The proteins Pma1 and Afg1 were both identified in the analysis of the yeast mit
ID: 177824 • Letter: T
Question
The proteins Pma1 and Afg1 were both identified in the analysis of the yeast mitochondrial proteome but their sequences do not contain any well-characterized mitochondrial targeting sequence. An in-vitro mitochondrial import assay was used to confirm their intracellular distribution. The genes coding for both proteins were used for in-vitro synthesis of radiolabeled Pma1 and Afg1 in rabbit reticulocyte lysate. An aliquot of purified mitochondria was then mixed with the radioactive proteins. After a 30 min incubation, trypsin was added to the reaction mixture. Half of the mixture (Fig. 1, T0) was immediately heat denatured in the presence of SDS, whereas the other half of the mixture (Fig. 1, T30) was incubated at 37°C for 20 min and then heat denatured in the presence of SDS. The heat denatured samples were then resolved on a 1D-SDS PAGE and the gel was placed in contact with an X-ray film to visualize the radiolabeled proteins.
Figure 1: Autoradiogram showing radiolabeled proteins Pma1 and Afg1 after trypsin treatment as described above (panel A) and with mitochondria pre-treated with valinomycin (Panel B)
a) The exposed X-ray film corresponding to the assay described above is presented in the panel A of figure 1. Based on these data, are Pma1 and Afg1 both mitochondrial proteins? Briefly explain
b) In a separate import assay, mitochondria treated with valinomycin were mixed with radiolabeled proteins and then processed as described before. In figure 1 panel B, carefully draw, for each condition, the bands corresponding to the radiolabeled proteins.
c) By mistake, the experimenter used a preparation of mitochondria that has been previously treated with digitonin a mild detergent that can only solubilize the mitochondrial outer membrane. Radioactive protein was not detected in the Pma1 and Afg1 T30 fractions. What is the likely explanation?
Pma1 To Afg1 To Panel A Afg1 Pma1 Tao 130 To To Panel BExplanation / Answer
According to the data available, we can find out that Pma1 Is not an mitochondria protein, since it cannot be seen in gel at T30 slot.
Where as the Afg1 is seen at both the timeline, which proves that it is an mitochondrial protein.
Explanation:
In the experiment we had used radiolabeled protein to e reacted in mitochrondial. Only the proteins in mitochondria will be attached with radiolabeled Afg1 since these antibodies against Afg1 is available in mitochondria whereas Pma1 is not available in purified mitochondria is absent due to which band is missing in the gel.
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