1. Biochemistry and Chemical Biology
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Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes

  1. Daniela G Vitali
  2. Sandro Käser
  3. Antonia Kolb
  4. Kai S Dimmer
  5. Andre Schneider  Is a corresponding author
  6. Doron Rapaport  Is a corresponding author
  1. University of Tübingen, Germany
  2. University of Bern, Switzerland
Research Article
Cite this article as: eLife 2018;7:e34488 doi: 10.7554/eLife.34488
8 figures and 1 table

Figures

Figure 1 with 2 supplements
pATOM36 forms native-like complexes in the yeast mitochondrial OM.

(A) Mitochondria isolated from WT or mim1Δ/mim2Δ cells expressing pATOM36-HA were left intact or lysed with Triton X-100 (TX) before they were subjected to treatment with proteinase K (PK). Alternatively, other samples were subjected to alkaline extraction followed by separation by centrifugation to pellet (P) and supernatant (S) fractions. All samples were analysed by SDS-PAGE followed by immunodecoration with antibodies against the HA-epitope, the OM receptor protein Tom70, or the matrix soluble protein Hep1. (B) Mitochondria were isolated from yeast WT cells transformed with an empty plasmid (-) or from WT and mim1Δ/mim2Δ (ΔΔ) cells expressing pATOM36-HA (+). Isolated yeast organelles and mitochondria-enriched fraction from T. brucei (Tryp.) cells expressing pATOM36-HA were lysed with 1% digitonin. All samples were then subjected to BN-PAGE followed by immunodecoration with an antibody against the HA-tag. pATOM36-containing complexes are indicated with an asterisk.

https://doi.org/10.7554/eLife.34488.002
Figure 1—figure supplement 1
Topologies and protein sequence alignments of Mim1, Mim2 and pATOM36.

Left panels: Schematic representation of the experimentally determined and predicted topologies of Mim1, Mim2 and pATOM36. The transmembrane segment (TMS) of Mim1 has been experimentally characterised (solid line), whereas those of Mim2 and pATOM36 are predicted by PolyPhobius with TOPCONS (dashed lines). The amino acid positions of the TMSs are illustrated and the protein sizes are in brackets. CYT, cytosol; OM, outer mitochondrial membrane; IMS, intermembrane space. Right panels: Multiple protein sequence alignments of Mim1, Mim2 and pATOM36. Verified and predicted TMSs are highlighted by solid and dashed lines, respectively. Conserved glycine residues are marked in green. scer, Saccharomyces cerevisiae; ncra, Neurospora crassa; spom, Schizosaccharomyces pombe; tbru, Trypanosoma brucei; lmaj, Leishmania major; tcru, Trypanosoma cruzi.

https://doi.org/10.7554/eLife.34488.003
Figure 1—figure supplement 2
pATOM36-HA is expressed in the transformed cells.

Whole cell lysate of wild type (WT), mim1Δ (1Δ), mim2Δ (2Δ) and mim1Δmim2Δ (ΔΔ) cells transformed with either an empty plasmid (Ø) or a plasmid encoding for pATOM36-HA were obtained. The samples were analysed by SDS-PAGE and immunodecoration with the indicated antibodies.

https://doi.org/10.7554/eLife.34488.004
Figure 2 with 1 supplement
pATOM36 rescues the growth defects of cells lacking Mim1, Mim2 or both.

(A) The indicated strains transformed with an empty plasmid (Ø) or with a plasmid expressing pATOM36 or its HA-tagged variant were tested at three different temperatures by drop-dilution assay for growth on synthetic medium containing either glucose (SD-Leu) or glycerol (SG-Leu). For comparison, plasmid-encoded Mim1 or Mim2 were transformed into mim1Δ or mim2Δ cells, respectively. All dilutions are in fivefold increment. (B) Cells deleted for both MIM1 and MIM2 (mim1Δ/mim2Δ) were transformed with the empty plasmid (Ø) or a plasmid encoding either native pATOM36 or pATOM36-HA. Transformed cells were analysed by drop-dilution assay at the indicated temperatures on synthetic medium containing either glucose (SD-Leu) or glycerol (SG-Leu). All dilutions are in fivefold increment.

https://doi.org/10.7554/eLife.34488.005
Figure 2—figure supplement 1
pATOM36 rescues the growth defect of mim1Δmim2Δ cells.

The indicated strains transformed with an empty plasmid (Ø), a plasmid expressing pATOM36, or its HA-tagged variant were tested at three different temperatures by drop-dilution assay for growth on rich media containing either glucose (YPD) or glycerol (YPG). All dilutions are in fivefold increment.

https://doi.org/10.7554/eLife.34488.006
Figure 3 with 1 supplement
pATOM36 can compensate for the reduced steady state levels and assembly defects in cells lacking both Mim1 and Mim2.

(A) Mitochondria were isolated from WT or mim1Δ/mim2Δ cells transformed with either an empty plasmid (-) or with a plasmid encoding pATOM36-HA (+). The specified amounts were analysed by SDS-PAGE and immunodecoration with antibodies against either the indicated mitochondrial proteins or the HA-tag. (B) The intensity of the bands from three independent experiments such as those presented in (A) was monitored. The amounts of Tom70, Ugo1 and Tom20 in the various mitochondria samples are presented as mean percentage of their levels in control organelles (WT+ Ø). The levels of Fis1 were taken as loading control. Error bars represent ± SD. **p≤0.005, ***p≤0.0005. (C) Whole cell lysates were obtained from WT, mim1Δ (1Δ), mim2Δ (2Δ), or the double deletion mim1Δ/mim2Δ (ΔΔ) cells transformed with either an empty plasmid (Ø) or with a plasmid encoding pATOM36-HA. Samples were analysed by SDS-PAGE and immunodecoration with antibodies against the indicated mitochondrial proteins. The precursor form of mitochondrial Hsp60 is indicated with an arrowhead. (D) The mitochondria described in (A) were solubilised in a buffer containing 1% digitonin and then analysed by BN-PAGE followed by western blotting. The membranes were immunodecorated with antibodies against the TOM subunits, Tom40 (long and short exposures) and Tom22. The TOM complex is signposted. A Tom40-containing low molecular weight complex is indicated with an arrowhead. (E) Mim1 and pATOM36 interact directly with Tom70. Radiolabelled Mim1 or pATOM36 (input, I) were incubated with glutathione beads (-) or with beads that were pre-bound to recombinant GST alone or to GST fused to the cytosolic domain of Tom70 (GST-Tom70). After washing, bound material was eluated and proteins were analysed by SDS–PAGE followed by blotting onto a membrane, and detection with either autoradiography (upper panel) or Ponceau staining (lower panel).

https://doi.org/10.7554/eLife.34488.007
Figure 3—source data 1

pATOM36 can compensate for the reduced steady state levels in cells lacking both Mim1 and Mim2.

Orinigal data used for quantification in Figure 3B.

https://doi.org/10.7554/eLife.34488.009
Figure 3—figure supplement 1
pATOM36-HA does not rescue biogenesis defects in mas37Δ cells.

(A) Mitochondria isolated from wild type (WT) and mas37Δ (37Δ) cells transformed with either an empty plasmid (-) or a plasmid encoding for pATOM36-HA (+) were solubilised in 0.2% Triton X-100. Samples were analysed by BN-PAGE and immunodecoration with an antibody against Tob55. (B) Isolated mitochondria as in (A) were subjected to SDS-PAGE and immunodecoration with the indicated antibodies.

https://doi.org/10.7554/eLife.34488.008
pATOM36 can rescue some of the import defects of cells lacking the MIM complex.

(A) Mitochondria were isolated from WT cells transformed with an empty plasmid (WT-) or from mim1Δ/mim2Δ cells transformed with either an empty plasmid (-) or with a plasmid encoding pATOM36-HA (+). Radiolabelled Tom20ext molecules (5% input, I) were incubated with the indicated isolated organelles for the specified time periods. Then, mitochondria were treated with PK and analysed by SDS-PAGE and autoradiography. A proteolytic fragment of Tom20ext, which reflects correct membrane integration, is indicated by an arrowhead. (B) Radiolabelled Tom20 was incubated with isolated mitochondria as in (A). At the end of the import reactions, mitochondria were solubilised with 0.2% digitonin and samples were analysed by BN-PAGE followed by autoradiography. The migration of Tom20 molecules assembled into the TOM complex is indicated. (C) Radiolabelled Ugo1 was incubated with isolated mitochondria as in (A). Then, mitochondria were treated with trypsin and analysed by SDS-PAGE and autoradiography. A proteolytic fragment of Ugo1, which reflects correct membrane integration, is indicated by an arrowhead. (D) Radiolabelled Fis1-TMC (5% input, I) was incubated with isolated mitochondria as in (A). Then, mitochondria were subjected to an IASD assay, re-isolated and analysed by SDS-PAGE and autoradiography. Bands representing correctly integrated Fis1-TMC are marked by an arrowhead. (E) Radiolabelled pSu9-DHFR (5% input, I) was incubated with isolated mitochondria as in (A). Then, mitochondria were re-isolated and analysed by SDS-PAGE and autoradiography. The precursor and mature forms are indicated by p and m, respectively.

https://doi.org/10.7554/eLife.34488.010
mim1Δ and mim2Δ cells expressing pATOM36 do not show altered mitochondrial morphology.

(A) WT, mim1Δ, mim2Δ, and mim1Δ/mim2Δ cells harbouring mitochondria-targeted GFP (mito-GFP) were transformed with either an empty plasmid (Ø) as a control (left panels) or a plasmid encoding pATOM36 (right panels). Cells were analysed by fluorescence microscopy and representative images of the predominant morphology for each strain are shown. Scale bar, 5 µm. (B) Statistical analysis of the cells described in (A). Average values with standard deviation bars of three independent experiments with at least n = 100 cells in each experiment are shown.

https://doi.org/10.7554/eLife.34488.011
Figure 5—source data 1

mim1Δ and mim2Δ cells expressing pATOM36 have normal mitochondrial morphology

Original data used for quantification in Figure 5B.

https://doi.org/10.7554/eLife.34488.012
Yeast Mim1 and Mim2 form a high-molecular-weight complex in mitochondria of T.

brucei. (A) Schematic representation of the insert of the pLew100-based vector that allows tetracycline-inducible expression of C-terminally myc-tagged Mim1 and HA-tagged Mim2 in T. brucei. Pro prom, procyclin promotor; tet, tetracycline operator; pro sas, procycline splice acceptor site; tub igr, α- and β-tubulin intergenic region; ald polyA, 3'-UTR of the aldolase gene. (B) Top panels: immunoblot analysis of whole cells (Tot), soluble (Cyt) and digitonin-extracted mitochondria-enriched pellet (Mit) fractions of a tetracycline-inducible pATOM36-RNAi cell line expressing Mim1-myc and Mim2-HA. Duplicate blots were analysed for the expression of Mim1-myc (left panels) and Mim2-HA (right panels). ATOM40 and EF1a serve as mitochondrial and cytosolic markers, respectively. Bottom panels: Alkaline extraction of the mitochondria-enriched fraction (Mit) shown in the top panels. The pellet (P) and the supernatant (S) fractions corresponding to integral membrane and soluble proteins, respectively, were analysed by SDS-PAGE and immunodecoration. ATOM40 and CytC serve as markers for integral and peripheral membrane proteins, respectively. (C) Mitochondria-enriched fractions of the same cell line describe in (B) were left intact or lysed with Triton X-100 (TX) before they were subjected to treatment with proteinase K (PK). All samples were analysed by SDS-PAGE followed by immunodecoration with antibodies against myc and HA tags, the OM protein ATOM69, the IMS protein TbTim9, or the matrix protein mtHsp70. Note that mtHsp70 contains a folded core, which is protease resistant. A proteolytic fragment of Mim1 and Mim2 is indicated with an arrowhead. (D) Duplicate immunoblots from BN-PAGE analysis of mitochondria-enriched fractions of the same cell line describe in (B) were probed for Mim1-myc (left panels) and Mim2-HA (right panels). Sections of the coomassie-stained gels serve as loading control. (E) Immunoblots of a BN-PAGE analysis of mitochondria-enriched fractions of the T. brucei (T.b.) cell line simultaneously expressing myc-tagged Mim1 (Mim1-myc) and HA-tagged Mim2 (Mim2-HA) and isolated yeast (S.c.) mitochondria simultaneously expressing HA-tagged versions of Mim1 and Mim2. The immunoblots are probed with antibodies against HA- or myc-tag.

https://doi.org/10.7554/eLife.34488.013
Yeast Mim1 and Mim2 complement the mitochondrial OM biogenesis phenotype of T.

brucei cells ablated for pATOM36. (A) Left panel: growth in the presence and absence of tetracycline (black and grey lines, respectively) and loss of kDNA (red line) in the presence of tetracycline of the pATOM36-RNAi parent cell line. Right panel: as in the left but the analysis was done for the pATOM36-RNAi cell line that co-expresses Mim1-myc and Mim2-HA. (B) Whole cell lysates from the cell lines as in (A) were obtained after the indicated time of induction. Proteins of these samples were analysed by SDS-PAGE and immunodecoration with the indicated antibodies. ATOM46, ATOM19 and ATOM14 are subunits of the ATOM complex. Cytosolic EF1a serves as a loading control.

https://doi.org/10.7554/eLife.34488.014
Figure 8 with 1 supplement
Mim1 and Mim2 rescue the assembly defect of the ATOM complex and the altered mitochondrial morphology in cells lacking pATOM36.

(A) Mitochondria-enriched fractions from the cell lines as in Figure 7A were obtained after the indicated time of induction. Samples were analysed by BN-PAGE followed by immunodecoration with antibodies against the indicated subunits of the ATOM complex. The migration of the ATOM complex is signposted. Sections of the coomassie-stained gels serve as loading controls. Arrowhead indicates an ATOM40-containing lower molecular weight complex. (B) Left images: Immunofluorescence analyses of mitochondrial morphology in the pATOM36 RNAi cell line after 0 or 3 days of induction. Right images: as in the left panels but the analysis was performed with the RNAi cell line co-expressing Mim1-myc and Mim2-HA. ATOM40 is shown in green and DAPI-stained DNA is shown in blue. DIC, differential interference contrast. Scale bar, 5 µm.

https://doi.org/10.7554/eLife.34488.015
Figure 8—figure supplement 1
Complementing the biogenesis function of pATOM36 requires both Mim1 and Mim2.

Individual clones of a pATOM36-RNAi cell line transfected with plasmids encoding myc-tagged Mim1 and HA-tagged Mim2 were analysed by BN-PAGE and subsequent immunodecoration. Clones that primarily express either myc-tagged Mim1 (A) or HA-tagged Mim2 (B) were analysed. The BN-PAGE blots were probed with anti-ATOM40 (upper panel), anti-myc (middle panel), and anti-HA (bottom panel) antibodies. Days of tetracycline induction (+Tet (d)) are indicated. Bottom graphs: growth curve for the same clone as above analysed in the presence and absence of tetracycline. Days of induction with tetracycline (+Tet [d]) are indicated. Inset: whole cell lysates of the clones were analysed by SDS-PAGE and immunodecoration with the indicated antibodies.

https://doi.org/10.7554/eLife.34488.016

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Saccharomyces cerevisiae)WT; W303α; MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15NA
Strain, strain background (S. cerevisiae)mim1Δ; W303α; MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 MIM1::KanMXDOI: 10.1242/jcs.103804
Strain, strain background (S. cerevisiae)mim2Δ; W303α; MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 MIM2::HIS3DOI: 10.1242/jcs.103804
Strain, strain background (S. cerevisiae)mim1Δ mim2Δ; W303α; MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 MIM1::KanMX MIM2::HIS3DOI: 10.1242/jcs.103804
Strain, strain background (S. cerevisiae)WT; YPH499; MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1
Strain, strain background (S. cerevisiae)mas37Δ; YPH499; MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1 MAS37::HIS3DOI: 10.1074/jbc.M411510200
Cell line (Trypanosoma brucei)29–13, procyclic, pATOM36 RNAiPMID: 22787278
Transfected construct (S. cerevisiae)pATOM36 RNAi + mim1-myc/mim2-HA (Figures 6, 7 and 8)this papersee Materials and methods
Transfected constructs (S. cerevisiae)pATOM36 RNAi + mim1-myc/mim2-HA (Figure 8—figure supplement 1)this papersee Materials and methods
Antibodyanti-HA (polyclonal rat)Roche11867423001; AB_390918WB 1:15000
Antibodyanti-Tom70 (polyclonal rabbit)N/AWB 1:2000
Antibodyanti-Hep1 (polyclonal rabbit)N/AWB 1:3000
Antibodyanti-Ugo1 (polyclonal rabbit)N/AWB 1:500
Antibodyanti-Tom20 (polyclonal rabbit)N/AWB 1:1600
Antibodyanti-Fis1 (polyclonal rabbit)N/AWB 1:1000
Antibodyanti-Hsp60 (polyclonal rabbit)N/AWB 1:100000
Antibodyanti-Tom40 (polyclonal rabbit)N/AWB 1:4000
Antibodyanti-Aco1 (polyclonal rabbit)N/AWB 1:7000
Antibodyanti-Tom22 (polyclonal rabbit)N/AWB 1:2000
Antibodyanti-Tob55 (polyclonal rabbit)N/AWB 1:2000
Antibodyanti-Por1 (polyclonal rabbit)N/AWB 1:4000
Antibodyanti-rat (HRP coupled goat)Abcamab6845; AB_955449WB 1:3000
Antibodyanti-rabbit (HRP coupled goat)Bio-Rad1721019; AB_11125143WB 1:10000
Antibodyanti-myc (monoclonal mouse)Invitrogen132500WB 1:2000
Antibodyanti-HA (monoclonal mouse)Enzo Life Sciences AGCO-MMS-101 R-1000WB 1:5000
Antibodyanti-EF1a (monoclonal mouse)Merck Millipore05–235WB 1:10000
Antibodyanti-ATOM40 (polyclonal rabbit)N/AWB 1:10000, IF 1:1000
Antibodyanti-CytC (polyclonal rabbit)N/AWB 1:1000
Antibodyanti-ATOM69 (polyclonal rabbit, affinity purified)N/AWB 1:50
Antibodyanti-TbTim9 (polyclonal rabbit)N/AWB 1:20
Antibodyanti-mtHsp70 (mouse)N/AWB 1:1000
Antibodyanti-ATOM46 (polyclonal rabbit; affinity purified)N/AWB 1:50
Antibodyanti-ATOM19 (mouse)N/AWB 1:500
Antibodyanti-ATOM14 (polyclonal rabbit)N/AWB 1:500
Antibodyanti-pATOM36 (polyclonal rabbit; affintiy purified)N/AWB 1:250
Antibodyanti-rabbit Alexa488ThermoFisher ScientificIF 1:1000
Antibodyanti-rabbit IRDye 800CWLI-COR BiosciencesP/N 925–32211WB 1:20000
Antibodyanti-mouse IRDye LT680LI-COR BiosciencesP/N 925–68020; AB_2687826WB 1:20000
Antibodyanti-mouse (HRP-coupled goat)Sigma AldrichAP308PWB 1:5000
Antibodyanti-rabbit (HRP coupled goat)Sigma AldrichAP307PWB 1:5000
Recombinant DNA reagentØ; pYX142 (plasmid)
Recombinant DNA reagentpATOM36; pYX142-pATOM36 (plasmid)this paperpATOM36 ORF was amplified from pFT33 and cloned in pYX142 between EcoRI and BamHI
Recombinant DNA reagentpATOM36-HA; pYX142-pATOM36-3HA (plasmid)this paperpATOM36 ORF was amplified from pFT33 and cloned in pYX142 between EcoRI and BamHI
Recombinant DNA reagent35S-Mim1; pGEM4-Mim1-4M (plasmid)DOI: 10.1038/sj.embor.7400318
Recombinant DNA reagent35S-pATOM36; pGEM4-pATOM36 (plasmid)this paperpATOM36 ORF was subcloned from pYX142-pATOM36 in pGEM4 with EcoRI and BamHI
Recombinant DNA reagent35S-Tom20ext; pGEM4-Tom20ext (plasmid)DOI: 10.1074/jbc.M410905200
Recombinant DNA reagent35S-Tom20; pGEM3-Tom20 (plasmid)DOI: 10.1074/jbc.M410905200
Recombinant DNA reagent35S-Ugo1; pGEM4-Ugo1 (plasmid)DOI: 10.1083/jcb.201102041
Recombinant DNA reagent35S-Fis1; pGEM4-Fis1-TMC (plasmid)DOI:10.1242/jcs.024034
Recombinant DNA reagent35S-pSu9-DHFR; pGEM4-pSu9-DHFR (plasmid)PMID: 2892669
Recombinant DNA reagentmito-GFP; pRS426-TPI-pSu9-eGFP (plasmid)this paperpSu9-eGFP was subcloned from pYX142-pSu9-GFP (Westermann B. and Neupert W. Yeast, 2000) to pRS426 with EcoRI and HindIII
Peptide, recombinant protein GSTGSTDOI:10.1128/MCB.00227–13
Peptide, recombinant protein GST-Tom70GST-Tom70DOI:10.1128/MCB.00227–13

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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