The ATP synthase is a highly conserved protein complex found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. The complex consists of two functionally distinct portions: the hydrophilic F₁ and the membrane-bound F_o (Walker, 2013). The mechanism of this molecular motor is best understood for the mitochondrial ATP synthases of yeast and mammals. Within their mitochondria, the proton gradient generated by the electron transport chain (ETC) drives the rotation of a ring of c subunits in F_o and of the attached central stalk within F₁. This rotation causes the conformational changes in the α and β subunits of F₁ that mediate catalysis of ATP from ADP and inorganic phosphate (P_i) (Jonckheere et al., 2012). The stator, also known as the lateral stalk, is an essential component of the ATP synthase because it counteracts the rotation of the α and β subunits, enabling ATP synthesis (Dickson et al., 2006). It is therefore surprising that despite general conservation of the central subunits, the lateral elements of protozoan ATP synthases are structurally diverse, and these organisms appear to lack homologs for the stator subunits of yeast and mammals (Lapaille et al., 2010).

The composition of the mitochondrial ATP synthase has mainly been determined from detailed studies of purified Saccharomyces cerevisiae and Bos taurus mitochondria, both members of the eukaryotic clade Opisthokonta. In these two species, the architecture of the ATP synthase is virtually identical, and sequence analysis and proteomics have identified homology for nearly all of the subunits that constitute the mitochondrial ATP synthase (Wittig and Schägger, 2008). In contrast, recent studies in different protozoan species have reported unique structural and functional features in their ATP synthases. Proteomic and biochemical studies have identified several potential stator subunits in Tetrahymena thermophila and Euglena gracilis, although none appear conserved among protists (Balabaskaran Nina et al., 2010; Perez et al., 2014). Similarly, the ATP synthases of algae like Chlamydomonas reinhardtii and Polytomella spp. and kinetoplastids like Trypanosoma brucei lack canonical stator subunits but possess phylum-specific proteins that likely perform that function (Šubrtová et al., 2015; van Lis et al., 2007).

Apicomplexans comprise a large phylum of obligate intracellular eukaryotic parasites that infect animals and cause significant human mortality and morbidity. Comparative genomics suggests that there is extensive metabolic diversity within this phylum, reflecting the variety of life cycles and host environments the various species have adapted to. Several core subunits of the ATP synthase (α, β, γ, δ, ε, c, and oligomycin sensitivity-conferring protein (OSCP)) can be found in most apicomplexan genomes (Vaidya and Mather, 2009). However, within the Cryptosporidium genus, drastic reductions in mitochondrial metabolism have coincided with preservation of only the α and β subunits in certain species (Liu et al., 2016; Makiuchi and Nozaki, 2014). Notably, the stator subunits and key components of the membrane-bound portion of this complex have not been identified in any apicomplexan species.

Reliance on mitochondrial metabolism can also vary over the course of a parasite’s life cycle. For instance, the rapidly replicating forms of T. gondii catabolize glucose via the tricarboxylic acid (TCA) cycle (MacRae et al., 2012), whereas slow replicating stages seem to lack a functional TCA cycle and rely on glycolysis of stored amylopectin (Denton et al., 1996; Uboldi et al., 2015). A comparable metabolic shift seems to occur when T. gondii parasites egress from host cells and rely on oxidative phosphorylation for the majority of their ATP production (Lin et al., 2011; MacRae et al., 2012). Plasmodium spp. increase their TCA cycle activity as they transition from the asexual blood stages to the sexual cycle (MacRae et al., 2013). Consequently, deletion of the ATP synthase β subunit marginally affects blood stages but is essential for the sexual stages in the mosquito (Sturm et al., 2015). Together with pharmacological assays, these results confirm that the apicomplexan ATP synthase is active and essential in at least some life cycle stages (Balabaskaran Nina et al., 2011; Sturm et al., 2015). Direct measurements of ATP synthase activity in T. gondii (Vercesi et al., 1998) further suggest that these parasites must employ highly divergent stator subunits to form a functional enzyme.

To systematically examine gene function in T. gondii, our lab previously performed a CRISPR-based genome-wide screen that identified genes contributing to parasite fitness (Sidik et al., 2016). Approximately 360 of these fitness-conferring genes were broadly conserved among apicomplexans, but not other eukaryotes, and most lacked functional annotation; we referred to them as indispensable conserved apicomplexan proteins (ICAPs). Of the 17 ICAPs we determined a subcellular localization for, eight localized to the mitochondrion. This preponderance of ICAPs in the mitochondrion motivated further investigation.

Using unbiased proteomic and cryptic-homology analyses, we show that two mitochondrial ICAPs—ICAP2 and ICAP18—share homology with the ATP synthase stator subunits of opisthokonts. We show that both proteins interact with known and novel components of the ATP synthase, consistent with their role as putative subunits of the complex. Conditional depletion of ICAP2 leads to aberrant mitochondrial morphology and the disassembly of the ATP synthase complex, consistent with its proposed role in the stator. Our study reveals new information regarding the unconventional apicomplexan stator and provides a perspective on the divergent nature of the ATP synthase in this important group of pathogens.

The elegant rotary mechanism of the mitochondrial ATP synthase requires a stator to impart the conformational changes on F₁ that drive ATP catalysis. It has therefore been a long-standing conundrum that most apicomplexan genomes appear to code for only the core subunits of the ATP synthase but none of the stator subunits. Here, we propose that ICAP2 (TGME49_231410) and ICAP18 (TGME49_268830) fulfill the roles of the stator b and d subunits, respectively. Both proteins localize to the mitochondrion of T. gondii and share distant homology with the respective subunits of the bovine ATP synthase, despite their unusual length and domain composition. Immunocapture of these putative subunits showed that they stably interact with the ATP synthase complex, along with at least nine other proteins that are conserved among apicomplexans. Moreover, conditional knockdown of ICAP2 leads to abnormal mitochondrial morphology, slow replication, and parasite death. ICAP2 was also necessary for the activity of the ATP synthase, and its absence caused a reduction in oxygen consumption by the mitochondrion and disassembly of the complex. Our results reveal highly divergent aspects of the apicomplexan ATP synthase and demonstrate their importance for parasite viability.

We previously identified ICAP2 as one of eight novel mitochondrial proteins, which we localized because of their roles in parasite fitness, a lack of functional domains, and conservation among apicomplexans (Sidik et al., 2016). Pattern hidden Markov models and secondary structure predictions—implemented in HHPRED or Phyre2 (Kelley et al., 2015; Zimmermann et al., 2018)—first hinted at the homology between ICAP2 and the b subunit of the mammalian ATP synthase. These methods have also helped us assign putative functions to other subunits we isolated as part of the ATP synthase complex of T. gondii, including the putative d subunit (ICAP18; TGME49_268830), the putative a subunit (TGME49_310360), and the putative inhibitor (TGME49_215350). In opisthokonts, the a subunit is a multi-span membrane protein with a conserved arginine residue critical for its function (Mitome et al., 2010). Topology predictions of the putative a subunit suggest the presence of three TM domains and an arginine residue that is conserved in apicomplexans and other alveolates (Figure 3—figure supplement 2). We have also identified other mitochondrial domains among the novel subunits. TGME49_258060 and TGME49_285510 bear homology to CHCH domain-containing proteins, which localize to the mitochondrial intermembrane space and play roles in biogenesis and bioenergetics (Modjtahedi et al., 2016). ICAP15 has a low-probability Bcl-like domain, common among proteins involved in apoptosis and regulation of the electron transport chain (Gross and Katz, 2017).

During the revision of this manuscript, a similar set of proteins was identified as part of a proteomic analysis of the purified T. gondii ATP synthase complex (Salunke et al., 2018). In addition to the novel subunits we described, the authors describe ten additional subunits, which our proteomic analysis identified, but we excluded on the basis of lack of conservation in either C. muris, P. falciparum, or C. velia (Supplementary file 1). The novel subunits we propose may be closer to the core set of ATP synthase subunits present in alveolates; however, further studies will be needed to elucidate the function of most novel subunits regardless of their degree of conservation. ATP synthase complexes in other protists are characterized by a combination of conserved and phylum-specific subunits (Balabaskaran Nina et al., 2010; Šubrtová et al., 2015; van Lis et al., 2007). Our work demonstrates that distant homology searches can complement the molecular characterization of protein complexes in apicomplexans as in other divergent eukaryotes.

The length and extraneous domains present in ICAP2 make it a highly unusual b subunit, which has motivated the characterization we present. In particular, ICAP2 is nearly twice as long as its mammalian counterparts and appears to have an EF-like domain in its N terminus. EF domains bind cations like Ca²⁺ or Mg²⁺ and can act to modulate protein-protein interactions or enzymatic activity (Kawasaki and Kretsinger, 2017). Mitochondrial Ca²⁺ uptake is known to regulate several metabolic processes (De Stefani et al., 2016), and the presence of an EF-like domain suggests that ATP synthase activity may be somehow coordinated with mitochondrial Ca²⁺. Treatment of T. gondii with the ATP synthase inhibitor oligomycin causes an immediate increase in cytosolic Ca²⁺ levels (Moreno and Zhong, 1996). Although this may simply reflect the use of ATP by the endoplasmic-reticulum Ca²⁺-ATPase, it also suggests a need for strong coupling between ATP production and Ca²⁺ sequestration. Divergent stator paralogs are expressed in the Drosophila testis and underlie tissue specific properties of the ATP synthase that impact mitochondrial morphology and activity (Sawyer et al., 2017). Having provided strong evidence for the role of ICAP2 in the ATP synthase, more work will be needed to characterize the structure of the complex and understand how its unusual features mediate the specific adaptations of apicomplexan mitochondria.

ICAP2 is one of 11 novel subunits we believe are part of the apicomplexan ATP synthase. Orthologs of all of these subunits are found exclusively among alveolates, although they are absent from the genomes of species with reduced mitochondrial pathways. Several Cryptosporidium spp. (C. hominis, C. parvum, and C. ubiquitum) exhibit extreme reductions in the TCA cycle and electron transport chains, and concomitantly lack most of the known subunits of the ATP synthase along with the novel subunits we have identified (Liu et al., 2016). However, all Cryptosporidium spp. retain the α and β subunits, which can convert ATP^4- to ADP^3-, fueling the generation of a membrane potential (Δψ) across the inner mitochondrial membrane through the action of the ADP/ATP carrier (AAC). A similar mechanism has been proposed for the F_o-independent generation of Δψ in Trypanosoma brucei strains carrying mutations in the γ subunit (Dean et al., 2013). Even in the absence of an ATP synthase, Δψ is needed for the import of proteins into the matrix of mitochondria and mitosomes (Fox, 2012). Intriguingly, the genome of Gregarina niphandrodes, a divergent apicomplexan that infects beetles, lacks all of the mentioned ATP synthase subunits, including α and β along with homologs of the AAC (CryptoDB), and would require an alternative mechanism to generate Δψ if mitosomes are present and active in this organism.

We present the first direct evidence for the important role of the ATP synthase in T. gondii metabolism. Pharmacological and metabolomics approaches had previously demonstrated the presence of ATP synthase activity in T. gondii mitochondria and the importance of oxidative phosphorylation in supplying ATP to the parasite (Lin et al., 2011; MacRae et al., 2012; Vercesi et al., 1998). Consistent with these observations, depletion of ICAP2 reduces the rate of parasite replication and ultimately leads to a loss of parasite viability. This is in contrast to the mild effect of knocking out the β subunit in blood stages of P. berghei, although such mutants are subsequently impaired during the sexual stages of the life cycle (Sturm et al., 2015). The variable impact of the ATP synthase on the various stages of different apicomplexans may reflect the ability of these parasites to adapt to the changing environmental conditions they encounter across their life cycles.

ICAP2 depletion appears to damage T. gondii mitochondria leading to fragmentation and loss of volume. In other eukaryotes, mitochondrial fission segregates damaged parts of the organelle (Youle and van der Bliek, 2012). Mitochondrial fragmentation has been previously observed in T. gondii in response to exogenous stresses, including starvation (Ghosh et al., 2012) or monensin treatment (Charvat and Arrizabalaga, 2016). The effect of monensin was attributed to its uncoupling activity and to the induction of oxidative stress. Both effects could resemble defects in the ATP synthase like those predicted to result from loss of the stator; uncoupling would dissipate the Δψ used by the complex, and mutations in the F_o subunits can lead to increased reactive oxygen species (Baracca et al., 2007). Saccharomyces cerevisiae stator subunit mutants have punctate, rounded mitochondria that display an altered ultrastructure (Bornhövd et al., 2006; Weimann et al., 2008). These defects result from the involvement of the stator subunits in cristae formation (Cogliati et al., 2016; Wittig and Schägger, 2008) and the uncoupling of ATP hydrolysis from proton translocation (Paul et al., 1989; Soubannier et al., 2002). Although ICAP2-depleted mitochondria did not show the smooth concentric membranes seen in S. cerevisiae stator mutants, they did have fewer cristae than wild-type mitochondria. To further understand the effects that lead to mitochondrial fragmentation, it will be interesting to compare uncoupling mutations, like ICAP2 loss, to disruption of the catalytic or proton-transporting functions of the T. gondii ATP synthase.

Without a functioning ATP synthase, the ETC is presumably inhibited by a buildup of Δψ, which would normally be dissipated through the rotating F_o (Mueller, 2000). We observed that the mOCR is reduced in ICAP2-depleted parasites and can be restored by the addition of a proton ionophore that dissipates Δψ. In the accompanying paper by Seidi, Muellner-Wong, and Rajendran, et al., disruption of a novel apicomplexan component of the ETC complex IV also reduced the mOCR, although this effect could not be reversed by proton ionophore treatment. This indicates a general ETC defect in the complex IV mutant distinct from that observed upon ICAP2 depletion. Notably, mutations in complex IV appear to have a milder effect on parasite viability and did not alter mitochondrial morphology. We also observed normal morphology upon ETC inhibition with atovaquone (a complex III inhibitor) and fragmented mitochondria following ATP synthase inhibition with oligomycin (data not shown).

The contribution of the mitochondrial ATP synthase to cellular ATP pools can be directly measured in T. gondii following repression of glycolysis by 2-DG and utilization of glutamine for oxidative phosphorylation (Lin et al., 2011). As expected, ICAP2-depletion severely compromised the production of ATP from glutamine, while its production from glycolysis remained largely unchanged. ICAP2 is therefore necessary for the proper function of the mitochondrial ATP synthase.

Together, these data indicate that the defects in mitochondrial morphology upon ICAP2 knockdown are not the result of general defects in the ETC, but rather a specific effect of ATP synthase impairment. In mammalian cells, inhibition of complexes I, III, or V (ATP synthase) resulted in distinct metabolic signatures (Chen et al., 2016). Similarly, in blood-stage malaria, it appears that the main purpose of the ETC is the regeneration of ubiquinone for pyrimidine biosynthesis (Painter et al., 2007). Therefore, inhibiting oxidative phosphorylation at different steps is expected to have different consequences, which should be investigated further as we explore these pathways as therapeutic targets in apicomplexans.

Our work identifies novel and highly divergent features of the apicomplexan ATP synthase, including two proteins that we believe comprise the complex’s stator—ICAP2 and ICAP18. Previous studies had hypothesized the existence of a divergent stator in apicomplexans, and our observations confirm the presence and identity of such elements. We demonstrate the importance of these features by knocking down ICAP2, which leads to breakdown of the complex, defects in mitochondrial function and morphology, and loss of parasite viability. The conservation of these proteins in other apicomplexan species will also motivate their consideration as therapeutic targets. Future studies into the structure and function of these divergent features will help us understand their contributions to apicomplexan adaptation. Unlike most organisms, all of the ATP synthase subunits are encoded in the nuclear genome, making T. gondii a highly tractable organism to study the evolution of this important protein complex.

Proteomics data have been deposited in ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009799 and 10.6019/PXD009799. All other data are included in the manuscript and supporting files.

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Author details

1. Diego Huet

   Whitehead Institute for Biomedical Research, Cambridge, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing—original draft

   Competing interests

   No competing interests declared

2. Esther Rajendran

   Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Giel G van Dooren

   Research School of Biology, Australian National University, Canberra, Australia

   Contribution

   Funding acquisition, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2455-9821

4. Sebastian Lourido

   1. Whitehead Institute for Biomedical Research, Cambridge, United States
   2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Visualization, Writing—original draft, Writing—review and editing

   For correspondence

   lourido@wi.mit.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5237-1095

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