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Fig. 5. High ergosterol content reduces the efficiency of insertion of Fis 1 into the membrane of lipid vesicles. (A) Radiolabeled precursor of Fis1-TMC was incubated for the indicated time periods with either mitochondria (50 µg protein) or an equivalent amount of lipid vesicles (33 µg lipids). Further treatment was as described in Fig. 2D. (B) Radiolabeled precursor of Fis1-TMC was incubated with lipid vesicles containing the indicated mol% of ergosterol. Further treatment was as described in Fig. 2D. The band that represents unlabeled Fis1 molecules in the presence of IASD (lower band in +IASD) was quantified by densitometry. Insertion was calculated as the intensity of this band in comparison to the amount of precursor protein added to the reaction. (C) Radiolabeled precursor of Tom20ext (Ahting et al., 2005) was incubated for 20 minutes at 25°C with either mitochondria or an equivalent amount of lipid vesicles containing the indicated mol% of ergosterol. After incubation, the samples were halved and in one half mitochondria or liposomes were pelleted and solubilized directly in sample buffer (–proteinase K). The other aliquots were treated with PK (500 µg/ml) before solubilization in sample buffer (+proteinase K). The specific membrane-inserted proteolytic fragment (Ahting et al., 2005) is indicated with an arrowhead. (D) Radiolabeled precursor of Fis1-TMC was incubated with a mixture of mitochondria (50 µg protein) and lipid vesicles (33 µg). Modification with IASD was as described in Fig. 2D. Mitochondria were separated from lipid vesicles by differential centrifugation and proteins were analyzed by SDS-PAGE and autoradiography. The bands were quantified and the intensity of the band corresponding to the unmodified protein was taken as a measure for protein insertion. For each mixture, the amount of protein inserted into mitochondria was set to 100%.
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