Figure 6.

Import of AtNDI1 into isolated soybean (Glycine max) cotyledon mitochondria. A, AtNDI1 precursor protein was produced in an in vitro transcription-translation system and incubated with mitochondria to assess its ability to be imported into mitochondria. Lane 1, Translation mixture alone; the precursor protein is seen as a prominent band with an apparent molecular mass of 60 kD. Lane 2, Translation mixture incubated with mitochondria; an additional band with an apparent molecular mass of 56 kD is evident. Lane 3, As in lane 2 but treated with Proteinase K after the import assay. Lanes 4 and 5, As in lanes 2 and 3 but with valinomycin added to the import assay before the addition of precursor protein. Lanes 6 to 9, As in lanes 2 to 4 except that after the import assay, mitochondria were pelleted and their outer membranes ruptured to generate intermembrane space-depleted mitochondria by resuspending in water. B, Western-blot analysis of mitochondria (mit), mitochondria treated with PK (mit + PK) intermembrane space-depleted mitochondria (mp), and intermembrane space-depleted mitochondria treated with PK (mp + PK), using antibodies against Tom20, an outer membrane protein, cytochrome c, an intermembrane space protein, and HSP70, a matrix protein.