Mitochondrial protein import

The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit β (F1β) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane

Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19

MOM19, an import receptor for mitochondrial precursor proteins

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit β. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor

Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix

Targeting of the master receptor MOM19 to mitochondria

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP

Import of ADP/ATP carrier into mitochondria

We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria

Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix