Author response: Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes
2018-05-03

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged. https://doi.org/10.7554/eLife.34488.001 Introduction Mitochondrial outer membrane (MOM) proteins include a diverse set of enzymes, components of protein import machineries, pore forming proteins, as well as proteins mediating mitochondrial fusion, fission, and motility. In addition, the MOM harbours proteins that regulate apoptosis and mitophagy and hence are of central importance for the fate of the organelle and the whole cell. All these MOM proteins are nuclear-encoded and synthesised on cytosolic ribosomes. Therefore, they have to bear appropriate signals that ensure both their correct targeting to the organelle and their ability to acquire different topologies in the lipid bilayer. Despite their well-recognised importance, the diverse molecular mechanisms by which MOM proteins are specifically targeted to the organelle and inserted into their target membrane remain incompletely defined (Dukanovic and Rapaport, 2011). MOM proteins can be divided into several topological groups (Dukanovic and Rapaport, 2011). Some of them span the lipid bilayer with one transmembrane segment (TMS), while others transverse the membrane with multiple β-strands or α-helical structures. Depending on their orientation, single-span proteins can be classified into three groups: the first two are signal- or tail-anchored proteins, which face the intermembrane space (IMS) with either the N- or C-terminus, respectively. These proteins typically expose the bulk of the protein to the cytosol and only a very short segment faces the IMS. A third subclass of single-span proteins exposes soluble domains towards both the IMS and the cytosol. Other integral MOM proteins span the bilayer either with several α-helical TMSs or as β-barrel structures. Whereas the import pathway taken by β-barrel precursor proteins has been studied in some detail (Becker et al., 2008b; Endo and Yamano, 2009; Walther et al., 2009), much less is known about the factors and the mechanisms that assure the membrane integration of MOM proteins with helical TMSs. MOM helical multispan proteins follow a unique import pathway in yeast cells (Becker et al., 2011; Papic et al., 2011). Precursors of these proteins are integrated into the membrane in a process where the MOM protein mitochondrial import 1 (Mim1) cooperates with the import receptor Tom70 in binding precursor proteins and facilitating their insertion into the lipid bilayer. Interestingly, it appears that neither other subunits of the translocase of the outer membrane (TOM) nor components residing in the mitochondrial IMS are involved in this process. Currently, it is unresolved whether the MIM complex has only a receptor-like function or it acts also as an insertase (Vögtle et al., 2015). In addition to mediating the membrane integration of multi-span proteins, Mim1 is also involved in the biogenesis of the import receptors Tom20 and Tom70 and therefore the protein is also required for the proper assembly of the TOM complex (Becker et al., 2008a; Dimmer et al., 2012; Hulett et al., 2008; Lueder and Lithgow, 2009; Thornton et al., 2010; Waizenegger et al., 2005). Mim1 is known to interact with Mim2, another protein of the MOM that has a crucial role in the biogenesis of α-helical multispan proteins (Dimmer et al., 2012; Krüger et al., 2017). Both proteins form a high-molecular-weight complex (MIM complex). They transverse the MOM once and expose their N-terminal domains to the cytosol whereas their C-terminal regions are facing the IMS (Dimmer et al., 2012; Ishikawa et al., 2004; Lueder and Lithgow, 2009; Waizenegger et al., 2005). Considering their multifaceted functions, it is not surprising that the absence of Mim1 and/or Mim2 results in severe growth retardation and multiple cellular defects like hampered assembly of the TOM complex, alteration in mitochondrial morphology, and accumulation of unprocessed mitochondrial precursor proteins (Dimmer et al., 2012; Ishikawa et al., 2004; Mnaimneh et al., 2004; Popov-Celeketić et al., 2008; Waizenegger et al., 2005). Mim1 and Mim2 are conserved among various fungi but homologues in any other eukaryotes were not identified so far (Dimmer et al., 2012; Ishikawa et al., 2004; Otera et al., 2007; Waizenegger et al., 2005). This situation raises the question which factor(s) facilitate the membrane integration of helical MOM proteins in non-fungal organisms. Recently, a first candidate for such a factor was reported in the parasitic protozoan Trypanosoma brucei. It was shown that the integral MOM protein, peripheral archaic translocase of the outer membrane 36 (pATOM36), in analogy to the MIM complex, is involved in the assembly and/or membrane insertion of a small subset of MOM proteins including subunits of the main trypanosomal outer membrane protein translocase (ATOM complex) (Bruggisser et al., 2017; Käser et al., 2016). However, in contrast to the MIM complex, pATOM36 is also directly required for the inheritance of the single unit mitochondrial genome of trypanosomes, termed kinetoplast DNA (kDNA). A fraction of the protein localises to the tripartite attachment complex (TAC) (Käser et al., 2016), which connects the kDNA across the two mitochondrial membranes with the basal body of the flagellum (Schnarwiler et al., 2014). Although pATOM36 and Mim1/2 do not share any sequence or topological similarities (Figure 1—figure supplement 1), we wondered whether convergent evolution allowed these unrelated proteins to fulfil similar tasks in the biogenesis of MOM proteins. To address this question, we expressed pATOM36 in yeast cells. Remarkably, introduction of pATOM36 could complement the deletion of MIM1, MIM2, or even of both genes. Accordingly, the presence of pATOM36 in the deletion strains could reverse the known alterations resulting from the absence of the MIM complex. Importantly, the reciprocal complementation was also successful and co-expression of Mim1 and Mim2 in T. brucei cells ablated for pATOM36 could rescue all phenotypes associated with the MOM protein biogenesis function of pATOM36. Taken together, we present the first reciprocal functional rescue of two evolutionary unrelated mitochondrial biogenesis complexes between eukaryotic supergroups. Results pATOM36 forms a native-like complex in yeast cells To better understand the functional relation between yeast Mim1/2 and T. brucei pATOM36, we wanted to investigate whether the trypanosomal protein can complement the phenotypes observed in yeast cells lacking the MIM complex. To that aim, plasmids encoding for pATOM36 or its C-terminally 3xHA-tagged version (pATOM36-HA), as well as an empty plasmid (Ø) as a control, were transformed into wild type (WT), mim1Δ, mim2Δ or mim1Δ/mim2Δ cells. In T. brucei, pATOM36 is an integral MOM protein with the C-terminus exposed to the cytosol (Pusnik et al., 2012). Blue native (BN)-PAGE analysis has shown that the endogenous protein occurs in two groups of protein complexes of unknown composition with molecular weights of approximately 140–250 kDa and larger than 480 kDa (Käser et al., 2016; Pusnik et al., 2012). Initially, we verified that pATOM36-HA can be expressed in the aforementioned yeast strains (Figure 1—figure supplement 2). Next, we isolated mitochondria from either control or mim1Δ/mim2Δ cells harbouring pATOM36-HA. We observed that the C-terminally HA-tagged pATOM36, similar to the yeast import receptor Tom70, is accessible to added proteinase K in isolated mitochondria, whereas the matrix protein Hep1 was protected as would be expected for intact organelles (Figure 1A). Alkaline extraction of the isolated organelles showed that pATOM36, as Tom70 but unlike the soluble matrix protein Hep1, was detected in the pellet fraction indicating that it is an integral membrane protein (Figure 1A). Finally, a BN-PAGE analysis demonstrated that pATOM36 expressed in yeast forms complexes of similar size to the 140 and 250 kDa complexes observed in T. brucei mitochondria (Figure 1B). However, the higher molecular weight complex, which likely corresponds to a TAC subcomplex required for kDNA maintenance (Käser et al., 2016), was not detected. In summary, these results suggest that pATOM36 expressed in yeast cells behaves essentially identical to the endogenous protein: it is embedded into the MOM with its C-terminus facing the cytosol and it forms oligomeric complexes of ca. 140–250 kDa. Figure 1 with 2 supplements see all Download asset Open asset 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 pATOM36 can replace the MIM complex in yeast We next asked whether pATOM36 can rescue the growth defect on respiratory carbon sources of mim1Δ or mim2Δ cells. To that aim, plasmids encoding for pATOM36 or its HA-tagged version, as well as MIM1 or MIM2 and an empty plasmid (Ø) as a control, were transformed into wild type, mim1Δ and mim2Δ strains. The growth of the transformed cells was analysed by drop dilution assays on synthetic fermentative glucose-containing (SD-Leu) and respiratory glycerol-containing media (SG-Leu) at three different temperatures (15°C, 30°C and 37°C). Of note, the expression of pATOM36 and its HA-tagged version did not alter the growth of WT cells. Under all the tested conditions, pATOM36 and pATOM36-HA were able to rescue the growth defect caused by the absence of either Mim1 or Mim2 (Figure 2A). Of note, the rescue capacity of pATOM36 was similar to that of Mim1 or Mim2 in the corresponding deletion strains. Figure 2 with 1 supplement see all Download asset Open asset 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 These results suggest that pATOM36 is active in yeast cells but it remained unclear whether pATOM36 can function alone or if it requires one of the remaining Mim proteins. To address this question, we monitored the capacity of pATOM36 to rescue the growth retardation of the double deletion mim1Δ/mim2Δ cells. We observed that pATOM36 could functionally compensate for the absence of both Mim1 and Mim2, since it was able to rescue the growth defect on non-fermentable carbon sources, a condition which requires fully functional mitochondria (Figure 2B and Figure 2—figure supplement 1). The absence of Mim1 and/or Mim2 in yeast cells results in a variety of mitochondrial defects including reduction in the steady-state levels of Mim1/2 substrates like the outer membrane proteins Ugo1, Tom20 and Tom70 (Dimmer et al., 2012; Ishikawa et al., 2004; Popov-Celeketić et al., 2008; Waizenegger et al., 2005). We therefore monitored whether expression of pATOM36 restores the reduced levels of these MIM substrates. To that aim we isolated mitochondria from WT and mim1Δ/mim2Δ cells transformed with either an empty plasmid or a plasmid encoding pATOM36-HA and monitored the levels of the proteins by immunodecoration. The results indicate that, whereas expression of pATOM36-HA in WT cells did not alter the abundance of the tested proteins or did it only to a minor extent, it did restore the levels of Mim1/2 substrates Tom20 and Tom70 in mitochondria from the double deletion cells (Figure 3A and B). Interestingly, the effect of pATOM36 on the levels of Ugo1 was only marginal, suggesting that pATOM36 has preferences to certain MIM substrates. Figure 3 with 1 supplement see all Download asset Open asset 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 Download elife-34488-fig3-data1-v1.xlsx A further phenotype of cells lacking Mim1/2 is the accumulation of mitochondrial precursor proteins due to hampered assembly of the TOM complex (Ishikawa et al., 2004; Mnaimneh et al., 2004; Waizenegger et al., 2005). To test whether pATOM36 is able to reverse this situation, we obtained whole cell lysates from the cells described above. As can be seen in Figure 3C, the presence of pATOM36-HA in the deletion strains completely eliminated the appearance of the precursor form of mitochondrial Hsp60. The presence of pATOM36-HA in the deletion cell lines resulted also in enhanced levels of Tom40 whereas the amounts of aconitase (Aco1) were not affected (Figure 3C). These results suggest that the function of the MIM complex in TOM complex assembly can be replaced by pATOM36. To substantiate this assumption, we used digitonin-solubilised mitochondria, which were isolated from control and deletion strains, and analysed them by BN-PAGE. To detect the TOM complex, the corresponding immunoblots were probed with antibodies against either Tom40 or Tom22. Of note, pATOM36-HA did not affect the assembly of the TOM complex in WT cells (Figure 3D). As expected, in the absence of Mim1/2, a dramatic reduction in the amount of assembled TOM complex and an appearance of an unassembled Tom40-containing species can be observed. Strikingly, these alterations completely disappeared upon the introduction of pATOM36-HA into these cells (Figure 3D). To investigate the specificity of the complementation by pATOM36, we asked whether it can functionally replace another import factor that mediates the biogenesis of other MOM proteins. To that goal, pATOM36 was introduced into cells lacking Mas37/Sam37, a subunit of the TOB/SAM complex that facilitates membrane integration of β-barrel proteins and the TOM subunit Tom22 (Chan and Lithgow, 2008; Dukanovic et al., 2009; Wiedemann et al., 2003). Figure 3—figure supplement 1 shows that pATOM36 could revert neither the drop in the steady-state levels of the TOB complex and its altered assembly behaviour nor the reduced levels of either the β-barrel proteins Tom40 and Porin or the single-span protein Tom22. Thus, the effect of pATOM36 is specific for MIM substrates. These findings further support the notion that the single-span protein Tom22 follows an import pathway that is distinct from that taken by the signal-anchored subunits Tom20 and Tom70. Previous reports suggested that Tom70 works together with Mim1 in the biogenesis of multi-span helical MOM proteins (Becker et al., 2011; Papic et al., 2011). To test whether pATOM36 can also interact with Tom70, we utilised a recombinant protein composed of the cytosolic domain of Tom70 fused to GST moiety (GST-Tom70). When this protein was incubated with newly synthesised radiolabelled pATOM36, or with Mim1 as a control, we observed a specific binding to both proteins (Figure 3E). Although we cannot exclude the possibility that Tom70, as an import receptor for MOM proteins, recognises Mim1 and pATOM36 as substrates, it can be envisaged that, similarly to Mim1, pATOM36 can also cooperate with Tom70 in the biogenesis of MOM proteins. The aforementioned results indicate that pATOM36 can compensate for the loss of the MIM machinery. To demonstrate directly a role of pATOM36 in protein import into the outer membrane of yeast mitochondria, we performed in vitro import assays. To that aim, we tested whether the presence of pATOM36 in mitochondria lacking the MIM complex can rescue the reduced import capacity of the MIM substrates Tom20 and Ugo1 observed for these organelles. To monitor the import efficiency of radiolabelled Tom20 into isolated organelles, we employed an established assay based on the formation of a proteolytic fragment of an N-terminally extended variant of Tom20 (Ahting et al., 2005). This assay clearly demonstrated that the presence of pATOM36 is sufficient to improve dramatically the capacity of organelles lacking Mim1/2 to import radiolabelled Tom20 molecules (Figure 4A). Along the same line, the assembly of newly synthesised Tom20 molecules into pre-existing TOM complexes was markedly improved when pATOM36 was present in mitochondria lacking the MIM complex (Figure 4B). Similarly to its minor effect on the steady state levels of Ugo1, the presence of pATOM36 did not improve the capacity of isolated mitochondria to import radiolabelled Ugo1 (Figure 4C). As a control, we checked the effect of pATOM36 on the import of proteins that are not known as MIM substrates like the matrix-targeted model protein pSu9-DHFR or the MOM tail-anchored protein Fis1. In both cases, we did not observe altered import upon expression of pATOM36 (Figure 4D and E). Collectively, pATOM36 can support the biogenesis of MIM substrates but appears to have preferences to certain ones. Figure 4 Download asset Open asset 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 Finally, we tested whether the trypanosomal protein is able to rescue the mitochondrial fragmentation that is observed in cells lacking Mim proteins. To that goal, we transformed a plasmid encoding pATOM36 into WT, mim1Δ, mim2Δ, or mim1Δ/mim2Δ cells expressing mitochondrial targeted GFP (mito-GFP). Analysis of mitochondria from the resulting cell lines by fluorescence microscopy revealed that pATOM36 is able to revert the mitochondrial fragmentation observed in cells lacking Mim1 and/or Mim2 to the tubular-like morphology of organelles in control cells (Figure 5A and B). Figure 5 Download asset Open asset 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 or a plasmid encoding pATOM36 Cells were analysed by fluorescence microscopy and of the morphology for are 5 (B) analysis of the cells described in (A). with bars of three independent experiments with at cells in are Figure data 1 mim1Δ and mim2Δ cells expressing pATOM36 have mitochondrial morphology data used for quantification in Figure Download Mim1/2 form a native-like MIM complex in trypanosomes the rescue capacity of pATOM36 in yeast cells, we asked whether the functional between Mim1/2 and pATOM36 the yeast proteins to replace the function pATOM36 has in the biogenesis of trypanosomal MOM proteins. To that we a plasmid for the co-expression of Mim1 and HA-tagged Mim2 in T. brucei (Figure Next, this plasmid was introduced into a cell of pATOM36. In these cells, addition of the of the pATOM36 as well as the expression of the Mim1 and Mim2. Figure Download asset Open asset Mim1 and Mim2 form a high-molecular-weight complex in mitochondria of T. brucei. (A) of the of the that expression of C-terminally Mim1 and HA-tagged Mim2 in T. brucei. and of the (B) analysis of whole cells soluble and mitochondria-enriched pellet of a cell expressing and were analysed for the expression of and and as mitochondrial and cytosolic respectively. Alkaline extraction of the mitochondria-enriched fraction shown in the The pellet (P) and the supernatant (S) corresponding to integral membrane and soluble proteins, were analysed by SDS-PAGE and immunodecoration. and as for integral and peripheral membrane proteins, respectively. (C) of the same cell 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 and the OM protein the IMS protein or the matrix protein that a which is A proteolytic fragment of Mim1 and Mim2 is indicated with an arrowhead. (D) immunoblots from BN-PAGE analysis of mitochondria-enriched of the same cell in (B) were probed for and of the as loading control. (E) of a BN-PAGE analysis of mitochondria-enriched of the T. brucei cell expressing Mim1 and HA-tagged Mim2 and isolated yeast mitochondria expressing HA-tagged of Mim1 and Mim2. The immunoblots are probed with antibodies against or of cells showed that both proteins are expressed like the mitochondrial protein they are in the mitochondrial fraction (Figure Alkaline extraction of the revealed that, as the endogenous proteins in both Mim1 and Mim2 are in the together with the integral membrane protein whereas the soluble protein was present in the supernatant (Figure To monitor whether Mim1 and Mim2 are inserted into the membrane in their native orientation, mitochondria-enriched were treated with proteinase This treatment resulted for both proteins in the formation of C-terminal (Figure Thus, Mim1 and Mim2 their native in T. brucei mitochondria with their exposed to the cytosol and the C-terminus in the IMS. Mim1 and Mim2 of yeast cells form a complex of kDa (Dimmer et al., 2012; Ishikawa et al., 2004; Waizenegger et al., 2005). BN-PAGE shows that similar complexes of ca. which both and could be detected in T. brucei (Figure Importantly, these complexes similarly to complexes harbouring and of yeast mitochondria (Figure The higher molecular weight than that observed for native complexes in yeast can be by the