Figure 1.
Internal dimensions of the inner and outer membrane protein import channels. (A) Precursors containing a single Cys at their C termini were modified with monomaleimido Undecagold (20 Å gold), monomaleimido Nanogold (26 Å gold), Texas-Red-C2-maleimide (Texas Red), or fluorescein-5-maleimide (Fluorescein) or were left unmodified (unmodified) and then imported into mitochondria at 20°C as described in Materials and Methods. Samples were taken at the indicated times [in seconds (“) or minutes (’)], and import was stopped in normal stop buffer, which leaves the outer membrane intact. Mitochondria were reisolated, and the samples were analyzed for imported precursors by SDS/PAGE and autoradiography. For the total lanes, equivalent samples were taken before incubation with mitochondria. The autoradiograms show that precursor modification leads to decreased mobility in SDS/PAGE. Bands representing modified and unmodified precursor are labeled accordingly. The 20-Å gold-, fluorescein-, and Texas-Red-modified precursors are imported into mitochondria, whereas 26-Å gold-modified precursors are not. (B) To compare the import kinetics of the gold-modified and unmodified precursors, the import reactions described in A were quantified by electronic autoradiography. The amount of imported precursor is plotted as a percentage of the total amount of precursor presented to the mitochondria. (C) Conditions were the same as described for A, except that import was stopped in mitoplasting buffer to rupture the outer membrane by hypoosmotic shock. Mitoplasts were reisolated, and the samples were analyzed for imported precursors by SDS/PAGE and autoradiography. Bands represent protein imported across the inner mitochondrial membrane. The gels with 20-Å and 26-Å gold-modified precursors show additional bands that run faster than unmodified precursor and that presumably represent partially imported precursor from which the modification has been cleaved by the protease in the mitoplasting buffer. (D) A comparison was made in the same way described for B, except that import across the inner mitochondrial membrane is plotted.