Figure 1.
In vitro import of BH3-only protein. Import of in vitro-translated BimEL (a), tBid (b), Puma (c), NoxaT103 M (d) or Bad (e) into isolated yeast mitochondria, as described in Materials and Methods. Radiolabelled proteins were incubated with mitochondria isolated from S. cerevisiae cells. Then samples were divided. One part was left untreated (−) and one was treated with 100 μg/ml trypsin (T) for 30 min on ice. Another part was subjected to hypotonic swelling and trypsin treatment (SW/T). The two last parts were subjected to alkaline extraction without or with pre-treatment with trypsin resulting in pellet fractions (P, integral membrane proteins) and supernatant fractions (S, soluble and peripherally attached proteins). For comparison, 10% of the total input of radiolabelled precursors was included (L). Reactions were analysed by SDS-PAGE and autoradiography (f, proteolytic fragments). Molecular weight markers are indicated. Some lanes were digitally removed