Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Abstract

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

Figure 1.

Tom13 and Tom38 are mitochondrial outer membrane proteins. Mitochondria were prepared from yeast strains expressing Tom13-HA or Tom38-FLAG. Mitochondria and mitoplasts generated by osmotic swelling (SW) were treated with 500 μg/ml PK for 30 min on ice. Mitochondria were treated with either 0.1 M Na₂CO₃ or 1% Triton X-100 with 500 mM NaCl (TX-100), and then pellets (ppt) and supernatants (sup) were separated by centrifugation. Proteins were detected by immunoblotting with the antibodies against the HA epitope (for Tom13-HA mitochondria), the FLAG epitope (for Tom38-FLAG mitochondria), or indicated proteins (for Tom38-FLAG mitochondria).

Figure 2.

Tom13 and Tom38 are involved in mitochondrial protein import in vivo. (A) Yeast strains in which Tom13 or Tom38 is down-regulated slowed their growth on galactose-free medium. Wild-type cells (WT) and those carrying the TOM13 (GAL-TOM13) or TOM38 gene (GAL-TOM38) under control of the GAL7 promoter were first grown on galactose-containing medium and then transferred to galactose-free medium. (B) One of the two chromosomal TOM13 (top) or TOM38 (bottom) genes in a yeast diploid strain, W303-AB, was disrupted, the diploid cells were sporulated, and six different asci were dissected. The four spores recovered from each asci were allowed to germinate and to grow on YPD for 45 h at 30°C. (C) Total lysates were prepared from WT, GAL-TOM13, and GAL-TOM38 cells, which were grown at 23°C for12 h in lactate medium (+0.1% galactose), diluted, and then grown at 23°C for the indicated hours in lactate medium (+0.1% glucose). Total proteins were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The arrowheads indicate the accumulated precursor forms of mtHsp60 and Mdj1p.

Figure 3.

Depletion of Tom13 or Tom38 does not affect import of mtHsp60 or AAC into mitochondria in vitro. Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled mtHsp60 precursor and AAC were incubated with Tom13↓ (lanes 4–6 and open circles), Tom38↓ (lanes 7–9 and open squares), and WT mitochondria (lanes 1–3 and closed circles) at 23°C for the indicated times. The mitochondria were treated with PK, and the imported proteins were analyzed by SDS-PAGE and radioimaging. The amounts of radiolabeled proteins added to each reaction are set to 100%.

Figure 4.

Depletion of Tom13 or Tom38 affects assembly of Tom40 and porin in the mitochondrial outer membrane in vitro. (A) Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled yeast Tom40 was incubated with WT, Tom13↓, and Tom38↓ mitochondria at 23°C for the indicated times. The mitochondria were reisolated by centrifugation, solubilized with 1% digitonin (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol) for 10 min on ice, and subjected to BN-PAGE analyses followed by radioimaging. Assembly I, the assembly intermediate I; Assembly II, the assembly intermediate II. (B) Radiolabeled Tom40 was imported into WT, Tom13↓, and Tom38↓ mitochondria for 60 min at 23°C. After reisolation, the mitochondria were solubilized with 1% digitonin; incubated with the antibodies against Tim23 (αTim23), Mas37 (αMas37), and Tom22 (αTom22) or without antibodies (none); and analyzed by BN-PAGE followed by radioimaging. (C) Radiolabeled Tom40 was imported into WT and Tom38↓ mitochondria for 90 min at 23°C and subjected to osmotic swelling (SW), trypsin treatment (100 μg/ml for 30 min on ice), or alkaline extraction (Na₂CO₃, as Fig. 1). As a control, translated Tom40 was treated with (lane 2) or without (lane 1) trypsin. Relative amounts of radiolabeled Tom40 imported into Tom38↓ mitochondria were compared with those into wild-type mitochondria (set to 100%): TOM40 complex (BN-PAGE), lane 15/lane 5 in A; Trypsin treatment, −SW, lane 4/lane 3 in C; Trypsin treatment, +SW, lane 6/lane 5 in C; Alkaline extraction, ppt, lane 8/lane 7 in C. Endogenous Tom40 was also subjected to similar trypsin treatment followed by immunoblotting with anti-Tom40 antibodies. Asterisks indicate a clipped form of Tom40. (D) In vitro import of porin into WT (closed circles), Tom13↓ (open circles), and Tom38↓ (open squares) mitochondria were performed at 23°C as described in Fig. 3. The amounts of imported, PK-protected protein were plotted.

Figure 5.

Tom38 forms a complex with Mas37 and Sam50. (A) Mitochondria containing Tom38-FLAG or Sam50-FLAG were solubilized as in Fig. 4 A and incubated with preimmune serum (Shift, preimmune) or anti-Mas37 antibodies (Shift, αMas37) for 1 h on ice. Solubilized protein complexes were analyzed by BN-PAGE and immunoblotting with the anti-FLAG antibody. The band marked with an asterisk is variable in different experiments. (B) Mitochondria containing Tom38-FLAG (top) or Tom13-FLAG (bottom) were solubilized with 0.8% digitonin (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 50 mM 6-aminohexanoic acid, and 10% glycerol) in the presence (+Shift) or absence (−Shift) of the anti-FLAG antibody for 1 h on ice. Solubilized protein complexes were analyzed by glycerol density gradient centrifugation (10–30% glycerol and 0.2% digitonin in the same buffer for solubilization) at 200,000 g for 6 h at 4°C. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using antibodies against the FLAG epitope, Tom38, Sam50, and Tim23. Numbers indicate fractions (from top to bottom). The asterisk indicates IgG.