cytosol, not in peroxisome.

This HDH enzyme fused with protein containing thiolase protein.

Hence if protein fused with HDH contains peroxisomal targeting sequence then it will move in the peroxisome with HDH.

And HDH also moved in peroxisome with that protein.

as already written in question if HDH move into peroxisome then it is not able to function.

But if protein fused with HDH enzyme lacks peroxisomal targeting sequence, then it is not able to move into peroxisome.

And Enzyme HDH fused with this protein also remain in cytosol and performs their function, because it is functional in cytosol.

In this question 1 to 125 region of fused protein with enzyme HDH shows negative (-) sign means enzyme HDH is absent in cytosol.

and we can assume that it is moved in peroxisome with fused protein because protein has peroxisomal targeting sequence.

And sequence 150 shows positive (+) sign means it is present in cytosol and protein lacks peroxisomal targeting sequence.