FIGURE 6: imsDre2, but not Dre2, can be imported into isolated mitochondria.

(A) Oxa1 and imsDre2 were synthesized in reticulocyte lysate in the presence of ³⁵S-methionine. The proteins were either directly loaded onto the gel (20%) or incubated with isolated wild type mitochondria in the absence (∆ψ↑) or presence (∆ψ↓) of valinomycin. Subsequently, mitochondria were incubated with or without proteinase K (PK) to remove non-imported material, reisolated and dissolved in Laemmli buffer. Precursor (p) and mature (m) forms of the proteins are indicated.

(B) Import reactions were carried out with Dre2, imsDre2 and Oxa1 using mitochondria of the indicated strains.

(C) Import reactions with wild type mitochondria were carried out with urea-denatured radiolabeled Dre2 and imsDre2 using the conditions described by the Banci and colleagues [21].

(D) Radiolabeled Tim9 or imsDre2 were imported into isolated mitochondria for 2 min. Mitochondria were treated with 150 mM NEM, lysed and either directly loaded on the gel (Total) or subjected to immunoprecipitation with Mia40-specific antibodies (αMia40) or preimmune serum (PIS). To preserve and dissociate disulfides, sample buffer without or with β-mercaptoethanol (β-ME) was used.