This Data Analysis Problem does not appear in the textbook.

Source:  Hurt, E. C., B. Pesold-Hurt, K. Suda, W. Opplinger. 1985. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 4: 2061–2068.

Corresponding chapter(s) in the textbook: Chapter 13

Review the following terms before working on the problem: mitochondria, protein import, fusion protein, fusion gene, mitochondrial matrix, cytosol, expression plasmid, [35S]methionine labeling, inner mitochondrial membrane, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, autoradiography

Experiment

The import of a fusion protein into mitochondria was studied in this experiment. A fusion gene containing a DNA segment coding for the N-terminal 22 amino acids of a mitochondrial matrix protein attached to the coding region of the mouse dihydrofolate reductase (DHFR; a cytosolic enzyme) gene was inserted into an expression plasmid. Yeast cells were transformed with this plasmid and labeled with [35S]methionine for 16 minutes in the presence of CCCP, a protophore molecule that makes the inner mitochondrial membrane permeable for protons, or for 4, 6, 8, and 16 minutes in the absence of CCCP. Cell extracts were prepared and immunoprecipitated with an antibody to DHFR. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Figure

Source: Hurt, E. C., B. Pesold-Hurt, K. Suda, W. Opplinger. 1985. The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. EMBO J. 4: 2061–2068.

Questions

1. What is the relationship between the p, m, and DHFR bands?

2. How does CCCP affect the synthesis of the fusion protein?

3. What element of the autoradiograph provides evidence of the mitochondrial import of the fusion protein?

4. Name the enzyme that is responsible for the appearance of band m.

5. Explain how CCCP affects the import of the fusion protein.

6. What is the first, direct consequence of CCCP treatment in mitochondria?

7. Explain the appearance of the weak DHFR bands.