Figure 2. Ternary complex of Mia40, Erv1, and substrate. (A) 35S-Tim9 was incubated with mitochondria or mitoplasts and analyzed by native electro- phoresis (lanes 1–8). Immunoblotting of mitochondria and mitoplasts (lanes 9–12) is shown. Asterisk, nonspeciﬁc band. (B) After import of 35S-precursors, mitoplasts were incubated with antisera and analyzed by native electrophoresis. Asterisk, nonspeciﬁc band. (C) 35S-Tim9 was incubated with mitochondria or mitoplasts for 30 min. SDS-PAGE ± 50 mM DTT. (D) After import of 35S-Tim9, Mia40, or Mia40His, mitochondria or mitoplasts were subjected to afﬁnity puriﬁcation, and eluates were analyzed by nonreducing SDS-PAGE. (E) After import of 35S-Tim9, mitoplasts (WT and mia40-3) or mitochondria (mia40-4) were subjected to immunoprecipitation. 198 JCB • VOLUME 183 • NUMBER 2 • 2008