Abstract
Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
| Original language | English |
|---|---|
| Pages (from-to) | 2464-2483.e18 |
| Number of pages | 39 |
| Journal | Cell Metabolism |
| Volume | 33 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - 7 Dec 2021 |
Keywords
- complexome
- copy numbers
- disease
- half-lives
- high-confidence proteome
- human cells
- Mitochondria
- protein translocation
- respiratory chain
- smORFs
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In: Cell Metabolism, Vol. 33, No. 12, 07.12.2021, p. 2464-2483.e18.
Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Quantitative high-confidence human mitochondrial proteome and its dynamics in cellular context
AU - Morgenstern, Marcel
AU - Peikert, Christian D.
AU - Lübbert, Philipp
AU - Suppanz, Ida
AU - Klemm, Cinzia
AU - Alka, Oliver
AU - Steiert, Conny
AU - Naumenko, Nataliia
AU - Schendzielorz, Alexander
AU - Melchionda, Laura
AU - Mühlhäuser, Wignand W.D.
AU - Knapp, Bettina
AU - Busch, Jakob D.
AU - Stiller, Sebastian B.
AU - Dannenmaier, Stefan
AU - Lindau, Caroline
AU - Licheva, Mariya
AU - Eickhorst, Christopher
AU - Galbusera, Riccardo
AU - Zerbes, Ralf M.
AU - Ryan, Michael T.
AU - Kraft, Claudine
AU - Kozjak-Pavlovic, Vera
AU - Drepper, Friedel
AU - Dennerlein, Sven
AU - Oeljeklaus, Silke
AU - Pfanner, Nikolaus
AU - Wiedemann, Nils
AU - Warscheid, Bettina
N1 - Funding Information: We thank Peter Rehling for experimental advice and discussion and the PRIDE team for data deposition to the ProteomeXchange Consortium. Work included in this study has also been performed in partial fulfillment of the requirements for the doctoral theses of M.M., P.L., C.D.P., and S.D. and the master theses of C.Kl. and O.A. at the University of Freiburg. This work was supported by the European Research Council (ERC) Consolidator grant 648235 (to N.W. and B.W.) and 769065 (to C.Kr.); the European Union Marie Curie Initial Training Networks (ITN) program PerICo grant agreement 812968 (to B.W.) and DRIVE grant agreement 765912 (to C.Kr.); and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project ID 403222702/SFB 1381 (to B.W., N.W., and C.Kr.), 259130777/SFB 1177 (to C.Kr.), 278002225/RTG 2202 (to B.W.), FOR 2743 (to B.W.), PF 202/9-1 – project ID 394024777 (to N.P.), WI 4506/1-1 – project ID 406757425 (to N.W.), project ID 409673687, and Germany’s Excellence Strategy CIBSS – EXC-2189 – project ID 390939984 to (B.W., N.W., N.P., and C.Kr.). This work reflects only the authors’ view and the European Union’s Horizon 2020 research and innovation program is not responsible for any use that may be made of the information it contains. Funding Information: For a direct analysis of interaction partners, we selected seven proteins of the MitoCoP identified/validated list and performed q-AP-MS studies (Table S5). (1) The smORF protein of 6.3 kDa, PIGB opposite strand 1 (PIGBOS1), copurified the hypoxia inducible domain family member 2A (HIGD2A), an ortholog of the yeast respiratory supercomplex III-IV assembly factor Rcf1, together with subunits of complex III, complex IV and metabolite transporters (Figure 6A), suggesting a relation of this smORF protein to respiratory chain assembly (Chen et al., 2012; Salvatori et al., 2020; Strogolova et al., 2012; Vukotic et al., 2012). (2) The membrane-associated protein nucleoside triphosphatase-cancer related (NTPCR) was associated with individual structural subunits of complex IV (COX5A, COX4I1 and MT-CO2) and with the assembly factor COX11 (Figure 6B), supporting a putative role in COX assembly (Nuebel et al., 2016; Tim?n-G?mez et al., 2018). (3) Metallo-beta-lactamase domain containing protein 2 (MBLAC2) copurified the mitochondrial ubiquitin ligase MARCH5, the cytoskeleton related proteins HAX1 and RMDN3, the signaling related protein MAVS, the TOM complex, all three VDAC isoforms, and further mitochondrial outer membrane proteins (Figure 6C), suggesting that this membrane protein may function at the outer membrane/cytosol interface. (4) The membrane-associated C22orf39 protein copurified the mitochondrial metallopeptidase and ATP synthase assembly factor homolog ATP23 together with the serine protease HTRA2/OMI, both located in the intermembrane space (Figure 6D; Osman et al., 2007; Zeng et al., 2007). We generated a CRISPR/Cas9 knockout cell line of C22orf39. The oxygen consumption of C22orf39 knockout cells, analyzed by a mitochondrial Seahorse assay, was impaired (Figure 6E). (5) LYRM9 is a member of the leucine-tyrosine-arginine motif (LYRM)-containing family of proteins. Several LYRM proteins have been shown to be involved in biosynthesis or assembly processes, including assembly of respiratory complexes and mitochondrial ribosomes (Angerer, 2015; Dibley et al., 2020), yet the function of LYRM9 has been unknown. LYRM9 copurified the respiratory chain complex I (NADH-ubiquinone oxidoreductase) N-module core subunit NDUFS1, the N-module assembly factor NDUFA2, the acyl carrier protein NDUFAB1 (which binds LYR motifs present in NDUFA6 and NDUFB9) and TMEM160 (Figure 6F; Dibley et al., 2020; Stroud et al., 2016). We generated a knockout cell line of LYRM9 and the oxygen consumption of LYRM9 knockout cells was strongly inhibited (Figure 6E). Analysis of the composition of mitochondrial protein complexes in LYRM9 knockout cells revealed a defect of complex I assembly (Figures S6H and S6I) accompanied with a decrease in complex I activity (Figure S6J). We conclude that C22orf39 and, in particular, LYRM9 are required for mitochondrial metabolic activity. (6) The membrane-integrated smORF protein NCBP2 antisense 2 (NCBP2-AS2) and (7) the transmembrane protein 256 (TMEM256) both copurified core subunits of the TIM23 complex as well as inner membrane members of the prohibitin-stomatin family of membrane scaffold proteins (PHB, PHB2, STOML2) (Figures 6G and 6H; Pfanner et al., 2019; Tatsuta and Langer, 2017). TMEM256 presumably localizes to the inner membrane (Kustatscher et al., 2019), and its complexome profile supports an interaction with the TIM23 core complex (Figure S6K). NCBP2-AS2 displays a high molecular mass form similar to PAM16 (TIMM16), DNAJC19 (PAM18/TIMM14), TIMM21, the complex III assembly factor OCIAD1 (Le Vasseur et al., 2021), the mitochondrial disease gene DNAJC30 (Figure S6L; Richter-Dennerlein et al., 2014; Tebbenkamp et al., 2018), the prohibitin protein family (Figure S6F), and the high molecular mass form of TIM23 (Figure S6M). The TIM23 complex can perform two different functions, distinguished by its association with the presequence translocase-associated motor (PAM) (Chacinska et al., 2005, 2010; Mick et al., 2012). TIM23-PAM promotes the import of proteins into the matrix, whereas the motor-free translocase is able to support lateral membrane protein insertion. TIMM21 shuttles between TIM23 and the mitochondrial translation regulation assembly intermediate of cytochrome c oxidase (MITRAC) (Mick et al., 2012; Pfanner et al., 2019; Richter-Dennerlein et al., 2016; Richter et al., 2019). Strikingly, NCBP2-AS2 and TMEM256 interact with different subcomplexes of TIM23. NCBP2-AS2 co-purified the motor subunits PAM16 and DNAJC19 (Figure 6G), indicating that NCBP2-AS2 was linked to the TIM23-PAM machinery. Immunodecoration of reverse TIMM23FLAG co-precipitates confirms the interaction of NCBP2-AS2 with TIM23 (Figure S6N). TMEM256 co-purified TIMM21, membrane integral subunits of complex I, subunits of cytochrome c oxidase and ATP synthase (Figure 6H), indicating a link of TMEM256 with TIM23 and the respiratory chain assembly line. Immunodecorations of TMEM256FLAG co-precipitates demonstrate the association with the core subunits TIMM23, TIMM17B, and TIMM50 of the presequence translocase as well as the MITRAC-linked TIMM21 and the absence of interaction with the PAM-subunits TIMM44 and mitochondrial HSP70 (GRP-75/Mortalin) (Figure 6I). A reverse pull-down with FLAG-tagged TIMM23 similarly showed the interaction of TMEM256 with TIM23 (Figure 6J). The yield for TMEM256 co-precipitation with TIMM23FLAG was lower than that for core subunits of TIM23, in line with the MS-based finding that TMEM256FLAG interacts with a number of further inner membrane proteins (Figure 6H). 2D-analysis of the TIMM23FLAG co-precipitate showed a native migration at ?200 kDa of both TIMM23FLAG and co-purified TMEM256 (Figure 6K), demonstrating that TIMM23-interacting TMEM256 migrated with TIM23. siRNA depletion of TMEM256 moderately impaired the biogenesis of ATP5F1C (ATP synthase subunit gamma) and COX4l precursors in import experiments with isolated mitochondria (Figure 6L; Figure S6O). We conclude that NCBP2-AS2 and TMEM256 both interact with inner membrane scaffold proteins and TIM23. To confirm the differential specificities of NCBP2-AS2 and TMEM256 for the PAM import motor of the presequence translocase, a PAM16FLAG motor subunit co-precipitate was analyzed, which specifically co-purified TIM23 and NCBP2-AS2MYC but virtually no TMEM256 (Figure 6M). Thus, TMEM256 is associated with the TIM23 core and NCBP2-AS2 with the import motor of TIM23, suggesting that they assist in distinct processes of protein biogenesis and assembly.We thank Peter Rehling for experimental advice and discussion and the PRIDE team for data deposition to the ProteomeXchange Consortium. Work included in this study has also been performed in partial fulfillment of the requirements for the doctoral theses of M.M. P.L. C.D.P. and S.D. and the master theses of C.Kl. and O.A. at the University of Freiburg. This work was supported by the European Research Council (ERC) Consolidator grant 648235 (to N.W. and B.W.) and 769065 (to C.Kr.); the European Union Marie Curie Initial Training Networks (ITN) program PerICo grant agreement 812968 (to B.W.) and DRIVE grant agreement 765912 (to C.Kr.); and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project ID 403222702/SFB 1381 (to B.W. N.W. and C.Kr.), 259130777/SFB 1177 (to C.Kr.), 278002225/RTG 2202 (to B.W.), FOR 2743 (to B.W.), PF 202/9-1 ? project ID 394024777 (to N.P.), WI 4506/1-1 ? project ID 406757425 (to N.W.), project ID 409673687, and Germany's Excellence Strategy CIBSS ? EXC-2189 ? project ID 390939984 to (B.W. N.W. N.P. and C.Kr.). This work reflects only the authors? view and the European Union's Horizon 2020 research and innovation program is not responsible for any use that may be made of the information it contains. M.M. P.L. C.S. I.S. C.Kl. O.A. N.N. B.K. J.D.B. S.B.S. S.D. C.L. M.L. and C.E. designed and performed the experiments and analyzed the data together with C.D.P. F.D. L.M. A.S. W.W.D.M. V.K.P. R.G. R.M.Z. M.T.R. C.Kr. S.D. S.O. N.P. N.W. and B.W.; B.W. S.O. N.W. and N.P. designed and supervised the project; M.M. C.D.P. I.S. P.L. A.S. N.N. S.O. B.W. and N.W. prepared the figures and tables; B.W. S.O. M.M. N.W. and N.P. wrote the manuscript together with input of the other authors; all authors discussed results from the experiments and commented on the manuscript. The authors declare no competing interests. Publisher Copyright: © 2021 The Authors
PY - 2021/12/7
Y1 - 2021/12/7
N2 - Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
AB - Mitochondria are key organelles for cellular energetics, metabolism, signaling, and quality control and have been linked to various diseases. Different views exist on the composition of the human mitochondrial proteome. We classified >8,000 proteins in mitochondrial preparations of human cells and defined a mitochondrial high-confidence proteome of >1,100 proteins (MitoCoP). We identified interactors of translocases, respiratory chain, and ATP synthase assembly factors. The abundance of MitoCoP proteins covers six orders of magnitude and amounts to 7% of the cellular proteome with the chaperones HSP60-HSP10 being the most abundant mitochondrial proteins. MitoCoP dynamics spans three orders of magnitudes, with half-lives from hours to months, and suggests a rapid regulation of biosynthesis and assembly processes. 460 MitoCoP genes are linked to human diseases with a strong prevalence for the central nervous system and metabolism. MitoCoP will provide a high-confidence resource for placing dynamics, functions, and dysfunctions of mitochondria into the cellular context.
KW - complexome
KW - copy numbers
KW - disease
KW - half-lives
KW - high-confidence proteome
KW - human cells
KW - Mitochondria
KW - protein translocation
KW - respiratory chain
KW - smORFs
UR - http://www.scopus.com/inward/record.url?scp=85120405223&partnerID=8YFLogxK
U2 - 10.1016/j.cmet.2021.11.001
DO - 10.1016/j.cmet.2021.11.001
M3 - Article
C2 - 34800366
AN - SCOPUS:85120405223
SN - 1550-4131
VL - 33
SP - 2464-2483.e18
JO - Cell Metabolism
JF - Cell Metabolism
IS - 12
ER -