Electron tomogrophy of mitochondria after the arrest of protein import associated with Tom19 depletion

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated three-dimensional reconstructions of 28 FPA-treated mitochondria at four time points (0-32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.