Distinct Roles of Mic12 and Mic27 in the Mitochondrial Contact Site and Cristae Organizing System

    Distinct roles of Mic12 and Mic27 in the mitochondrial contact site and cristae organizing system Ralf M. Zerbes, Philipp H¨oß, Nikolaus Pfanner, Martin van der Laan, Maria Bohnert PII: DOI: Reference:

S0022-2836(16)00161-3 doi: 10.1016/j.jmb.2016.02.031 YJMBI 65017

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

2 February 2016 26 February 2016 29 February 2016

Please cite this article as: Zerbes, R.M., H¨oß, P., Pfanner, N., van der Laan, M. & Bohnert, M., Distinct roles of Mic12 and Mic27 in the mitochondrial contact site and cristae organizing system, Journal of Molecular Biology (2016), doi: 10.1016/j.jmb.2016.02.031

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ACCEPTED MANUSCRIPT Communication

Distinct Roles of Mic12 and Mic27 in the Mitochondrial Contact Site and Cristae

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Organizing System

Ralf M. Zerbes1,2, Philipp Höß1, Nikolaus Pfanner1,3, Martin van der Laan1,3,4 and

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Maria Bohnert1,†

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1 - Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany

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2 - Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany 3 - BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104

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Freiburg, Germany

4 - Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg,

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Germany

to

Nikolaus

Pfanner

and

Martin

van

der

Laan:

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Correspondence

[email protected]; [email protected]

†

Present address: Department of Molecular Genetics, Weizmann Institute of Science,

Rehovot 7610001, Israel

Abbreviations used: MICOS, mitochondrial contact site and cristae organizing system; MicX, mitochondrial contact site and cristae organizing system protein of ~X kDa; SAM, sorting and assembly machinery; TOM, translocase of outer membrane.

Running title: Mitochondrial membrane organization

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ACCEPTED MANUSCRIPT Abstract

The mitochondrial inner membrane consists of two morphologically distinct domains,

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the inner boundary membrane and large invaginations termed cristae. Narrow

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membrane structures, the crista junctions, link these two domains. Maintenance of this elaborate architecture depends on the evolutionarily conserved mitochondrial contact site and cristae organizing system (MICOS), a multi-subunit inner membrane protein complex. MICOS consists of two functional modules, a Mic60-Mic19 subcomplex

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forming Mic60-mediated contact sites with the outer mitochondrial membrane and a Mic10-Mic12-Mic26-Mic27 membrane-sculpting subcomplex that contains large Mic10

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oligomers. Deletion of MIC10 or MIC60 results in the loss of most crista junctions. Distinct views have been discussed how the MICOS modules cooperate with each

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other. We searched for components required for the structural organization of MICOS and identified Mic12 and Mic27 as crucial factors with specific roles in MICOS

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complex formation. Mic27 promotes the stability of the Mic10 oligomers in the membrane-sculpting subcomplex, whereas Mic12 is required for coupling of the two

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MICOS subcomplexes. We conclude that in addition to the MICOS core components Mic10 and Mic60, Mic12 and Mic27 play specific roles in the organization of the

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MICOS complex.

Keywords: Crista junction; MICOS; Mitofilin; QIL1; Saccharomyces cerevisiae

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ACCEPTED MANUSCRIPT Introduction

Crista junctions are characteristic narrow tubular or slit-like membrane structures of

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the mitochondrial inner membrane. They connect the inner boundary membrane, the

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inner membrane domain that is directly adjacent to the outer membrane, to the cristae, heterogeneously shaped membrane invaginations that protrude into the central mitochondrial matrix. The mitochondrial contact site and cristae organizing system (MICOS; previously also termed MINOS or MitOS) is a multimeric protein

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complex required for the maintenance of crista junctions [1-4]. In the yeast Saccharomyces cerevisiae, the complex consists of six subunits: Mic10, Mic12,

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Mic19, Mic26, Mic27, and Mic60 (previously termed mitofilin/Fcj1) [5]. Except for the peripherally membrane-bound Mic19, all MICOS components are integral inner

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membrane proteins. MICOS has been conserved from fungi to humans, where it consists of MIC10, MIC19, MIC25 (a paralog of MIC19), MIC26, MIC27, MIC60, and

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the recently identified component QIL1 [6-15]. MICOS subunits are enriched at crista junctions [1,13,16,17]. Loss of Mic10 or

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Mic60 leads to a dramatic loss of crista junctions, resulting in the detachment of cristae membranes from the inner boundary membrane and the formation of large

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internal membrane stacks [1-3,16]. According to the severity of the mutant phenotypes, Mic10 and Mic60 have been termed MICOS core components. As for the remaining non-core subunits, mutants lacking Mic12, Mic19 or Mic27 show a similar, yet less pronounced morphology phenotype, whereas lack of Mic26, a paralog of Mic27, results in only minor alterations [1-3]. MICOS exhibits a modular organization: one subcomplex contains the core component Mic60 together with Mic19, and a second subcomplex is composed of the core component Mic10 together with Mic12, Mic26 and Mic27 [13,18,19]. Mic60 forms inner membrane–outer membrane contact sites through interaction with several resident outer membrane components: the protein translocase of the outer membrane (TOM complex), the sorting and assembly machinery (SAM/TOB complex), Ugo1, a component of the mitochondrial fusion machinery, and the abundant -barrel protein porin [1-3,7,11,20-23]. Mic10 is a small,

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ACCEPTED MANUSCRIPT hydrophobic protein with two transmembrane domains that each contains a conserved glycine motif crucial for the formation of Mic10 oligomers. Mic10 oligomerization is required for the maintenance of crista junctions in vivo and Mic10 oligomers have the

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ability to deform membranes in vitro, indicating that multiple copies of Mic10 may form

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large membrane-sculpting scaffold complexes [18,24]. Overexpression of either Mic10 or Mic60 leads to formation of large amounts of crista junction-like structures [16,18]. However, in each case the inner membrane has a grossly aberrant appearance [16,18], indicating that both Mic60-dependent contact site formation and Mic10-

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dependent membrane scaffolding have to be tightly coordinated for formation of regular crista junctions and cristae membranes. It has been suggested that a key

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functional role of non-core MICOS components may be to mediate cooperation of the Mic60-Mic19 and the Mic10-Mic12-Mic26-Mic27 modules [13,18,19]. However, the

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exact molecular function of the non-core components in MICOS organization is unknown and different views have been proposed how the two MICOS modules are

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connected [13,18,19].

In this study, we analyzed the role of non-core MICOS components in Mic10

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oligomerization and in cooperation of the two MICOS subcomplexes. We find that Mic27 promotes Mic10 oligomerization, whereas Mic12 is required for connecting the

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Mic60-Mic19 and Mic10-Mic12-Mic26-Mic27 subcomplexes. The latter finding is reminiscent of the molecular function described for human QIL1 [13] and provides experimental support for a recent bioinformatics study that reported a remote homology between QIL1 and yeast Mic12 [25], suggesting that QIL1 may be the human Mic12 ortholog.

Mic10 oligomers are connected to Mic60 in the absence of Mic19

MICOS functionality depends on the balanced activity of the contact site forming Mic60-Mic19 module and the membrane-sculpting Mic10-Mic12-Mic26-Mic27 module [18,19]. We reasoned that a functional cooperation of the two MICOS modules should rely on their structural connection, and that a loss of this interplay should result in a

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ACCEPTED MANUSCRIPT morphology phenotype similar to the one observed for the loss of the core components Mic10 and Mic60. Three yeast genes are known to date, deletion of which results in a phenotype similar to mic10∆ or mic60∆ mutants including loss of

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crista junctions and accumulation of lamellar crista stacks in the mitochondrial matrix.

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These genes encode the MICOS components Mic12, Mic19, and Mic27 [1-3,5]. We therefore isolated mitochondria from S. cerevisiae strains with single deletions of these genes [20] and analyzed the structural integrity of MICOS. No obvious effects on the protein levels of other MICOS subunits were observed in these mutants with

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the exception of mic12∆ mitochondria, which showed a strong reduction of Mic27 levels upon growth of the mutant cells on glycerol-containing medium (Fig. S1, lanes

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4-6) [3], suggesting that Mic12 may have an important role in MICOS complex formation.

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We assessed MICOS integrity by affinity purification of the complex using mitochondria isolated from single gene deletion strains that additionally expressed a

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Mic60 variant fused to a C-terminal protein A-moiety (Mic60ProtA) from the endogenous MIC60 locus [3]. Comparison of the elution fractions by SDS-PAGE showed

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differential effects of the deletion of MIC12, MIC19 and MIC27 on MICOS stability; the loss of MIC12 resulted in the most pronounced effects (Fig. 1a, lanes 7, 8 and 15) in

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agreement with von der Malsburg et al. [3]. For comparison, deletion of the core component MIC10 led to a virtually complete loss of co-purification of other MICOS subunits with Mic60ProtA as expected (Fig. 1a, lane 16) [3]. We then directly analyzed co-isolation of Mic10 oligomers, which have been proposed to be the main structural and functional unit of the Mic10-Mic12-Mic26-Mic27 subcomplex and to form a backbone structure of MICOS [18,24], with protein A-tagged Mic60, the main determinant for contact site formation. Elution fractions of affinity purifications were subjected to blue native polyacrylamide gel electrophoresis (blue native-PAGE). Coisolation of Mic10 oligomers with Mic60ProtA reflected by formation of a ladder band pattern in the native gel was virtually unaffected by deletion of MIC19, whereas deletion of either MIC12 or MIC27 led to a dramatic loss of high molecular weight Mic10-containing complexes in the elution fractions (Fig. 1b). We thus considered

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ACCEPTED MANUSCRIPT Mic12 and Mic27 as candidates in the search for MICOS subcomplex-connecting subunits.

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Differential roles of Mic12 and Mic27 in MICOS integrity

The loss of Mic10 oligomers in Mic60ProtA isolation fractions upon deletion of MIC12 or MIC27 could result either from reduced coupling of the Mic10- and Mic60-containing MICOS modules or from a primary defect in Mic10 oligomerization. We looked for

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independent approaches in order to discriminate between these two possibilities. Cterminal tagging of Mic60 has been reported to affect Mic60 function [1]. To exclude

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that a partial loss of Mic60 function may contribute to the observed effects on MICOS integrity in mic12 and mic27 mitochondria, we fused a protein A-tag to the C-

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terminus of Mic26 in the mic12∆ and mic27∆ strains. Mic26 is the only MICOS component that can be ablated without substantial alterations of mitochondrial

Mic10

oligomers.

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ultrastructure [1-3] and Mic26 is part of the MICOS subcomplex that contains the We

performed

affinity

chromatography

experiments

with

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mitochondria isolated from Mic26ProtA mic12∆ and Mic26ProtA mic27∆ strains and analyzed the samples using SDS-PAGE and blue native-PAGE. With Mic26ProtA as

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bait, deletion of MIC12 and MIC27 resulted in differential effects. Upon deletion of MIC12, co-purification of Mic10, the only further component of the Mic10-Mic12-Mic26-Mic27 module present at substantial amounts in these mitochondria, was not affected (Mic27 levels are very low in mic12∆ mitochondria; Fig. 2a, lane 3, and Fig. S1, lanes 4-6). However, co-purification of both subunits of the contact site-module, Mic60 and Mic19, was strongly impaired (Fig. 2a, lane 6), pointing toward a role of Mic12 in coupling of the MICOS subcomplexes. In contrast, deletion of MIC27 had no adverse effect on the co-purification of any other MICOS component with Mic26ProtA (Fig. 2b, lane 6). When analyzing the elution fractions by blue native-PAGE, however, Mic10-containing high molecular weight complexes were strongly diminished in the absence of Mic27, whereas the levels of low molecular weight Mic10 complexes were increased (Fig. 2b, lane 9).

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ACCEPTED MANUSCRIPT Moreover, additional Mic10-containing complexes not present in the elution fractions obtained from Mic26ProtA wild-type mitochondria were detected in the mic27∆ strain (Fig. 2b, compare lanes 8 and 9, marked with arrowheads). Consistent with the

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reduction of Mic27 levels in mic12 mitochondria, the co-purification of high molecular

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weight Mic10 oligomers with Mic26ProtA was also considerably impaired with mic12 mitochondria (Figure 2a, lane 9). Taken together, these results indicate that Mic27 stabilizes high molecular weight Mic10 oligomers, but is not crucial for the connection of the MICOS subcomplexes.

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As further approach, we performed in organello crosslinking using the homobifunctional amino-reactive reagent disuccinimidyl glutarate (DSG) to covalently link

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Mic10 oligomers [18]. We generated mic12∆ and mic27∆ strains expressing a Mic10 variant with a deca-histidine tag fused to the C-terminus of the protein (Mic10His).

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Mic10-containing crosslinking products were purified by Ni2+-NTA chromatography under denaturing conditions. Higher order Mic10 oligomers were considerably

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diminished upon deletion of MIC12 or MIC27 (Fig. 2c, lanes 6 and 12), underscoring the findings obtained by native PAGE.

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We conclude that a lack of Mic12, but not of Mic27, leads to an impaired connection between the Mic60-Mic19 and Mic10-Mic12-Mic26-Mic27 subcomplexes,

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supporting a role of Mic12 as molecular linker between the two MICOS modules. Mic27 is required for the stable formation of higher-order Mic10 oligomers.

Mic12 and Mic27 are required for structural organization of MICOS

Several studies independently observed a dissociation of MICOS into Mic60-Mic19 and Mic10-Mic12-Mic26-Mic27 subcomplexes in micos mutants [13,18,19]. However, in wild-type mitochondria, Mic60 and Mic10 can be crosslinked using DSG, which has a short spacer length of 7.7 Å, indicating that these two proteins are in close proximity in organello [18]. We asked if Mic12 or Mic27 were required for keeping the two MICOS core components close together in intact membranes. We performed in organello crosslinking with mitochondria isolated from mic12∆ and mic27∆ strains

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ACCEPTED MANUSCRIPT expressing Mic10His and analyzed the samples by denaturing Ni2+-NTA isolation and SDS-PAGE. Decoration with an antibody directed against Mic60 revealed the previously described Mic60XMic10 crosslinking product (Fig. 3a and b, lanes 8) [18].

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The crosslinking product was virtually absent in mitochondria lacking Mic12, indicating

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that the physical proximity of Mic10 and Mic60 is disturbed in the absence of Mic12 (Fig. 3a, lane 7). In contrast, the crosslinking product was still detectable in mic27∆ mitochondria (Fig 3b, lane 7) (the crosslinking yield was moderately reduced in the mutant, indicating that destabilization of the Mic10 oligomers affects, but does not

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block the Mic10-Mic60 interaction in organello).

Taken together, we conclude that both Mic12 and Mic27 have important

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functions in MICOS organization. Upon depletion of Mic27 (observed in mic27∆ and mic12∆ strains), the abundance of higher order Mic10 oligomers was decreased as assessed by affinity chromatography and blue native-PAGE as well as by in organello

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chemical crosslinking (Fig. 2). In mic27∆ cells, we observed additional Mic10-

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containing complexes on blue native-PAGE that were absent in wild-type cells (Fig. 2b, lane 9). These complexes were not observed in mic12∆ mitochondria, in which

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small amounts of Mic27 are still present. Importantly, although strongly reduced, high molecular weight Mic10 oligomers were not completely absent upon loss of Mic27,

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indicating that Mic10 is able to oligomerize independently of Mic27 (Fig. 2a and b, lanes 9). This is in agreement with previous studies showing that overexpression of Mic10 alone leads to accumulation of Mic10 oligomers and membrane deformation [18] and that purified Mic10 forms oligomers and is sufficient to tubulate model membranes [24]. We conclude that Mic27 is not essential for Mic10 oligomerization but exerts a stabilizing effect. In support of a close functional relationship between Mic27 and Mic10, Bohnert et al. [18] reported that Mic27 forms a ladder pattern on blue native-PAGE similar to the one observed for Mic10. Deletion of MIC12 results in dissociation of the two MICOS subcomplexes Mic60-Mic19

and

Mic10-Mic12-Mic26-Mic27

when

assessed

by

affinity

chromatography upon solubilization of mitochondrial membranes with the mild detergent digitonin (Fig. 2a). Though deletion of MIC12 has a profound effect on the

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ACCEPTED MANUSCRIPT co-isolation of MICOS modules, the ultrastructural phenotype of mic12∆ mitochondria is milder than the phenotypes of mic10∆ and mic60∆ mitochondria [1-3]. A loss of copurification upon lysis with detergent does not necessarily imply that the proteins do

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not interact at all in intact membranes, but can reflect a reduced stability of the

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interaction. Thus, further factors may contribute to the coupling of the two MICOS subcomplexes and may partially take over the function of Mic12 in vivo resulting in the milder morphology phenotype of mic12∆ mitochondria compared to mic10 and mic60 mitochondria. Friedman et al. [19] reported that the peripheral membrane

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protein Mic19 plays a role in mediating the interplay of the MICOS modules. However, the co-localization of MICOS components from both subcomplexes at crista junctions

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was not affected in mic19∆ cells [19], suggesting that further components are involved in MICOS subcomplex coupling. We propose that Mic12 is the membrane-integral

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linker between the contact site and membrane-sculpting modules of MICOS. Mic19 may regulate their interactions by binding MICOS components at their intermembrane

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space-exposed surface. A recent study suggested that an intramolecular disulfide bond in Mic19 may be important for such a regulatory function [26].

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Mic12 is the only MICOS component that has been considered to be fungalspecific as primary structure analysis did not readily reveal a metazoan ortholog

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[15,27]. Our finding that yeast Mic12 is required for the cooperation between the two MICOS subcomplexes provides a functional similarity to the human MICOS subunit QIL1; depletion of QIL1 leads to MICOS disassembly with the accumulation of a MIC60-MIC19/MIC25 subcomplex [13]. A refined bioinformatics analysis employing sequence-profile to sequence-profile searches recently detected remote similarities that indicate an evolutionary relationship of Mic12 and QIL1 [25]. Together with the functional characterization reported here and by Guarani et al. [13], it is thus plausible that QIL1 may represent the metazoan MIC12.

Acknowledgements We thank Drs. Susanne Horvath, Heike Rampelt and Nils Wiedemann for discussion and Inge Perschil for expert technical assistance. Work included in this study has also

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ACCEPTED MANUSCRIPT been performed in partial fulfillment of the requirements for the doctoral thesis of R.M.Z. at the University of Freiburg. This work was supported by the Deutsche Forschungsgemeinschaft (PF 202/8-1), the Sonderforschungsbereich 746, and the

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Excellence Initiative of the German federal and state governments (EXC 294 BIOSS;

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GSC-4 Spemann Graduate School).

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ACCEPTED MANUSCRIPT [11] C. Ott, K. Ross, S. Straub, B. Thiede, M. Götz, C. Goosmann, et al., Sam50 functions in mitochondrial intermembrane space bridging and biogenesis of respiratory complexes, Mol. Cell. Biol. 32 (2012) 1173–1188.

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ACCEPTED MANUSCRIPT Figure legends

Fig. 1. Role of individual subunits for MICOS stability. (a) The stability of the MICOS

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complex in the absence of either Mic10, Mic12, Mic19 or Mic27 was assessed by

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affinity purification of MICOS subunits together with C-terminally protein A-tagged Mic60 (Mic60ProtA) that contained a tobacco etch virus (TEV) protease cleavage site. The open reading frames of MIC10, MIC12, MIC19 or MIC27 have been replaced by a kanMX4 cassette via homologous recombination in a Mic60ProtA S. cerevisiae strain

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(YPH499 background) [3]. Cells were grown on YPG medium (yeast extract, bactopeptone, glycerol) at 30°C [3]. Mitochondria were isolated by differential centrifugation

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[28] and subsequently solubilized under non-denaturing conditions in digitonin buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [v/v] glycerol, 1% [w/v]

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digitonin, 2 mM PMSF, 1x Roche protease inhibitor cocktail). After a clarifying spin, mitochondrial extracts were incubated with human immunoglobulin G (IgG)-coupled

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Sepharose. To remove unbound material, beads were washed extensively with washing buffer (20 mM Tris-HCl, pH 7.4, 60 mM NaCl, 0.5 mM EDTA, 10% [v/v]

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glycerol, 0.3% [w/v] digitonin, 2 mM PMSF). Bound material was eluted by the addition of TEV protease, which released Mic60 from the protein A-tag, and subsequently

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analyzed by SDS-PAGE and Western blotting. Load, 4%; eluate, 100%. Asterisks, nonspecific bands. Harner et al. [1] reported that the function of Mic60 can be affected by C-terminal tagging, indicated here by moderate effects on the levels of Mic12 and Mic27 in some load fractions. (b) Samples were prepared as in (a), but the elution fractions were analyzed by blue native (BN)-PAGE and Western blotting. The indicated antibodies were used for immunodecoration. Su e, subunit e of F1Fo-ATP synthase; Su g, subunit g of F1Fo-ATP synthase; Su k, subunit k of F1Fo-ATP synthase; WT, wild-type.

Fig. 2. Mic12 and Mic27 affect Mic10 oligomerization and MICOS stability. (a) MICOS stability was assessed in the absence of Mic12. Wild-type (WT), Mic26ProtA and Mic26ProtA mic12 mitochondria were subjected to affinity purification and analyzed by

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ACCEPTED MANUSCRIPT SDS-PAGE or BN-PAGE and Western blotting as described in the legend of Figure 1. Mic26ProtA contained a TEV protease cleavage site [20]; by the addition of TEV protease, Mic26 was released from the protein A-tag. Load, 4%; eluate, 100%. It has

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been reported that tagged Mic60, but neither tagged Mic12 nor tagged Mic26 co-purify

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Tom40 [3,20], suggesting that TOM-interacting Mic60 molecules are not stably associated with the Mic10-Mic12-Mic26-Mic27 subcomplex. Sam50 (Tob55) is copurified by tagged Mic26 as well as tagged Mic60 [20,22], indicating that SAMinteracting Mic60 molecules are typically associated with the Mic10-Mic12-Mic26-

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Mic27 subcomplex (though the SAM-Mic60 interaction does not require the other MICOS subunits [21,22]). (b) To unravel the contribution of Mic27 to MICOS integrity,

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WT, Mic26ProtA and Mic26ProtA mic27 mitochondria were analyzed as in (a). Load, 4%; eluate, 100%. Arrowheads indicate additional Mic10-containing complexes observed

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in the absence of Mic27. (c) The oligomerization state of Mic10 was analyzed in the presence or absence of either Mic12 or Mic27 by chemical crosslinking followed by

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denaturing immunoprecipitation. Mic10His mic12 and Mic10His mic27 strains were generated by replacing the open reading frames of either MIC12 or MIC27 by a

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kanMX4 cassette via homologous recombination in the previously described Mic10His strain [18]. WT, Mic10His, Mic10His mic12 and Mic10His mic27 mitochondria were

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incubated in the presence of 1 mM disuccinimidyl glutarate (DSG) on ice. Subsequently, mitochondria were solubilized in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [v/v] glycerol, 1% [w/v] SDS, 10 mM imidazole, 2 mM PMSF). Non-solubilized material was removed by centrifugation and the supernatant was diluted 1:10 in dilution buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [v/v] glycerol, 0.2% [v/v] Triton X-100, 10 mM imidazole, 2 mM PMSF). Mic10His containing crosslinking products were purified by incubation of the diluted mitochondrial extracts with Ni2+-NTA agarose beads. Beads were washed extensively with low imidazole washing buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [v/v] glycerol, 0.2% [v/v] Triton X-100, 40 mM imidazole, 2 mM PMSF) and bound proteins were eluted with high imidazole elution buffer (20 mM TrisHCl, pH 7.4, 50 mM NaCl, 0.1 mM EDTA, 10% [v/v] glycerol, 0.2% [v/v] Triton X-100,

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ACCEPTED MANUSCRIPT 250 mM imidazole, 2 mM PMSF). Eluted material was analyzed by SDS-PAGE and immunoblotting using anti-Mic10 antibodies. Arrowheads mark Mic10 containing

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crosslinking products.

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Fig. 3. Mic12 is required for linking MICOS subcomplexes. (a) Mic12 is required for generating a crosslinking product between Mic10 and Mic60. Mitochondria from wildtype (WT), Mic10His and Mic10His mic12 cells were treated with DSG and analyzed as described in the legend of Figure 2c using anti-Mic60 antibodies. Load, 2%; eluate

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100%. (b) Crosslinking between Mic10 and Mic60 in the absence of Mic27. Samples from WT, Mic10His and Mic10His mic27 mitochondria were prepared and analyzed as

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in (a). Load, 2%; eluate, 100%. Arrowheads indicate Mic60XMic10 crosslinking

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Graphical abstract

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ACCEPTED MANUSCRIPT Distinct Roles of Mic12 and Mic27 in the Mitochondrial Contact Site and Cristae

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Organizing System

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Zerbes et al.

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Functional dissection of mitochondrial contact site and cristae organizing

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system

Mic27 promotes the stability of membrane-sculpting Mic10 oligomers

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Mic12 is required for coupling of MICOS subcomplexes

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Mic12 and Mic27 differentially organize the MICOS complex

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