You are a molecular biologist studying sorting sequences and have the ability to add, delete and mutate at will. In all problems, assume the protein is made and is stable.
1.You have a normally soluble cytosolic protein and add the following signals. Where does the mutant protein end up?
A.An ER signal sequence to the amino-terminal end.
B.An ER signal sequence to the amino-terminal end and change the hydrophobic amino acids into charged amino acids.
C.An ER signal sequence to the amino-terminal end and change the hydrophobic amino acids into other, hydrophobic, amino acids.
D.An ER signal sequence to the middle.
2.You are trying to identify the peroxisome-targeting sequence in the thiolase enzyme, an enzyme that normally resides in the peroxisome. To identify the targeting sequences, you create a set 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. If the fusion proteins are imported into the peroxisome, the HDH portion of the protein cannot function and the yeast cells cannot grow on media lacking histidine. You obtain the results below. What region of the thiolase protein contains the peroxisomal targeting sequence? Explain your answer.
3.Protein A contains a typical nuclear localization signal but is usually found in the cytosol of cells. When the cell is exposed to hormones, protein A moves from the cytosol into the nucleus. When you purify protein A from cells that have not been treated with hormones, you find that protein B is always complexed with it. To determine the function of protein B, you engineer cells lacking the gene for protein B. You compare normal and defective cells by separating the nuclear fraction from the cytoplasmic fraction and examining the proteins by gel electrophoresis. The gel you run is shown below. Propose a function for protein B. Explain your answer.
4.You have a normally soluble cytosolic protein and add the following two (and only two) conflicting signal sequences that specify different compartments. Predict which signal would win out for the following combinations. Explain your answers.
A.Signals for import into the nucleus and import into the ER.
B.Signals for export from the nucleus and import into the mitochondria.
C.Signals for import into mitochondria and retention in the ER.
5.You have discovered two vesicular membrane proteins, Y and Z, that you believe are involved in membrane fusion. You design an ingenious assay for the fusion of vesicles utilizing alkaline phosphatase. The protein alkaline phosphatase is made in a pro form that must be cleaved in order for the protein to be active. You have designed two different strains of yeast: strain A produces the pro form of alkaline phosphatase (pro-AP), while strain B produces the protease that can cleave pro-AP into the active form (AP). Neither strain has the active form of the alkaline phosphatase, but when vesicles from the strains A and B are mixed, fusion of vesicles generates active alkaline phosphates, whose activity can be measured and quantified.
Vesicle A (pro-AP) + Vesicle B (protease) ?fusion?Vesicle AB (active AP)
You take each of these yeast strains and further engineered them so that they express only Y, Z, both (the normal situation), or neither. You isolate vesicles from all strains and tests the ability of each variant form of strain A to fuse with each variant form of strain B, using the alkaline phosphatase assay. The data are shown in the table.
Alkaline Phosphatase Activity
Strain A
Strain BYZYZ-
YZ10080800
Y800500
Z805000
-0000
What does this data suggest about the function for Y and Z in the vesicles? Be sure to comment on whether it is important to have a specific protein (that is, Y or Z) on each vesicle.Need aProfessionalWriter to Work on this Paper?