The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae

Abstract

The mitochondrion is an essential organelle for the production of cellular ATP in most eukaryotic cells. It is extensively studied, including in parasitic organisms such as trypanosomes, as a potential therapeutic target. Recently, numerous additional subunits of the respiratory-chain complexes have been described in Trypanosoma brucei and Trypanosoma cruzi. Since these subunits had apparently no counterparts in other organisms, they were interpreted as potentially associated with the parasitic trypanosome lifestyle. Here we used two complementary approaches to characterise the subunit composition of respiratory complexes in Euglena gracilis, a non-parasitic secondary green alga related to trypanosomes. First, we developed a phylogenetic pipeline aimed at mining sequence databases for identifying homologs to known respiratory-complex subunits with high confidence. Second, we used MS/MS proteomics after two-dimensional separation of the respiratory complexes by Blue Native-and SDS-PAGE to both confirm in silico predictions and to identify further additional subunits. Altogether, we identified 41 subunits that are restricted to E. gracilis, T. brucei and T. cruzi, along with 48 classical subunits described in other eukaryotes (i.e. plants, mammals and fungi). This moreover demonstrates that at least half of the subunits recently reported in T. brucei and T. cruzi are actually not specific to Trypanosomatidae, but extend at least to other Euglenozoa, and that their origin and function are thus not specifically associated with the parasitic lifestyle. Furthermore, preliminary biochemical analyses suggest that some of these additional subunits underlie the peculiarities of the respiratory chain observed in Euglenozoa.Liège, february 06 th 2014Dear Editor, Please find the fully revised version of our manuscript "The mitochondrial respiratory chain of the secondary green alga Euglena gracilis shares many additional subunits with parasitic Trypanosomatidae."We are grateful to you for having encouraged us to resubmit a modified manuscript. We have addressed all the comments raised by the reviewers on our first submission. A point to point answer has been submitted along with the manuscript. Main changes are : (i) additional explanations about the phylogenic/bioinformatic strategy including some phylogenetic trees (supplemental figures 1 and 2); (ii) all data that were previously not shown (mass spectrometry data, in vivo respiratory assays) are now included in the manuscript as supplemental figures/tables.We hope that we have now significantly improved our manuscript so you'll find it suitable for publication in the special issue of Mitochondrion dedicated to "Plant Mitochondria Biology". R: Primary MS data are now given in Supplemental Table 3 for each polypeptide for which the score was > 60. (BLAST alignments, Evalues, etc.) are actually presented in the paper to support the inference that a particular E. gracilis protein is a true homolog of a given kinetoplastid protein. The most important conclusion of t...

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

“…This unusual arrangement of catalytic subunits is surprising, given the otherwise strictly conserved pseudo-sixfold symmetry and catalytic mechanism of the (αβ) 3 hexamer in all other known F-type ATPases, including those of bacteria and chloroplasts (4,24,40). The observed alterations in the T. brucei and E. gracilis F 1 region are in line with previous biochemical studies reporting a complete cleavage of the α subunit into two fragments, both of which remain bound to the ATP synthase (10)(11)(12)(13)(14). Furthermore, it has been shown that despite the α-subunit fragmentation, the T. brucei ATP synthase remains catalytically active throughout the life cycle and is essential for parasite survival in both the procyclic and bloodstream forms (10,16,18,19); as mentioned above, it is the reverse function as a proton-pumping ATPase that is essential for survival in the mammalian host.…”

“…In contrast, there seem to be no subunits in the protozoan-type ATP synthases that correspond to the peripheral stalk or dimer interface in the metazoan type. Instead, proteomics and biochemical analysis have identified several unique subunits, none of which are homologous among the various phyla (10,11,36,37). This variation in subunit composition correlates with the observed diversity in structure, which may reflect adaptations to different energetic requirements or environmental conditions.…”

“…an N-terminal 14-kDa fragment (α N ) and a C-terminal 44-kDa fragment (α c ) (10)(11)(12)(13)(14). Even though the α subunit is cleaved, the complex still hydrolyzes and synthesizes ATP, which is critical for the survival of Trypanosoma brucei, the sleeping sickness parasite (10,11,(14)(15)(16)(17)(18)(19).…”

“…Even though the α subunit is cleaved, the complex still hydrolyzes and synthesizes ATP, which is critical for the survival of Trypanosoma brucei, the sleeping sickness parasite (10,11,(14)(15)(16)(17)(18)(19). How this cleavage affects the structure and molecular mechanism of the enzyme is unknown.…”

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

“…However, recent work with bovine mitochondria elegantly suggests that there is just one common pool of ubiquinone/ubiquinol and cytochrome c, which is free to interact with any OXPHOS components found in the protein-dense mt inner membrane (60). Whereas the interactions between respiratory supercomplexes appear to be quite delicate, the detection of the dimeric F o F 1 -ATP synthase has been more pervasive throughout the eukaryotic supergroups studied so far, including the protists of the supergroups Excavata and Alveolata (T. brucei, Euglena gracilis, Plasmodium falciparum, and Tetrahymena thermophila) (13,(61)(62)(63)(64). This dimerization is proposed to be crucial for crista formation within the mt inner membrane (65), and the commonality of this structural organization in a wide variety of eukaryotes suggests that it was an early attempt to create microenvironments for components of the OXPHOS pathway.…”