bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 MICOS and F1FO-ATP synthase crosstalk is a fundamental 2 property of mitochondrial cristae 3 4 Lawrence Rudy Cadena1,2, Ondřej Gahura1, Brian Panicucci1, Alena Zíková1,2, Hassan 5 Hashimi1,2,# 6 7 8 1 Institute of Parasitology, Biology Center, Czech Academy of Sciences, České Budějovice, 9 Czech Republic 10 2 Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic 11 # Address correspondence to Hassan Hashimi, [email protected] 12 13 Running title: MICOS and ATP synthase crosstalk in Trypanosoma brucei 14 15 16 17 18 19 20 21 22 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 23 Abstract 24 Mitochondrial cristae are polymorphic invaginations of the inner membrane that are the fabric 25 of cellular respiration. Both the Mitochondrial Contact Site and Cristae Organization System 26 (MICOS) and the F1FO-ATP synthase are vital for sculpting cristae by opposing membrane 27 bending forces. While MICOS promotes negative curvature at cristae junctions, dimeric F1FO- 28 ATP synthase is crucial for positive curvature at cristae rims. Crosstalk between these two 29 complexes has been observed in baker’s yeast, the model organism of the Opisthokonta 30 supergroup. Here, we report that this property is conserved in Trypanosoma brucei, a member 31 of the Discoba supergroup that separated from Opisthokonta ~2 billion years ago. 32 Specifically, one of the paralogs of the core MICOS subunit Mic10 interacts with dimeric 33 F1FO-ATP synthase, whereas the other core Mic60 subunit has a counteractive effect on F1FO- 34 ATP synthase oligomerization. This is evocative of the nature of MICOS-F1FO-ATP synthase 35 crosstalk in yeast, which is remarkable given the diversification these two complexes have 36 undergone during almost 2 eons of independent evolution. Furthermore, we identified a 37 highly diverged trypanosome homolog of subunit e, which is essential for the stability of 38 F1FO-ATP synthase dimers in yeast. Just like subunit e, it is preferentially associated with 39 dimers, interacts with Mic10 and its silencing results in severe defects to cristae and 40 disintegration of F1FO-ATP synthase dimers. Our findings indicate that crosstalk between 41 MICOS and dimeric F1FO-ATP synthase is a fundamental property impacting cristae shape 42 throughout eukaryotes. 43 44 45 46 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 47 Importance 48 Mitochondria have undergone profound diversification in separate lineages that have radiated 49 since the last common ancestor of eukaryotes some eons ago. Most eukaryotes are unicellular 50 protists, including etiological agents of infectious diseases like Trypanosoma brucei. Thus, 51 the study of a broad range of protists can reveal fundamental features shared by all eukaryotes 52 and lineage-specific innovations. Here we report that two different protein complexes, 53 MICOS and F1Fo-ATP synthase, known to affect mitochondrial architecture, undergo 54 crosstalk in T. brucei, just as in baker’s yeast. This is remarkable considering that these 55 complexes have otherwise undergone many changes during their almost two billion years of 56 independent evolution. Thus, this crosstalk is a fundamental property needed to maintain 57 proper mitochondrial structure even if the constituent players considerably diverged. 58 59 Introduction 60 Mitochondria are ubiquitous organelles that play a central role in cellular respiration in 61 aerobic eukaryotes alongside other essential processes, some of which are retained in 62 anaerobes (1). These organelles have a complex internal organization. While the 63 mitochondrial outer membrane is smooth, the inner membrane is markedly folded into 64 invaginations called cristae, the morphological hallmark of the organelle in facultative and 65 obligate aerobes (2, 3). Cristae are enriched with respiratory chain complexes that perform 66 oxidative phosphorylation (4, 5), and eukaryotes that cannot form these complexes lack these 67 ultrastructures (1, 6). 68 69 Mitochondrial morphology differs between species, tissues, and metabolic states. 70 Nevertheless, these variations are derived from common structural features, some of which 71 were likely inherited from endosymbiotic -proteobacterium that gave rise to the organelle (1, bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 7, 8). Cristae contribute to this variety as they can assume different shapes, such as plate-like 73 lamellar cristae, which decorate yeast and animal mitochondria, and the paddle-like discoidal 74 cristae seen in discoban protists (9). These shapes are at least in part determined by protein 75 complexes embedded within the cristae membranes. 76 77 The Mitochondrial Contact Site and Cristae Organization System (MICOS) is a hetero- 78 oligomeric protein complex that is responsible for the formation and maintenance of cristae 79 junctions, narrow attachment points of cristae to the rest of the inner membrane (10, 11). 80 Cristae junctions serve as diffusion barriers into and out of cristae, as these structures cease to 81 act as autonomous bioenergetic units upon MICOS ablation (12). The two core MICOS 82 subunits Mic10 and Mic60, which are both well conserved throughout eukaryotes (13, 14), 83 have demonstrated membrane modeling activity that contributes to constriction at cristae 84 junctions (15-18). 85 86 The F1FO-ATP synthase (henceforth ‘ATP synthase’) dimers also influence cristae shape (19). 87 Dimeric ATP synthase assemble into rows or other oligomeric configurations that promote 88 positive curvature at crista rims (20, 21). In yeast and animals, both belonging to the 89 eukaryotic supergroup Opisthokonta (22), ATP synthase dimerization depends on membrane- 90 embedded FO moiety subunits e and g (23-25). Although these subunits do not directly 91 contribute to intermonomer contacts, deletion of either of them hinders dimer formation (20) 92 and subsequently results in the emergence of defective cristae (25, 26). 93 94 Crosstalk between the crista shaping factors MICOS and ATP synthase has been 95 demonstrated in yeast. A fraction of Mic10 physically interacts with ATP synthase dimers, 96 presumably via subunit e, and overexpression of Mic10 leads to stabilization of ATP synthase bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 97 oligomers (27, 28). Even more pronounced accumulation of ATP synthase oligomers was 98 observed upon deletion of the Mic60 gene, but no physical interaction has been reported 99 between Mic60 and any ATP synthase subunits (29). This interplay between MICOS and 100 ATP synthase is a remarkable and still unexplained phenomenon, as both complexes play 101 critical, yet apparently antithetical roles in shaping the inner membrane. 102 103 Here, we ask whether crosstalk between MICOS and ATP synthase dimers is a fundamental 104 property of cristae. We employed the protist Trypanosoma brucei, a model organism that is 105 part of supergroup Discoba, which diverged from Opisthokonta ~1.8 billion years ago (22, 106 30). Because of their extended independent evolution, discoban MICOS (30-32) and ATP 107 synthase (33-36) differ radically from their opisthokont counterparts. Discoban MICOS has 108 two Mic10 paralogs and an unconventional Mic60, which lacks the C-terminal mitofilin 109 domain, characteristic for other Mic60 orthologs (13-14). Furthermore, unlike opisthokont 110 MICOS, which is organized into two integral membrane sub-complexes, each assembled 111 around a single core subunit, trypanosome MICOS is composed of one integral and one 112 peripheral sub-complex. Discoban ATP synthase exhibits type IV dimer architecture, different 113 from the canonical type I dimers found in opisthokonts (20-33), and has a dissimilar FO 114 moiety, which lacks obvious homologs of subunit e or g (37). 115 116 Results 117 Depletion of trypanosome Mic60 affects oligomerization of F1FO-ATP synthase 118 independent of other MICOS proteins 119 In T. brucei, ablation of conserved MICOS components, Mic60 or both Mic10 paralogs 120 simultaneously, resulted in impaired submitochondrial morphology characterized by elongated 121 cristae adopting arc-like structures (31). Because in budding yeast, the knockout of Mic60 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.01.438160; this version posted April 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 122 affects oligomerization