Reversible Assembly of the ATP-binding Cassette Transporter Mdl1 with the F1F0-ATP Synthase in Mitochondria

Dominik Galluhn,Thomas Langer

doi:10.1074/jbc.m405871200

Abstract

The half-ABC transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins. The physiological role of the peptides released from mitochondria is currently not understood. Here, we have analyzed the oligomeric state of Mdl1 in the inner membrane and demonstrate nucleotide-dependent binding to the F(1)F(0)-ATP synthase. Mdl1 forms homo-oligomeric, presumably dimeric complexes in the presence of ATP, but was found in association with the F(1)F(0)-ATP synthase at low ATP levels. Mdl1 binds membrane-embedded parts of the ATP synthase complex after the assembly of the F(1) and F(0) moieties. Although independent of Mdl1 activity, complex formation is impaired upon inhibition of the F(1)F(0)-ATP synthase with oligomycin or N,N'-dicyclohexylcarbodiimide. These results are consistent with an activation of Mdl1 upon dissociation from the ATP synthase and suggest a link of peptide export from mitochondria to the activity of the F(1)F(0)-ATP synthase and the cellular energy metabolism.

Highlights

Whereas the vast majority of mitochondrial proteins are imported into the organelle after their synthesis in the cytosol, a small number of proteins is encoded within the mitochondrial genome
The half-ATP-binding cassette (ABC) transporter Mdl1 is localized in the inner membrane of mitochondria and mediates the export of peptides generated upon proteolysis of mitochondrial proteins
On the other hand, are exported from mitochondria along two pathways: peptides generated by the i-associated with various cellular activities (AAA) protease in the intermembrane space can traverse the outer membrane presumably through porins or protein-translocating translocase of the outer membrane complexes; peptides generated by the m-AAA protease in the matrix space, on the other hand, have first to be transported across the inner membrane

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Cloning Procedures—Yeast strains used in this study are derivatives of W303. ⌬yta10, ⌬yta, yta10E559Qyta12E614Q and various mdl mutant strains were described elsewhere (12, 22). ATP-dependent Dimerization of Mdl1—After completion of protein import and protease digestion of non-imported precursor proteins, wild type and mdl1His mitochondria (500 ␮g) were solubilized at a concentration of 5 mg/ml in buffer H (200 mM KCl, 1 mM magnesium acetate, 20 mM HEPES/KOH (pH 7.4), 10% glycerol, 1 mM PMSF) containing 0.2% (w/v) Triton X-100 and, when indicated, 1 mM ATP or ATP␥S. After a clarifying spin of the mitochondrial extract for 30 min at 90,000 ϫ g the supernatant was loaded onto a HiTrapTM Chelating HP column (1 ml; Amersham Biosciences) equilibrated with buffer T containing only 0.05% (w/v) Triton X-100 and 20 mM imidazole. Blue Native Polyacrylamide Gel Electrophoresis—Mitochondria were solubilized at a concentration of 5 mg/ml in 0.5% Triton X-100, and extracts were analyzed by BN-PAGE using a 3–13% gradient gel essentially as described (26)

RESULTS

DISCUSSION

Dominik Galluhn and Thomas Langer