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. 2005 Jan;6(1):57-62.
doi: 10.1038/sj.embor.7400318.

Mim1, a protein required for the assembly of the TOM complex of mitochondria

Thomas Waizenegger  1 , Simone Schmitt, Jelena Zivkovic, Walter Neupert, Doron Rapaport
Affiliations

Mim1, a protein required for the assembly of the TOM complex of mitochondria

Thomas Waizenegger et al. EMBO Rep. 2005 Jan.

Abstract

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.

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Figures

Figure 1
Figure 1
Mim1 is a mitochondrial protein integrated into the outer membrane. (A) Mim1 is conserved among fungi. Amino-acid sequences of Saccharomyces cerevisiae (S.c.), Schizosaccharomyces pombe (S.p.), Neurospora crassa (N.c.) and Candida albicans (C.a.) Mim1 are shown. Identical residues appear in white on a black background, and similar residues are in grey. The putative transmembrane segment of the S. cerevisiae protein is underlined. (B) Mitochondrial (M) and postmitochondrial fractions (P) were obtained from yeast cells and were subjected to SDS–PAGE and immunoblotting using antibodies against Mim1, hexokinase, a marker protein for the cytosol, and the mitochondrial protein Tom20. (C) Mitochondria were treated with proteinase K (PK) at the indicated concentrations for 15 min on ice. Samples were analysed by SDS–PAGE and immunoblotting with antibodies against Mim1, the outer membrane proteins Tom70 and Tom40 and the IMS protein Cytb2. Other samples were subjected to alkaline extraction. Untreated mitochondria (total, T), pellet (P) and a supernatant fraction (S) were analysed as above. (D) A strain containing an N-terminally His-tagged variant of Mim1 grows like WT. Cells containing His-tagged Mim1 and isogenic WT cells were tested for their ability to grow at 30°C on YPGal medium (dilution in tenfold increments). (E) Mitochondria containing N-terminally His-tagged Mim1 were treated with PK at the indicated concentrations for 15 min on ice. Samples were analysed by SDS–PAGE and immunoblotting with antibodies against the His tag, Tom70 and ADP/ATP carrier (AAC).
Figure 2
Figure 2
Depletion of Mim1 results in reduced levels of mitochondrial proteins. (A) Downregulation of Mim1 affects cell growth. WT cells and cells expressing Mim1 under control of the GAL10 promoter (GAL-MIM1) were shifted from galactose-containing medium to glucose-containing medium at time zero. (B) Depletion of Mim1 causes accumulation of mitochondrial precursor protein. Whole-cell lysates were prepared from GAL-MIM1 cells, which were grown first at 30°C on lactate medium containing 0.1% galactose, washed, diluted and then grown at 30°C on lactate medium containing 0.1% glucose for the indicated time periods. Cell lysates were analysed by SDS–PAGE and immunodecoration with the indicated antibodies. P and m represent the precursor and mature forms of Hsp60, respectively.
Figure 3
Figure 3
Mim1 is necessary for TOM complex assembly and is present in a high-molecular-mass complex. (A) Levels of various proteins in mitochondria from cells depleted of Mim1 for 15 h (Mim1↓), as determined by immunodecoration. (B) TOM complex in Mim1-depleted cells. Mitochondria as in (A) were lysed with 0.5% Triton X-100 (Tob55) or 1% digitonin and subjected to BNGE and immunoblotting with the indicated antibodies. Short and long exposures of the immunodecoration with antibodies against Tom40 are presented. The Tom40-containing low-molecular-mass species is indicated with an arrowhead. (C) Mitochondria from WT cells were lysed in 1.5% digitonin and subjected to size-exclusion chromatography on a Superose6 column. Fractions of 300 μl were analysed by immunoblotting for Tom22, Mim1 and Tob38. The elution peaks of marker proteins are indicated with arrows.
Figure 4
Figure 4
Mim1 is not required for the stability of the TOM complex. (A) Mitochondria isolated from either WT cells or cells depleted of Mim1 for 15 h (Mim1↓) were lysed with 1% digitonin in the presence of the indicated concentrations of n-dodecyl β-D-maltoside (DDM). The lysates were analysed by BNGE, blotted and immunodecorated with antibodies against Tom40. (B) Mitochondria as in (A) were lysed with 1% digitonin in the presence of the indicated concentrations of urea. (C) Mitochondria as in (A) were lysed with 1% digitonin and incubated for 10 min at the indicated temperatures.
Figure 5
Figure 5
Mim1 acts in a step after binding of the Tom40 precursor to the TOB complex. (A) Mitochondria isolated from WT and cells depleted of Mim1 for 8 h (Mim1↓) were analysed by SDS–PAGE and immunodecoration with the indicated antibodies. (B) Mitochondria as in (A) were incubated at 25°C with radiolabelled Tom40 precursor for various time periods. Mitochondria were lysed with 1% digitonin and analysed by BNGE followed by autoradiography. P, precursor protein; I and II, assembly intermediates I and II of Tom40 and the assembled TOM complex (TOM) are shown. (C) Quantification of the bands corresponding to the various intermediates presented in (B).
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