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

Figure 3. Targeting proteins to ER membranes and mitochondria in the same reactions.

Figure 3

(A) Translation reactions containing Myc–Cb5A, Cb5AΔ and pOCTgPA were added to mitochondrial import reactions containing mitochondria (50 μg of total protein) and equivalents of microsomes as indicated (Eq). After incubation at 30 °C for 60 min, EDTA and potassium acetate were added to final concentrations of 50 and 500 mM respectively, and the ER and mitochondria were separated by differential centrifugation. Equivalent amounts of all fractions were analysed by SDS/PAGE, and the relative intensity of the resulting bands was determined using a phosphoimager (data presented in B and C). Arrows at the side of the panels indicate the migration positions of the specified proteins in SDS/PAGE. pOCTgPA is the precursor containing a mitochondrial targeting signal that is cleaved to generate the mature form of the protein (OCTgPA) following import into mitochondria. (B) Relative amount of Myc–Cb5A co-fractionating with ER (grey squares) and mitochondria (black diamonds). (C) Relative amount Cb5AΔ co-fractionating with ER (grey squares) and mitochondria (black diamonds).