Figure 1.

The DHFR domain of pSu9-DHFR is unfolded at stage B but not at stage A on the mitochondrial surface. pSu9-DHFR was incubated with CCCP-treated mitochondria for 10 min at 0°C (lanes 1–3 and 7–9) or at 30°C (lanes 4–6 and 10–12). After 5-fold dilution with MSC buffer containing 10 mM KCl, 1 μM methotrexate, and 1 mM NADPH, the mitochondria were reisolated by centrifugation and were resuspended in the same buffer. The samples were divided into halves. For lanes 1–6, one aliquot was kept at 0°C (lanes 1 and 4) and the other was treated with 100 μg/ml proteinase K for 15 min at 0°C, which was inactivated by the subsequent addition of 1 mM phenylmethylsulfonyl fluoride (lanes 2, 3, 5, and 6). For lanes 7–12, one aliquot was kept at 0°C (lanes 7 and 10) and the other was treated with 2 μg/ml MPP and 1 mM MnCl₂ for 5 min at 30°C, and MPP was inactivated by the subsequent addition of 5 mM EDTA (lanes 8, 9, 11, and 12). The samples were centrifuged, and proteins in the pellet (p) and those in the supernatant (s), which were precipitated with trichloroacetic acid, were analyzed by SDS/PAGE. TEMP, temperature during the binding reaction; p, pellet; s, supernatant; pre and m, precursor and mature forms of pSu9-DHFR, respectively; f, proteinase K-resistant DHFR domain.