To obtain functional information about identified or poorly characterized mitochondrial proteins ( Tables S4 A and S4B), we analyzed the organization of selected proteins in protein complexes and networks (summarized in Figures 7 and S7 , and Table S2 J). For an overview, mitochondria containing tagged proteins or importedS-labeled proteins were lysed with digitonin and analyzed by blue native electrophoresis, revealing distinct high-molecular-weight complexes ( Figures 7 A and S7 A). Δψ-dependent formation of protein complexes demonstrates that the precursor proteins were translocated into or across the inner membrane to form a complex (shown here for Dpa10, Mco14, Dpi29, and Dpi34), whereas Δψ-independent complex formation typically points to an outer membrane/intermembrane space location (Min6, in agreement with its localization to the outer membrane [ Figure 5 D; Table S6 ]).

(C–E) Rcf3, Rci37, and Rci50 interaction networks identified by SILAC q-AP-MS (n = 2 each). In addition, IgG chromatography eluates were analyzed by SDS-PAGE and immunoblotting using the indicated antisera. Load = 5% (C and D) or 0.4% (E); eluate = 100%. (D, lanes 5–14) 35 S-labeled Yil077c was imported into mitochondria isolated from wild-type (WT) or yil077cΔ strains for the indicated periods at 25°C and subsequently treated with proteinase K. Where indicated (−Δψ), the membrane potential was dissipated prior to import reactions. Samples were solubilized with 1% digitonin and analyzed by blue native gel electrophoresis and digital autoradiography.

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Römpler et al., 2016 Römpler K.

Müller T.

Juris L.

Wissel M.

Vukotic M.

Hofmann K.

Deckers M. Overlapping role of respiratory supercomplex factor Rcf2 and its N-terminal homolog Rcf3 in Saccharomyces cerevisiae.

Römpler et al., 2016 Römpler K.

Müller T.

Juris L.

Wissel M.

Vukotic M.

Hofmann K.

Deckers M. Overlapping role of respiratory supercomplex factor Rcf2 and its N-terminal homolog Rcf3 in Saccharomyces cerevisiae.

van der Laan et al., 2016 van der Laan M.

Horvath S.E.

Pfanner N. Mitochondrial contact site and cristae organizing system.

Römpler et al., 2016 Römpler K.

Müller T.

Juris L.

Wissel M.

Vukotic M.

Hofmann K.

Deckers M. Overlapping role of respiratory supercomplex factor Rcf2 and its N-terminal homolog Rcf3 in Saccharomyces cerevisiae.

Rugarli and Langer, 2012 Rugarli E.I.

Langer T. Mitochondrial quality control: a matter of life and death for neurons.

Dunn et al., 2008 Dunn C.D.

Tamura Y.

Sesaki H.

Jensen R.E. Mgr3p and Mgr1p are adaptors for the mitochondrial i-AAA protease complex.

Rugarli and Langer, 2012 Rugarli E.I.

Langer T. Mitochondrial quality control: a matter of life and death for neurons.

To define interaction partners of the selected proteins, we performed SILAC labeling of wild-type yeast and yeast strains containing protein A-tagged proteins with a tobacco etch virus (TEV) cleavage site. After lysis with digitonin, interacting proteins were identified by q-AP-MS. In addition, interaction partners were analyzed by affinity purification and immunoblotting using specific antibodies. (1) Ybr230w-a, which was imported into the mitochondrial matrix in a Δψ-dependent manner ( Figure 4 C), specifically co-purified the coenzyme Q biosynthesis enzymes Coq4, Coq5, Coq6, Coq9, Coq11, and Cat5/Coq7 ( Figure 7 B), and was thus named Coq21. (2) The mitochondrial class 1 protein of 10 kDa (Mco10) is an interaction partner of the F-ATP synthase, revealed by the specific enrichment of ten ATP synthase subunits ( Figure S7 B). (3) Ybl059w interacted with the altered inheritance rate of mitochondria protein Aim11, the genetic interactor of prohibitins Gep7, and the high-confidence class 1 protein Mtc3 ( Figure S7 C, left panel). The reverse pulldown via Aim11 similarly co-purified Ybl059w, Gep7, and Mtc3 ( Figure S7 C, right panel). Both pulldowns led to the co-purification of three subunits of respiratory complex IV (Cox2, Cox6, Cox20), the mitochondrial peculiar membrane protein Mpm1, and numerous subunits of the F-ATP synthase. Due to the specific interaction with Aim11, Ybl059w was named Iai11 (interactor of Aim11). The topology of Aim11 and Iai11 in the inner membrane was determined in Figure 5 F. We conclude that Aim11 and Iai11 are core components of an inner membrane complex that interacts with a set of complex IV components and the F-ATP synthase. (4) Tmh11 (TMEM14 homolog of 11 kDa) contains conserved GxxxG transmembrane segment interaction motifs in two of its predicted membrane anchors ( Figure S7 D, alignment).S-labeled Tmh11 was imported into isolated mitochondria in a Δψ-dependent manner and assembled into a high-molecular-weight complex in the megadalton range ( Figure S7 D, left panel). Tmh11 predominantly interacted with other GxxxG motif-containing mitochondrial inner membrane proteins such as the MICOS subunit Mic10, the F-ATP synthase dimerization subunit e (Tim11), and Fmp10 (with unknown function) ( Figure S7 D, middle panel). The matrix-facing subunits Sdh1 and Cor1 of respiratory complexes II and III also interacted with Tmh11. Remarkably, Tmh11 only co-purified Mic10, but not other MICOS subunits ( Figure S7 D, right panel), suggesting that Tmh11 interacts with an inner membrane pool of Mic10 that is distinct from the MICOS complex. (5) During the preparation of this manuscript, Ybr255c-a was identified as Rcf3, a homolog of the respiratory supercomplex factor Rcf2 (). Our co-purification analysis agrees with the interaction of Rcf3 with Rcf1, Rcf2, and respiratory complexes III and IV (). In addition, we observed that Rcf3 co-purified all six subunits Mic10, Mic12, Mic19, Mic26, Mic27, and Mic60 of the MICOS complex ( Figure 7 C, SILAC and immunoblotting) (). Rcf3 may thus function as mediator between respiratory chain III–IV supercomplexes and MICOS. (6) Yil077c assembled into large blue native gel complexes that are characteristic for III–IV supercomplexes ( Figures 7 A and 7D, right panel), in agreement with the co-purification of Yil077c with Rcf3 ( Figure 7 C) (). Tagged Yil077c, named Rci37 (respiratory chain interacting protein of 37 kDa), co-purified subunits of complexes III and IV and additionally the subunits Afg3 and Yta12 of the inner membrane m-AAA protease (), followed by the prohibitin complex ( Figure 7 D). (7) Ykl133c co-purified subunits of respiratory complexes III and IV and was named Rci50 (respiratory chain interacting protein of 50 kDa). Rci50 shows sequence similarity to the i-AAA adaptor Mgr3 (). SILAC and immunoblotting analysis indeed revealed that Rci50 co-purified the i-AAA protease Yme1 ( Figure 7 E) (). Thus, Rci37 and Rci50 are interaction partners of the two inner membrane AAA proteases: Rci37 of the matrix-exposed m-AAA protease and Rci50 of the intermembrane space-exposed i-AAA protease.