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. 2008 Oct 31;283(44):29723-9.
doi: 10.1074/jbc.M805356200. Epub 2008 Sep 8.

Mitochondrial biogenesis, switching the sorting pathway of the intermembrane space receptor Mia40

Agnieszka Chacinska  1 , Bernard Guiard, Judith M Müller, Agnes Schulze-Specking, Kipros Gabriel, Stephan Kutik, Nikolaus Pfanner
Affiliations

Mitochondrial biogenesis, switching the sorting pathway of the intermembrane space receptor Mia40

Agnieszka Chacinska et al. J Biol Chem. .

Abstract

Mitochondrial precursor proteins are directed into the intermembrane space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the intermembrane space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40(core)) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40(core) was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40(core), we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40(core) onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.

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Figures

FIGURE 1.
FIGURE 1.
The C-terminal half of recombinant Mia40 is sufficient for the binding of precursor proteins. Recombinant forms of S. cerevisiae Mia40 and Mia40core were purified and incubated with 35S-labeled Tim9 at 15 °C for the indicated time periods. The samples were analyzed by nonreducing SDS-PAGE and digital autoradiography.
FIGURE 2.
FIGURE 2.
The C-terminal half of Mia40 is functional. A, a schematic representation of Mia40 constructs fused to the cytochrome (Cyt.) b2 presequence (plus linker amino acids) is shown. The numbering of amino acid residues is according to the current assignment in the Saccharomyces Genome Database. TM, transmembrane segment. B, the growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown. C and D, the immunodecoration of yeast mitochondria (Mitoch.) and mitoplasts (Mitopl.) is shown. i, intermediate; m, mature; CCHL, cytochrome c heme lyase. E, 35S-labeled Tim9 was imported into isolated yeast mitochondria at 30 °C for the indicated time periods. The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and digital autoradiography. F, 35S-labeled Tim9 imported into isolated mitochondria was analyzed by nonreducing SDS-PAGE and digital autoradiography. G, 35S-labeled Tim8 imported into isolated mitochondria was analyzed by blue native electrophoresis and digital autoradiography.
FIGURE 3.
FIGURE 3.
Mia40 homologs. A, PHYLIP tree for Mia40. The tree was generated with ClustalW2 and visualized by TreeView (30). B, presequence prediction for Mia40 homologs. Mitoch., mitochondrial; aa, amino acid residues. C, complementation analysis of a yeast mia40 deletion mutant by hMIA40. The growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown.
FIGURE 4.
FIGURE 4.
Mia40 without a presequence is functional. A, the growth of yeast at 24 °C before and after removal of wild-type MIA40 by 5-fluoroorotic acid (5-FOA) treatment is shown. B, the immunodecoration of mitochondria (Mitoch.) and mitoplasts (Mitopl.) is shown. CCHL, cytochrome c heme lyase; Cyt., cytochrome. C, 35S-labeled Tim9 was imported into isolated mitochondria. The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and digital autoradiography. D, 35S-labeled Tim9 imported into isolated mitochondria was analyzed by nonreducing SDS-PAGE and digital autoradiography. E, imported Tim8 was analyzed by blue native electrophoresis.
FIGURE 5.
FIGURE 5.
Purified Mia40core is imported by the MIA pathway. A, the import of recombinant Mia40core into mitochondria (Mitoch.) was performed as described under “Experimental Procedures.” The mitochondria were treated with proteinase K (Prot. K) and analyzed by SDS-PAGE and immunodecoration. B, Ni-NTA affinity purification of Mia40core was performed upon import into mitochondria and immunodecoration with different antisera. Load, 1%; Eluate, 100%. Cyt., cytochrome. C, Mia40core was imported into wild-type (WT) and mia40-3 mitochondria and analyzed as described for A. D, the import of Mia40core into wild-type and erv1 mutant mitochondria is shown.
FIGURE 6.
FIGURE 6.
Mia40core depends on the MIA machinery in vivo. The steady-state protein levels of mitochondria (Mitoch.) isolated from wild-type (WT) and erv1-2 cells coexpressing Mia40core are shown. The mitochondria were analyzed by SDS-PAGE and immunodecoration. Cyt., cytochrome; CCHL, cytochrome c heme lyase. The asterisk indicates an unspecific reaction.
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