Journal of Molecular Biology
Volume 429, Issue 8, 21 April 2017, Pages 1162-1170
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Communication
Mic10, a Core Subunit of the Mitochondrial Contact Site and Cristae Organizing System, Interacts with the Dimeric F1Fo-ATP Synthase

https://doi.org/10.1016/j.jmb.2017.03.006Get rights and content

Highlights

  • Dual function of Mic10 of mitochondrial contact site and cristae organizing system

  • Mic10 interacts with the dimeric form of mitochondrial F1Fo-ATP synthase.

  • Mic10 supports the formation of larger oligomers of ATP synthase.

  • Mic10 mediates the functional crosstalk between MICOS and F1Fo-ATP synthase.

Abstract

The mitochondrial contact site and cristae organizing system (MICOS) is crucial for maintaining the architecture of the mitochondrial inner membrane. MICOS is enriched at crista junctions that connect the two inner membrane domains: inner boundary membrane and cristae membrane. MICOS promotes the formation of crista junctions, whereas the oligomeric F1Fo-ATP synthase is crucial for shaping cristae rims, indicating antagonistic functions of these machineries in organizing inner membrane architecture. We report that the MICOS core subunit Mic10, but not Mic60, binds to the F1Fo-ATP synthase. Mic10 selectively associates with the dimeric form of the ATP synthase and supports the formation of ATP synthase oligomers. Our results suggest that Mic10 plays a dual role in mitochondrial inner membrane architecture. In addition to its central function in sculpting crista junctions, a fraction of Mic10 molecules interact with the cristae rim-forming F1Fo-ATP synthase.

Introduction

Mitochondria rely on their complex inner membrane morphology to fulfill central functions in energy metabolism. While the outer membrane of mitochondria is smooth, the inner membrane is strongly folded and displays a pronounced functional asymmetry [1], [2], [3], [4], [5]. The membrane invaginations are termed cristae and harbor the oxidative phosphorylation machinery with the complexes of the respiratory chain and the F1Fo-ATP synthase.

The angular arrangement of dimers and dimer rows of the F1Fo-ATP synthase induces membrane bending. Oligomerization of the ATP synthase is thus crucial for the formation of cristae tips and rims [6], [7], [8], [9]. Dimerization-deficient mutants display a grossly altered mitochondrial morphology with onion-like or septated inner membranes and are functionally impaired [7], [10], [11], [12], [13], [14].

Cristae are connected to the flat inner boundary membrane by a tubular region, the crista junction, which represents a diffusion barrier between the two inner membrane subdomains [4], [5]. This highly curved membrane structure is stabilized by the mitochondrial contact site and cristae organizing system (MICOS), a conserved hetero-oligomeric membrane protein complex [15], [16], [17], [18], [19], [20], [21], [22], [23]. In the budding yeast Saccharomyces cerevisiae, MICOS consists of six inner membrane proteins, two of which are essential for MICOS integrity. Absence of Mic10 or Mic60 (mitofilin) causes a collapse of nearly all crista junctions, generating parallel stacks of internal cristae membranes without a stable connection to the inner boundary membrane [15], [16], [17], [24], [25]. The two MICOS core components play different roles. Mic60 is present in a subcomplex together with Mic19 and forms outer–inner membrane contact sites that likely anchor the crista junctions to the outer membrane [15], [16], [17], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35]. Recent studies indicate a connection between Mic60 and the fusion protein optic atrophy 1 (OPA1) in metazoa, suggesting a cooperation of Mic60 and OPA1 in modulating cristae morphology [14], [27], [36], [37], [38], [39], [40]. Mic10 forms a subcomplex with Mic12, Mic26, and Mic27 [31], [35], [41], [42], [43], [44]. Mic10 is a small, wedge-shaped protein that oligomerizes via conserved glycine-rich motifs in its two transmembrane domains. Mic10 oligomers exhibit intrinsic membrane-shaping activity and are required for crista junction stability [41], [42]. Mic10 represents the main scaffold of MICOS and stabilizes the local membrane curvature of crista junctions.

Mic60 and the dimerization subunits e (Su e/Atp21) and g (Su g/Atp20) of the F1Fo-ATP synthase play antagonistic roles [25]. Whereas Su e and Su g enable the dimerization and subsequent oligomerization of the F1Fo-ATP synthase [7], [10], [45], [46], [47], [48], Mic60 diminishes the oligomerization. Mitochondria lacking Mic60 contain increased amounts of F1Fo-ATP synthase oligomers [25]. Here, we asked if the second MICOS core component, Mic10, is also functionally connected to the F1Fo-ATP synthase. Surprisingly, we found Mic10, but not Mic60, in association with the dimeric F1Fo-ATP synthase. Mic10 supports the oligomerization of the F1Fo-ATP synthase and thus exerts a function opposite to that of Mic60. We conclude that a pool of Mic10 molecules is involved in a functional crosstalk between MICOS and the F1Fo-ATP synthase.

In addition to monomeric (V) and dimeric (V2) forms of the F1Fo-ATP synthase, higher oligomers (VO) can be observed when isolated yeast mitochondria are lysed with the mild detergent digitonin and analyzed by blue native electrophoresis and ATPase in gel activity staining (Fig. 1a) [6], [11], [25], [49], [50]. Rabl et al. [25] reported that the small amounts of VO oligomers observed in wild-type mitochondria were strongly increased upon deletion of MIC60, whereas overexpression of Mic60 led to a loss of VO oligomers. In mitochondria lacking Mic10, the amounts of VO oligomers were moderately increased compared to wild-type mitochondria and considerably less than in mic60Δ mitochondria (Fig. 1a), although mic10Δ mitochondria display the same severe morphological phenotype as mic60Δ mitochondria [15], [16], [17]. Remarkably, overexpression of Mic10 led to a moderate increase of the levels of VO oligomers (Fig. 1b, lanes 2 and 4) in contrast to the lack of VO oligomers upon overexpression of Mic60 [25]. The levels of Su e/g and the formation of ATP synthase dimers were neither affected by the lack nor the overexpression of Mic10 or Mic60 (Fig. 1a and b and Fig. S1) [25], [41]. We conclude that Mic10 and Mic60 differentially affect the organization of the F1Fo-ATP synthase into larger oligomeric forms.

The precursor of Mic10 is synthesized in the cytosol and imported into the mitochondrial inner membrane in a membrane potential-dependent manner [41]. To investigate the interaction of Mic10 with mitochondrial protein complexes, we imported 35S-labeled Mic10 precursor into isolated yeast mitochondria and upon lysis with digitonin, we analyzed Mic10-containing complexes by blue native electrophoresis. Imported Mic10 was observed in several blue native complexes (Fig. 2a and b), as expected from its reported presence in MICOS complexes and subcomplexes [31], [35], [41], [42], [44]. One of the blue native bands of imported [35S]Mic10, marked as Mic10*, showed the same blue native mobility as the dimeric F1Fo-ATP synthase (V2) marked by the 35S-labeled subunit γ of the ATP synthase (F1γ; Fig. 2a, lanes 1 and 3). To test the identity of these blue native bands, we performed an antibody shift assay using antibodies directed against ATP synthase subunit β (F1β). Mic10* was efficiently shifted like the ATP synthase complexes, whereas the other [35S]Mic10 bands were not affected by anti-F1β (Fig. 2a, lanes 2 and 4), demonstrating that Mic10* was associated with the F1Fo-ATP synthase. For comparison, the supercomplexes of the yeast mitochondrial respiratory chain, comprising the bc1-complex (complex III) and the cytochrome c oxidase (complex IV) [49], [51], were labeled by importing the complex III subunit Qcr8. Mic10* did not co-migrate with the III–IV supercomplexes (Fig. 2a, lanes 5 and 6). We conclude that a fraction of imported Mic10 molecules interacts with the dimeric F1Fo-ATP synthase.

To determine if an intact MICOS complex was required for the interaction of Mic10 with the F1Fo-ATP synthase, we used mic12Δ mitochondria since Mic12 is required for stably connecting the MIC10 subcomplex with the MIC60 subcomplex [35]. [35S]Mic10 associated with the dimeric ATP synthase despite the lack of Mic12 (Fig. 2b, lanes 4 and 5), demonstrating that the interaction of Mic10 with the Mic60-containing subcomplex was not crucial for the association of Mic10 with the ATP synthase. In addition, we imported the 35S-labeled precursor of Mic60 into isolated mitochondria and observed several blue native complexes upon lysis with digitonin. However, no association of Mic60 with the dimeric ATP synthase was observed (Fig. 2b, lanes 7–9), supporting the conclusion that Mic10 interacts with the F1Fo-ATP synthase independently of the other MICOS core component.

To analyze the interaction of endogenous Mic10 with the ATP synthase, we used a yeast strain containing His-tagged Mic10 [41]. Mitochondria were isolated and lysed under mild conditions. Tagged Mic10 selectively pulled down the dimeric form of the F1Fo-ATP synthase but neither the monomeric form nor the F1-portion (Fig. 3a and b), demonstrating a specific interaction of Mic10 with the ATP synthase dimer. We performed in organello crosslinking with the amine-reactive, membrane-permeable crosslinking reagent disuccinimidyl glutarate, leading to multiple Mic10-containing crosslinking products in both wild-type and Mic10His mitochondria (Fig. 3c, upper panel, lanes 3 and 4) [35], [41]. Mic10His and its crosslinking adducts were isolated by affinity chromatography under denaturing conditions (Fig. 3c, upper panel, lane 6). To find interaction partners at the ATP synthase, we performed immunoblotting against subunits of the ATP synthase and detected crosslinking products between Mic10His and Su e (Fig. 3c, lower panel, lane 6), indicating that Mic10 is in close proximity to this dimerization subunit. Taken together with the import experiments using 35S-labeled Mic10 (Fig. 2a and b), we conclude that in vitro imported Mic10 and endogenous Mic10 interact with the dimeric F1Fo-ATP synthase.

In mitochondria lacking Su g, dimerization of the F1Fo-ATP synthase is abolished [6], [7], [10], [46], and thus, imported 35S-labeled F1γ associates only with the monomeric ATP synthase and the F1-portion (Fig. 4a, lane 7). Mic10 was imported into su gΔ mitochondria (Fig. S2, lanes 4 and 5) but did not associate with the F1Fo-ATP synthase (Fig. 4a, lane 3), indicating that Su g and/or dimerization of the ATP synthase are required for the association with Mic10. The formation of ATP synthase dimers involves several assembly steps [50]. Subunit k of the F1Fo-ATP synthase (Su k/Atp19) assembles with the dimeric ATP synthase in a final stage of the assembly process, after Su e and Su g, and functions as a stabilizing factor [10], [46], [50]. In the absence of Su k, dimers are formed, but their conformation/stability and the formation of larger oligomers of the ATP synthase are disturbed [10], [50]. To determine if Mic10 associates with the ATP synthase before or after Su k, we imported [35S]Mic10 into su kΔ mitochondria (Fig. S2, lanes 7 and 8). Mic10 did not interact with the ATP synthase in the mutant mitochondria (Fig. 4b) although su kΔ mitochondria contain Su e and Su g [10], [50]. We conclude that the formation of the fully assembled dimer is necessary for the binding of Mic10 to the F1Fo-ATP synthase. Additionally, mitochondria were isolated from cells treated for 4 h with cycloheximide, an inhibitor of cytosolic translation, to prevent the formation of new assembly intermediates. Mic10 associated with the ATP synthase dimer independently of the prior treatment with cycloheximide (Fig. 4c), supporting the view that Mic10 interacts with the mature ATP synthase.

We conclude that Mic10 associates with the mature dimer of the F1Fo-ATP synthase. The interaction of Mic10 with the ATP synthase was enhanced upon overexpression of Mic10 (Fig. 4d). Since Mic10 overexpression leads to increased levels of ATP synthase oligomers (Fig. 1b, in gel activity assay), these results indicate that the binding of Mic10 to the F1Fo-ATP synthase supports the formation and/or stabilization of larger oligomers.

Our study reveals a connection between MICOS and the F1Fo-ATP synthase via the MICOS core component Mic10. The MICOS complex and the F1Fo-ATP synthase are mainly located in opposite areas of the cristae membrane, crista junctions versus rims [8], [9], [16], [25], [52], raising the question of how an interaction between the two machineries can take place. Our results indicate that not the entire MICOS complex interacts with the ATP synthase. Instead, a subpopulation of Mic10 molecules, which is not associated with the other MICOS core component Mic60, binds to the fully assembled F1Fo-ATP synthase. However, Mic10 is not a bona fide ATP synthase subunit and appears to bind to ATP synthase oligomers in substoichiometric amounts. Interestingly, overexpression as well as deletion of Mic10 lead to increased levels of ATP synthase oligomers. In the case of Mic60, only its deletion promotes the formation of ATP synthase oligomers [25]. The lack of Mic10 or Mic60 causes the collapse of crista junctions and the formation of large inner membrane stacks with a considerable expansion of membrane rims [15], [16], [17], [18], [25], [53], the main location of ATP synthase oligomers [8], [9], in agreement with the expansion of ATP synthase oligomers in the absence of Mic10/60. The formation of internal membrane stacks upon MICOS disruption depends on dimerization of the ATP synthase [17], [25], [54]. In contrast to Mic60, however, Mic10 binds to the mature dimeric F1Fo-ATP synthase and in this way directly supports the formation of larger oligomers of the ATP synthase. An excess of Mic10 molecules thus leads to increased levels of the cristae rim-shaping ATP synthase oligomers. In line with this observation, Mic10 overexpression causes an expansion and increased curvature of cristae membranes [41], likely resulting from both Mic10 oligomerization and Mic10-mediated stabilization of ATP synthase oligomers. The more pronounced stabilization of ATP synthase oligomers observed in mic60∆ cells relative to mic10∆ cells likely reflects an additive effect of MICOS disintegration and Mic10 binding to the ATP synthase in mic60∆ mutants, whereas in mic10∆ mutants, the Mic10-mediated stabilization of ATP synthase oligomers is lacking.

Immunoelectron microscopy labeling experiments revealed that Mic10 is preferentially but not exclusively localized at crista junctions [16]. Since Mic10 is difficult to tag [31], the exact distribution of Mic10 between the different inner membrane regions has not been determined. The interaction of Mic10 with F1Fo-ATP synthase dimers reported here suggests that a subpopulation of Mic10 molecules may be located outside crista junctions, possibly in the vicinity of cristae rims. We propose that MICOS and the F1Fo-ATP synthase control the architecture of the mitochondrial inner membrane not only by functioning in opposite areas of cristae membranes, but are also in a direct crosstalk via a dual localization and function of Mic10.

Section snippets

Acknowledgments

We thank Florian Wollweber for discussion and Inge Perschil for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (PF 202/8-1), the Sonderforschungsbereich 746, the Excellence Initiative of the German federal and state governments (EXC 294 BIOSS; GSC-4 Spemann Graduate School), and by postdoctoral fellowships of the Peter and Traudl Engelhorn Stiftung (to H.R.) and the German Academy of Sciences Leopoldina (LPDS 2013-08 to S.E.H.).

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    Present address: M. Bohnert, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.

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