Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Abstract

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

Figure 1

Mia40 is located in the mitochondrial IMS. (A) Predicted primary structure of the translation product of the full open reading frame of S. cerevisiae MIA40 (YKL195w). Box, hydrophobic segment. It has also been proposed that Mia40 starts at the second methionine (residue 25; Kellis et al, 2003). We synthesized both forms in vitro and imported them into isolated mitochondria. The efficiency of in vitro synthesis and the observed import signals were considerably stronger for the protein derived from the full open reading frame. (B) Sequence alignment of S. cerevisiae (Sc) Mia40 (residues 273–392) with homologous protein domains from Schizosaccharomyces pombe (Sp), Neurospora crassa (Nc), Candida albicans (Ca), Drosophila melanogaster (Dm) and Homo sapiens (Hs). Black, identical residues in at least four proteins; gray, similar residues. The conserved twin Cx₉C motif is marked. (C) Migration of Mia40 from wild-type S. cerevisiae mitochondria on SDS–PAGE and BN–PAGE, analyzed by Western blotting. Where indicated, 10 mM DTT or 0.2% SDS were added to the digitonin buffer prior to BN–PAGE. The sample containing SDS was boiled for 15 min at 95°C. (D) Cellular fractionation. Equal volumes of the fractions after differential centrifugation steps (numbers refer to 1000 g) were analyzed by Western blot. S, supernatant; P, pellet. (E) Separation of sonicated mitochondria into membrane-bound and soluble fractions by centrifugation. Sup., supernatant. Samples were subjected to Western blot analysis. (F) Treatment of mitochondria and mitoplasts with proteinase K (Prot. K), followed by Western blot analysis. (G) Import and processing of the radiolabeled precursor of Mia40 by isolated mitochondria. The reisolated mitochondria were subjected to SDS–PAGE and digital autoradiography. Mature Mia40 analyzed by Western blotting is shown in samples 11 and 12. p, precursor; i, intermediate; m, mature.

Figure 2

A temperature-sensitive yeast mutant of Mia40 contains reduced levels of small Tim proteins. (A) WT and mia40-3 S. cerevisiae cells were subjected to serial 10-fold dilutions, plated on YPD or YPG medium and incubated for 3 days at the temperatures indicated. (B) Mitochondria from WT and mia40-3 cells grown at 19°C were analyzed by SDS–PAGE and immunodecoration. Mia40, 15 and 45 μg mitochondrial protein, respectively; other proteins, 25 and 50 μg mitochondrial protein, respectively. (C) WT and mia40-3 cells were grown at 30 or 33°C and mitochondria were isolated. Odd-numbered lanes, 15 μg mitochondrial protein; even-numbered lanes, 45 μg mitochondrial protein. (D) Mitochondria (80 μg of protein) from cells grown at 19°C were solubilized by digitonin and subjected to BN–PAGE and immunodecoration.

Figure 3

mia40-3 mitochondria are inhibited in import of small Tim proteins, Cox17 and Cox19 into the IMS. (A) Energized mitochondria isolated from WT and mia40-3 cells grown at 19°C were incubated with the radiolabeled precursor of Tim10 at 30°C for the indicated timespans. The import reaction was stopped on ice. Nonimported precursors were removed by treatment with proteinase K. The reisolated mitochondria were subjected to digital autoradiography and Western blot analysis. (B–H) The radiolabeled precursors of Tim9, Tim8, Tim13, Cox17, Cox19, cytochrome c (Cyc1) and b₂(220)-DHFR were imported into WT and mia40-3 mitochondria in the presence of a Δψ unless indicated otherwise. The mitochondria were treated with proteinase K.

Figure 4

mia40-3 mitochondria are not impaired in protein import to the matrix, inner membrane or outer membrane. (A–D) The radiolabeled precursors of the Rieske Fe/S protein, F₁β, Su9-DHFR and b₂(167)_Δ-DHFR were imported into WT and mia40-3 mitochondria (isolated from cells grown at 19°C) at 30°C in the presence or absence of a Δψ. Where indicated, the mitochondria were treated with proteinase K. The mitochondria were reisolated and analyzed by SDS–PAGE and digital autoradiography. (E) The precursor of DIC was imported into WT and mia40-3 mitochondria. The mitochondria were treated with proteinase K and analyzed by BN–PAGE and digital autoradiography. DIC₂, mature dimeric form of DIC. (F) The precursor of porin was imported into WT and mia40-3 mitochondria at 30°C. The mitochondria were analyzed by BN–PAGE.

Figure 5

The precursors of small Tim proteins associate with Mia40. (A) The radiolabeled precursor of Tim9 was incubated with energized WT mitochondria for 45 min at the indicated temperatures. The mitochondria were reisolated, solubilized by digitonin and subjected to BN–PAGE and autoradiography. Mia40^*, transport intermediate bound to Mia40. (B) Tim9 was imported into WT mitochondria for 10 min at 30°C. The mitochondria were converted to mitoplasts and incubated with antibodies for 30 min on ice. After reisolation and solubilization by digitonin, the samples were analyzed by BN–PAGE and autoradiography. (C) Tim9 was imported into WT mitochondria for 45 min at 30°C in the presence of zinc chloride as indicated. The mitochondria were reisolated, solubilized and subjected to BN–PAGE and autoradiography. Arrowhead, unspecific band. (D) Tim9 was imported into WT mitochondria for 15 min at 15 or 30°C. The mitochondria were lysed in digitonin buffer in the presence or absence of β-mercaptoethanol (β-ME) and analyzed by BN–PAGE. (E) Tim9 was imported into WT mitochondria for 10 min at 30°C where indicated. The mitochondria were subjected to swelling and incubated with antibodies. After reisolation, the mitochondria were mixed as indicated, solubilized with digitonin and analyzed by BN–PAGE and autoradiography. (F) Tim9 was imported into WT mitochondria for 10 min at 30°C. The mitochondria were reisolated, incubated at 30°C as indicated and analyzed by BN–PAGE. (G) Mitochondria isolated from WT and mia40-3 cells grown at 19°C were incubated with the radiolabeled precursors of Tim9 and Tim10 at 30°C. After solubilization with digitonin, the samples were analyzed by BN–PAGE and autoradiography. For comparison, WT mitochondria were analyzed by BN–PAGE and immunodecoration (samples 7 and 8). (H) Tim8 was imported into WT and mia40-3 mitochondria as described in the legend to G.

Figure 6

Mia40 is involved in the assembly pathway of small Tim proteins. (A) Mitochondria from WT and mia40-4 cells grown at 19°C were analyzed by SDS–PAGE and immunodecoration. Mia40, Tim10, Mge1 and Tom40, 15 μg mitochondrial protein (odd-numbered lanes), 45 μg mitochondrial protein (even-numbered lanes); other proteins, 25 μg mitochondrial protein (odd-numbered lanes), 50 μg mitochondrial protein (even-numbered lanes). (B) Energized mitochondria isolated from WT and mia40-4 cells grown at 19°C were incubated with the radiolabeled precursor of Tim9 at 30°C. The mitochondria were solubilized with digitonin and analyzed by BN–PAGE and autoradiography. (C) Tim9 was imported into WT and mia40-4 mitochondria at 30°C, followed by treatment with proteinase K. The mitochondria were reisolated and subjected to SDS–PAGE and autoradiography. (D) Mitochondria (μg of protein) from WT and tim10-2 cells grown at 24°C were analyzed by SDS–PAGE and immunodecoration. (E) Tim9 was imported into WT and tim10-2 mitochondria at 30°C, followed by proteinase K treatment and SDS–PAGE. (F) Tim9 was imported into WT and tim10-2 mitochondria at 30°C and analyzed by BN–PAGE and digital autoradiography. (G) Radiolabeled Cox19 and Cox17 were imported into WT or mia40-4 mitochondria in the presence of 10 μM CuCl₂. After swelling and incubation with antibodies as indicated, the mitoplasts were reisolated and analyzed by BN–PAGE.