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. 2007 Jan 12;401(Pt 3):701–709. doi: 10.1042/BJ20060990

Figure 5. Protease sensitivity of targeting of Cb5A and Cb5AΔ to membranes.

Figure 5

(A) Translation reactions containing RCb5, Cb5A or Cb5AΔ were incubated either in the absence of microsomes (–), with mock-treated microsomes (M) or with trypsin-treated microsomes (T). Trypsin-treated microsomes were treated with 20 μg/ml trypsin at 4 °C. After 30 min, PMSF was added to inactivate the protease. Microsomes were washed twice and collected by centrifugation. Mock-treated microsomes were processed the same way as the trypsin-treated microsomes but without addition of trypsin. The indicated membranes were added to translation reactions and after incubation the targeting reactions were then mixed with 2.5 M sucrose gradient buffer to a final sucrose concentration of 1.6 M. Sucrose gradient buffers of 1.3 and 0.8 M sucrose were then layered successively on top of the sample. After centrifugation for 18 h at 440000 g, gradients were fractionated from the top into five fractions of equal volume with the solubilized pellet as the bottom fraction. Equivalent amounts of all fractions were analysed by SDS/PAGE and the relative intensity of the resulting bands was determined using a phosphoimager. Fractions 1 and 2 contain microsomes and microsome-bound proteins (arrowheads) and were used to determine the percentage of microsome-bound protein. Fraction 3 contains a portion of the sucrose gradient. Fraction 4 is the load fraction and contains untargeted proteins, while fraction 5 contains protein aggregates. The migration positions in SDS/PAGE of each protein examined are indicated by arrows at the sides of the panels. (B) Mitochondria were incubated with 0, 5 or 20 μg/ml trypsin (as indicated) at 4 °C and processed as above. Trypsin-treated mitochondria were incubated with translation reactions containing pOCTgPA, RCb5, Cb5AΔ, Cb5AΔ/Bcl or Bcl-2. Targeted molecules were recovered with membrane pellets after centrifugation and analysed by SDS/PAGE. The major band in the pOCTgPA-containing reactions (0 trypsin) is the processed mature OCTgPA protein.