The emerging picture of the mitochondrial protein import complexes of Amoebozoa supergroup

Currently, the division of eukaryotic organisms consists of six large supergroups, namely Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta [1, 2]. As summarized in Fig. 1, the Amoebozoa, regarded as a sister clade to the Opisthokonta which involves animals and fungi, include some organisms that are known to have mitosomes, such as Entamoeba histolytica, an intestinal pathogen of humans [3, 4], but also organisms that have aerobic mitochondria, such as the slime mold Dictyostelium discoideum, a free-living organism that inhabits soil and compost [5], as well as the amoeba Acanthamoeba castellanii, a soil free-living amoeba also known as a facultative human parasite [6]. The organisms represent different subclades and subdivisions of the Amoebozoa [1]. A. castellanii represents the Lobosa, whereas D. discoideum and E. histolytica belong to the Conosa and are included in the Mycetozoa and Archamoebae, respectively (Fig. 1). Thus, the Amoebozoa encompass taxa of both biomedical and evolutionary importance, yet their genomic and transcriptomic diversity remains largely unsampled. The same applies to the mitochondrial protein import complexes. Accordingly, the data concerning the subunit organization of the mitochondrial protein import complexes for members of the same supergroup classified into distinct subclades and divisions are not well known.

Studies of the mitochondrial protein import machinery have revealed that the machinery consists of complexes located in all mitochondrial compartments and have provided information about their organization, function, and interplay. The most commonly used model organism in the studies is the yeast Saccharomyces cerevisiae. Consequently, the S. cerevisiae protein import machinery, schematically shown in Fig. 2, is defined as a canon and applied as a reference model in studies of the machinery of other eukaryotic organisms, including plants and animals [7–9]. The canon includes the presence of at least three complexes in the outer membrane, four in the inner membrane, and three in the intermembrane space. The TOM (translocase of the outer membrane), TOB/SAM (topogenesis of the mitochondrial outer membrane β-barrel proteins/sorting and assembly machinery) and MIM (mitochondrial import machinery) complexes are located in the outer membrane. The TOM complex represents a general entry gate for most proteins imported into mitochondria, whereas the TOB/SAM complex and MIM complex enable the insertion of β-barrel proteins and proteins containing one or more transmembrane-spanning helices into the membrane, respectively. The TIM22 (translocase of the inner membrane 22), TIM23 (translocase of the inner membrane 23), PAM (presequence translocase-associated motor), and OXA (oxidase assembly factor) complexes are located in the inner membrane. The TIM22 and TIM23 complexes mediate the import of precursor proteins with targeting signals located within their sequence and at their N-terminus, respectively. The PAM complex cooperates with the TIM23 complex to drive protein translocation into matrix, whereas the OXA complex participates in the insertion of proteins from the matrix side into the inner membrane. The MIA (mitochondrial intermembrane space assembly) complex as well as small Tim chaperone-like protein complexes (small Tims), i.e. Tim8-Tim13 and Tim9-Tim10-Tim12 complexes, are located in the intermembrane space. The MIA complex is responsible for the import of small intermembrane space proteins with the multiple cysteine residues due to the thiol-disulfide exchange, whereas small Tims associate with the TOB/SAM and TIM22 complexes to protect precursor proteins from misfolding in the intermembrane space.

Available data indicate that the subunit organization of the complexes of animals and plants is more or less similar to those depicted in Fig. 2 for S. cerevisiae (for reviews, see e.g. [7, 9–11] although some new subunits have recently been identified for animals e.g. [10] and plants [11]. Nevertheless, in other eukaryotes, the differences are more pronounced. Consequently, the commonly occurring subunits of the TOM, TOB/SAM, and TIM22 complexes are their channel-forming subunits, Tom40, Sam50/Tob55, and Tim22, respectively, whereas the common presence of Tim50, Tim17, and Tim23 is reported for the TIM23 complex, Erv1 for the MIA complex, and Tim9 and Tim10 for the small Tim protein complexes [12, 13]. In the case of the MIM and OXA complexes, data are still missing for this kind of analysis. The data concerning the protein import machinery are also strongly limited in mitosome-carrying organisms. As summarized by Heinz and Lithgow [12] as well as by Makiuchi and Nozaki [13], they are available for Cryptosporidium parvum (Chromalveolata), Encephalitozoon cuniculi (Opisthokonta), Giardia intestinalis (Excavata), and E. histolytica (Amoebozoa). The organisms carry Tom40 and Sam50/Tob55, although the latter was not detected for G. intestinalis, and only two receptor subunits of the TOM complex, Tom70 and Tom60, were predicted for E. cuniculi and E. histolytica, respectively. Moreover, C. parvum and E. cuniculi have Tim17 and Tim23 (the TIM23 complex) as well as Tim22 (the TIM22 complex), but it is difficult to distinguish the proteins at the level of their sequences. Other subunits of the complexes, Tim50 (the TIM23 complex) and Tim18 (the TIM22 complex), were found for C. parvum and E. cuniculi with the exception of G. intestinalis Tim18. In the case of small Tims, only Tim8 and Tim13 seem to be expressed by C. parvum. As far as the PAM complex subunits are concerned, mtHsp70 was found for all the mentioned organisms, Mge1 for C. parvum, G. intestinalis, and E. histolytica, while Tim44 and Pam16 were detected only for C. parvum.

Our previous study, based on searching of the available genome and transcriptome data concerning amoebozoan subunits of the TOM and TOB/SAM complexes, indicated that the complexes may display structural variability in the Amoebozoa lineage and their subunit number reduction, as compared with the corresponding canonical complexes of S. cerevisiae [14]. Accordingly, the predicted number of subunits of the TOM and TOB/SAM complexes were different for A. castellanii, and representatives of slime molds and entamoebas as well as within the slime molds and entamoebas. Importantly, entamoebas are proposed to contain mitosomes and E. dispar, and E. nuttalii are now recognized as separate species from the well-known pathogenic E. histolytica and E. invadens, although their pathogenicity is still a matter of debate [15–17]. To study the issue further, we extended our research on the transcriptome and genome analysis of the complexes located in the inner membrane and intermembrane space of the amoebozoan mitochondria. As mentioned above, they include the TIM22, TIM23, PAM, and OXA complexes located in the inner membrane as well as the MIA complex and the complexes of small Tim proteins forming Tim8-Tim13, and Tim9-Tim10-Tim12 complexes, all located in the intermembrane space. The predicted subunits indicate that for amoebozoans with aerobic mitochondria, organization variability of the complexes may be less pronounced than in the case of the mitochondrial outer membrane complexes. Nevertheless, the subunits predicted for the inner membrane and the intermembrane space complexes display different levels of diversity in regard to phylogenetic position and some of them have isoforms. Moreover, elimination of the complexes in the putative mitosome-bearing amoebozoans is predicted to be much more conspicuous than in the case of the outer membrane complexes. The results appear to be important for discussion on the complex localization and functions as well as their contribution to the protein import and functions of mitochondrial membranes.

We investigated the presence of genes encoding subunits of the mitochondrial protein import complexes located in the intermembrane space and inner membrane using available genome and transcriptome sequences. As summarized in Table S1 (Additional file 1), among the studied amoebozoans the transcriptome data are not available for the slime mold Dictyostelium fasciculatum and both the entamoebas, i.e. Entamoeba nuttalli and Entamoeba dispar. Moreover, in the case of the amoeba Acathamoeba castellanii we applied two transcriptome datasets, one already available at GenBank [6] and the second (AC_RNAseq) assembled by us (see Methods). The reason is that we noticed some differences between the transcriptome datasets reflected in amino acid sequences of proteins predicted for the mitochondrial outer membrane [14]. The involvement of transcriptome data supplies more information regarding gene isoforms and gene structure giving more opportunity in prediction process of examined genes.

Subunits predicted for A. castellanii

The application of the available GenBank and AC_RNASeq enabled prediction of 17 A. castellanii possible subunits of mitochondrial protein import complexes located in the intermembrane space and the inner membrane (Table 1; for the complex organization see Fig. 2). Importantly, four of the subunits appeared to have isoforms (Tim9, Tim10, Pam16, and Pam18). All the predicted proteins displayed a high level of amino acid sequence identity to the GenBank data but also some differences were observed when AC_RNASeq was taken into account. The differences are shown in Additional file 1: Figures S1 and S2.

Subunit	Tim9	Tim10	Tim22	Sdh3	Tim17	Tim21	Tim23	Tim50	Mgr2
A.castellanii	A: XP_004340603  MF045879 B:MF045869 C: XP_ 004340763  MF045880	A: XP_004339376  MF045875 B: XP_004336134  MF045868	AAT66174 MF045866	XP_ 004367486 MF045878	XP_ 004347782 MF045865	XP_004340873	XP_004336406 MF045876	XP_004344840 MF045867	XP_004333275 MF045874
D.discoideum	XP_644845	XP_639927	XP_638851	XP_643757	XP_637129	XP_629896	XP_637678	XP_646611	XP_637862
D.fasciculatum	XP_004354109	XP_004358579	XP_004361430	XP_004362434	XP_00435582	XP_004350055	XP_004361430	XP_004354426	XP_004354259
D.purpureum	XP_003293366	XP_003286712	XP_003289095	XP_003290414	XP_003289186	XP_003287691	XP_003287004	XP_003284507	XP_003284359
P.pallidum	EFA81367	EFA78336	EFA79918	EFA81038	EFA79540	EFA79679	EFA84293	EFA78450	EFA75083
E.dispar/nuttalli
Subunit	Pam16	Pam18	Tim44	mtHsp70	Tim15	Mge1	Mia40	AIF	Erv1	Oxa1
A.castellanii	A: XP_ 004335678  MF045873 B: XP_ 004368030  MF045872	A: XP_ 004345808  MF084995 B: MF045881	XP_004348518.1	XP_004339232 MF045871	XP_004335035.1	MF045870			XP_004367724	XP_004344684.1
D.discoideum	Q54SV6	Q54QN1	EAL64247	Q8I0H7		XP_638912	XP_643501	XP_636815.1	XP_645302	A:EAL66882.2 B: EAL64905.1
D.fasciculatum	XP_004356346	EGG24887	XP_004367372	XP_004360938		XP_004362148	XP_004357088	XP_004357359.1	XP_004360441	A:EGG21561.1 B: EGG16308.1
D.purpureum	XP_003293468	EGC31346	XP_003293823	XP_003283943		XP_003294616	XP_003291222	EGC30677.1	XP_003286950	A:XP_003292615.1 B: XP_003287360.1
P.pallidum	EFA78034	EFA86354	EFA81857	EFA85858		EFA77076	EFA83431	XP_020436302.1	EFA83584	A: EFA84401.1 B: EFA75517.1
E.dispar				XP_001736890
E.nuttalli				XP_008855870

Underlined letters denote sequences determined in this study Not-underlined letters denote sequences stored in the GenBank as specifically annotated proteins

The amino acid sequences predicted for Tim21, Tim44, Erv1 and Oxa1 have been already specifically annotated so the existing accession numbers were added to Table 1. The sequences predicted for Tim9C, Tim10A, Sdh3, Tim17, Tim23, Tim15 and Pam18A have been named specifically (Table 1, underlined letters; Tim15 is under the process of annotation). To check the accuracy of the predicted A. castellanii Tim9 and Tim10 isoforms the phylogenetic analysis was performed (Fig. 3a). In the obtained phylogenetic tree, two clusters representing Tim9 and Tim10 families can be easily distinguished. Although bootstrap values were not very high for the internal branches within two clusters, they were separated by the middle branch, which was supported by the bootstrap value of 98%. Thus, the isoforms were predicted properly and they appeared to be orthologs. Accordingly, to estimate amino acid similarity between A. castellanii isoforms of Pam16 and Pam18 and their phylogenetic relationships, phylogenetic trees were built (Fig. 3b–c). The obtained results indicated that the slime mold clades of Pam16 and Pam18 grouped with A. castellanii clades of these proteins. Thus the A. castellanii proteins appeared to be orthologs.

For the rest of the predicted proteins some differences were observed between AC_RNAseq and the GenBank data (Additional file 1: Figure S1). Consequently, we proposed some corrections of sequences predicted for Tim9A, Tim10B, Tim22, Tim50, Mgr2, Pam16A, Pam16B, and mtHsp70. In the case of the predicted Tim9A, the protein already stored in the GenBank (XP_004340603) was mis-annotated, as it contained only part of the functional domain of Tim9 proteins, and was fused with EF-hand Ca²⁺-binding domain that was removed from the corrected Tim9A version. The predicted Tim10B, Tim22, and Pam16A were longer than cognate annotated proteins (XP_004336134, AAT66174 and XP_004335678, respectively), whereas Tim50 and Mgr2 were longer than hypothetical proteins (XP_004344840 and XP_004333275, respectively). The protein XP_004368030.1 appeared to be encoded by an erroneously fused gene that was eliminated from the corrected Pam16B version. In the case of predicted mtHsp70, the correction concerned two amino acid substitutions at the C-terminus, namely alanine to serine and leucyne to methionine. Finally, the genes encoding Tim9B, Pam18B, and Mge1 were not present in the GenBank data but were supported by hits found in tBLASTn alignment against AC_RNASeq (Additional file 1: Figure S2).

Summing up, as can be concluded from data shown in Table 1, organization of the A. castellanii TIM23 and PAM complexes may be identical to cognate complexes of S. cerevisiae whereas in the case of small Tims, TIM22, OXA and MIA complexes some subunits described for cognate S. cerevisiae complexes appear not to be present.

The differences in the subunit organization of the mitochondrial protein import complexes have been mainly reported for members of different phylogenetic lineages, while for members of the same clade the issue is poorly studied. Thus, the data are missing to address the evolutionary aspects of the mitochondrial protein import machinery. As mentioned in the Introduction, the Amoebozoa includes taxa of both biomedical and evolutionary importance. We have recently shown comprehensive analyses of the mitochondrial outer membrane import complexes, i.e. the TOM and TOB/SAM complexes, of members of this supergroup, based on searching of the available genome and transcriptome data [14], but the mitochondrial protein import machinery located in the inner membrane and intermembrane have not been addressed.

Accordingly, the PubMed data searching for the Amoebozoa protein import complexes located in these mitochondrial compartments presents only results of a hidden Markov-model-based analysis performed for the D. discoideum and E. histolytica Tim9-Tim10-Tim12, TIM22, TIM23, and PAM complexes [12, 13, 28]. They predict the presence of the following subunits of the complexes for D. discoideum: Tim22 and undefined small Tim proteins (the TIM22 and Tim9-10-12 complexes), Tim17, Tim21, and Tim23 (the TIM23 complex) as well as Pam16, Pam18, and mtHsp70 (the PAM complex), whereas in E. histolytica, only mtHsp70 and a possible homolog of Mge1 have been predicted. Moreover, AIF was identified for D. discoideum [29]. Thus, we performed the analysis of genome and transcriptome sequences available for different amoebozoans to obtain a more coherent picture. The analysis includes the Tim9-Tim10-Tim12, TIM22, TIM23, PAM, MIA, and OXA complexes (Fig. 2) of organisms representing different subclades and subdivisions of the Amoebozoa (Fig. 1). In general, all the predicted subunits are conserved between S. cerevisiae and the studied amoebozoans.

For E. dispar and E. nuttalli only one subunit of all the studied complexes was predicted, namely mtHsp70, a member of the PAM complex. It is assumed that all Entamoeba species have mitosomes, although convincing data are still missing (Graham Clark, personal communication). Similarly, in contrast to the well-known pathogenic E. histolytica and E. invadens, the pathogenicity of E. dispar and E. nuttalli is still a matter of debate [15–17]. Nevertheless, it cannot be concluded that the predicted extreme reduction of the protein import apparatus of the mitochondrial inner membrane and the intermembrane space is a marker of a parasitic lifestyle, as parasitic protists are known to express Tim and Pam proteins [4, 13]. On the other hand, for the studied entamoebas the reduction of the protein import complexes in the inner membrane and intermembrane space seems to be much more pronounced than that of the outer membrane ones, where at least channel-forming subunits are predicted to be present [14]. Accordingly, Tom40 and Tob55/Sam50 as well as Mge1 and mtHsp70 were detected for E. histolytica [13]. However, for other mitosome possessing organisms the higher numbers of the import complex subunits have been predicted, namely Tom55/Sam50, Tom70, Tim17, Tim21, Tim23, Tim22 for Encephalitozoon cuniculi and Tom40, Pam16, Pam18, mtHsp70, Mge1 for Giardia lamblia [13]. Thus, the reduction of the protein import complexes appears to be an indicative feature of mitosomes but it seems that the level of the reduction is multifarious. It should also be remembered that mitosomes may contain proteins that are too divergent to be homologs of known import complex subunits, and consequently cannot be found using only bioinformatic tools [30]. For example, the presence of a novel mitosome outer membrane β-barrel protein and other two inner membrane proteins of unknown function has been shown to be unique to E. histolytica mitosomes [4, 17]. These proteins do not display similarity to canonical import complex subunits. Moreover, the β-barrel protein named MBOMP30 represents seventh MBOMP subclass lacking any recognizable sequence similarity to any of the six previously identified ones. On the other hand the lack of the transcriptome datasets of E. dispar and E. nuttalli may impact the possibility of protein prediction.

For A. castellanii and the slime molds, the organization of the studied complexes is very similar with exception of the MIA complex (Table 1). Moreover, the predicted organization appears to be quite similar to the canonical one described for S. cerevisiae (Fig. 2), Thus it appears that also in these amoebozoan mitochondria the diversity of the inner membrane and the intermembrane space import complexes is less pronounced than in the case of the outer membrane ones [31]. Accordingly, the encoding genes of the slime molds contain similar numbers of predicted exons, being not distinctly different from the numbers predicted for A. castellanii putative genes, with the exception of genes encoding Tim50, Mgr2, Tim44, mtHsp70, and Erv1 (Additional file 1: Table S3).

However, a distinct difference is apparent when the predicted subunits of the A. castellanii and slime mold Tim9-Tim10-Tim12, PAM, and OXA complexes are compared. Namely, the predicted A. castellanii Tim9, Tim10, Pam16, and Pam18 and the slime mold Oxa1 appear to have isoforms encoded by separate genes. Interestingly, searching of available databases indicates the presence of Tim9, Tim10, and Pam protein isoforms in mitochondria of other organisms, mainly animals and humans [7, 9]. Isoforms were also found for human Tim17 [32] and Tim8 [33–35]. In the case of the OXA complex, two genes encoding Oxa1 and Oxa2 have been reported for Opisthokonta (animals and fungi) and more than two for Archaeplastida (plants) [36]. On the other hand, members of the Excavata seem to have isoforms of Oxa2, while Oxa1 is not present, whereas only one gene encoding Oxa protein and a lack of the protein isoforms have been shown for Chromalveolata [7, 36, 37]. Thus the diversity of the OXA complex organization is still to be explained. Accordingly, the precise roles of the paralogs, including Oxa1 and Oxa2, have yet to be clarified [7]. The difference concerning the presence of small Tims, Pam16, Pam18, and Oxa1 isoforms between the studied amoebozoans constitutes an interesting observation from the point of view of the supergroup phylogeny, as the Amoebozoa are regarded as a sister clade to the Opisthokonta [1, 2]. While the putative expression of the A. castellanii Tim9, Tim10, Pam16, and Pam18 isoforms resembles Opisthokonta cognate proteins, the possible presence of the slime mold Oxa1 isoforms is much more difficult to explain. Nevertheless, the differences may reflect the evolutionary history of the subclade formation.

As mentioned above, the difference is also observed for the MIA complex as only Erv1 protein is predicted for the A. castellanii MIA complex, whereas Erv1, Mia40 and AIF are predicted for all the studied slime molds. Interestingly, as in the case of plant and mammalian but not S. cerevisiae mitochondria [38], the predicted slime mold Mia40 appears to be a soluble protein (not shown). Therefore, one can suggest that AIF can serve as Mia40 receptor not only in mammalian mitochondria [39–41]. On the other hand, the apparent absence of Mia40 regarded as an essential protein (e.g. [38]) in some eukaryotes has also been reported. For example, the protein does not seem to be present in brown algae [31] and parasitic protists, such as Plasmodium falciparum, Leishmania tarentolae, and Trypanosoma cruzi [42]. Accordingly, A. castellanii is the only proven parasite among the studied amoebozoans, defined as an opportunistic pathogen responsible for amoebic keratitis and granulomatous amoebic encephalitis in humans [43, 44]. Yet, the conclusions in regard to the correlation between the lack of Mia40 and pathogenicity are at present unjustified. Importantly, the presence of small Tim proteins which undergo oxidative folding, indicate that an oxidative folding machinery exists in A. castellanii mitochondria [45]. It suggests that other protein(s) can replace the subunit. Accordingly, for the Trypanosoma brucei TOM complex, novel receptors with no homology to known TOM import proteins i.e. ATOM46 and ATOM69, have been identified [46]. Interestingly, these proteins were also found in all kinetoplastids (including the non-pathogenic ones), showing that the expression of the unique proteins is not an adaptation to the parasitic lifestyle of T. brucei [47].

Interestingly, amino acids sequence comparison (Table 2) indicates that the predicted subunits of the studied complexes display different levels of amino-acid sequence conservation. The lowest diversity is observed for subunits predicted for the TIM23 and MIA complexes whereas the highest diversity occurs for subunits predicted for the OXA complex, particularly in the case of the slime mold Oxa1B isoform that appears to be a homolog of A. castellanii Oxa1. The differences are not reflected in the encoding gene intron-exon structures (Additional file 1: Table S3), as it has been observed for the outer membrane complexes that excludes different regulation of protein expression at the level of splicing known to support an adaptation to a given lifestyle [14]. Nevertheless, it can be suggested that the different levels of amino acid sequence variability observed for subunits predicted for the inner membrane and intermembrane space import complexes and the outer membrane import complexes may result from natural selection, as the proteins appear to control efficiently organelle biogenesis and function, including both membranes. Accordingly, as it is speculated that differences in amino acid sequences observed between members of different supergroups reflect the early diversification of eukaryotes [47], it can be proposed that within a given supergroup, the variability may reflect emergence of species within its branches.

Here we present results of a comprehensive bioinformatic analysis of the protein import complexes located in the mitochondrial inner membrane (the TIM22, TIM23, PAM, and OXA complexes) and intermembrane space (the Tim9-Tim10-Tim12 and MIA complexes) of selected species of the Amoebozoa. This analysis was based on searching of the available genome and transcriptome sequences. Our results indicate that in A. castellanii and slime molds, the complex organization appears to be quite similar to the canonical ones described for S. cerevisiae. However, distinct differences are observed in amino acid sequences of some of the predicted proteins and in the numbers of some of the protein isoforms. Moreover, the performed analysis for entamoebas, i.e. E. dispar and E. nuttalli, indicates the absence of any subunit of all the studied complexes except mtHsp70 (the PAM complex), and that further supports the suggestion that all entamoebas have mitosomes. Importantly, the reduction of the protein import apparatus of entamoebas is much more pronounced than in the case of the outer membrane. It seems to be a rule for entamoebas, including E. histolytica, but not for all organisms possessing mitosomes. This constitutes an interesting issue from the evolutionary and biomedical perspective, as it addresses the problem of mitochondrial and/or mitosome protein import machinery variability within the currently defined supergroup of eukaryotes. On the other hand, it is becoming clear that the knowledge of mitochondrial protein import of model organisms cannot be generally transferred to all other eukaryotes.