Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

Kmita, Katarzyna; Wirth, Christophe; Warnau, Judith; Guerrero–Castillo, Sergio; Hunte, Carola; Hummer, Gerhard; Kaila, Ville R. I.; Zwicker, Klaus; Brandt, Ulrich; Zickermann, Volker

doi:10.1073/pnas.1424353112

Cited by 72 publications

(57 citation statements)

References 42 publications

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“…The prime examples are the accessory proteins in the OXPHOS system, which have been suggested to be involved in the organization, regulation, and biogenesis of the complex (35,37,39). The evolution of the mitochondrial ribosome has been studied in great detail previously (40), and it has been inferred that 19 accessory ribosomal proteins were present already in the mitochondrial ancestor (41).…”

Section: Discussionmentioning

confidence: 99%

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Mitochondrial genomes are retained by selective constraints on protein targeting

Björkholm

Harish

Hagström

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome. mitochondria | protein domain | evolution | endoplasmatic reticulum | signal recognition particle

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Section: Discussionmentioning

confidence: 99%

“…The functions of the accessory proteins are largely unknown, although a few have been implicated in the biogenesis of the OXPHOS complex (35,37). Some of the short STMDs are quite hydrophobic, and as such might help stabilize the respiratory protein complexes in the membrane.…”

Section: Phyletic Distribution Patterns Of Mitochondrial Protein Foldmentioning

confidence: 99%

Mitochondrial genomes are retained by selective constraints on protein targeting

Björkholm

Harish

Hagström

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, in the 5-Å resolution electron cryomicroscopy (cryo-EM) structure of the B. taurus enzyme, 18 supernumerary transmembrane helices (TMHs) were observed, and it was possible to propose assignments and partial models for 14 supernumerary subunits (2). One of these assignments (for the 13-kDa subunit) was confirmed recently in Y. lipolytica complex I using the anomalous diffraction from a bound zinc ion (13). Currently, 17 supernumerary subunits remain unassigned, and 11 supernumerary TMHs in the cryo-EM model have no subunits assigned to them.…”

mentioning

confidence: 86%

Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments

Zhu

King

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondrial complex I (proton-pumping NADH:ubiquinone oxidoreductase) is an essential respiratory enzyme. Mammalian complex I contains 45 subunits: 14 conserved "core" subunits and 31 "supernumerary" subunits. The structure of Bos taurus complex I, determined to 5-Å resolution by electron cryomicroscopy, described the structure of the mammalian core enzyme and allowed the assignment of 14 supernumerary subunits. Here, we describe the 6.8-Å resolution X-ray crystallography structure of subcomplex Iβ, a large portion of the membrane domain of B. taurus complex I that contains two core subunits and a cohort of supernumerary subunits. By comparing the structures and composition of subcomplex Iβ and complex I, supported by comparisons with Yarrowia lipolytica complex I, we propose assignments for eight further supernumerary subunits in the structure. Our new assignments include two CHCH-domain containing subunits that contain disulfide bridges between CX 9 C motifs; they are processed by the Mia40 oxidative-folding pathway in the intermembrane space and probably stabilize the membrane domain. We also assign subunit B22, an LYR protein, to the matrix face of the membrane domain. We reveal that subunit B22 anchors an acyl carrier protein (ACP) to the complex, replicating the LYR protein-ACP structural module that was identified previously in the hydrophilic domain. Thus, we significantly extend knowledge of how the mammalian supernumerary subunits are arranged around the core enzyme, and provide insights into their roles in biogenesis and regulation.CHCH domain | electron transport chain | LYR protein | mitochondria | NADH:ubiquinone oxidoreductase I n mammalian mitochondria, respiratory complex I (NADH: ubiquinone oxidoreductase) (1, 2) contains 45 subunits with a combined mass of 1 MDa (3-6). Fourteen core subunits, conserved from bacteria to humans, constitute the minimal complex I and catalyze the energy transducing reaction: NADH oxidation and ubiquinone reduction coupled to proton translocation across the inner membrane. Complex I is thus critical for mammalian metabolism: it oxidizes the NADH generated by the tricarboxylic acid cycle and β-oxidation, produces ubiquinol for the rest of the respiratory chain, and contributes to the proton-motive force that supports ATP synthesis and transport processes. Seven of the core subunits are hydrophilic proteins that are encoded in the nucleus and imported to mitochondria, and the other seven are hydrophobic, membrane-bound proteins (known as the ND subunits) that are encoded by the mitochondrial genome. These two sets of seven subunits form the two major domains of complex I, in the matrix and inner membrane, which confer its characteristic L shape.The protein composition of complex I from Bos taurus heart mitochondria has been characterized extensively, and is the blueprint for the human enzyme (3-6). In addition to the 14 core subunits it contains 31 supernumerary subunits (including two copies of the acyl-carrier protein, SDAP; ref. 2). Several supernumerary subunit...

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“…For follow-up validation of these proteins, we performed western blots of candidate proteins in purified nucleoid preparation or fluorescence microscopy with mtDNA co-stained, depending on the availability of antibodies and genetic constructs. For one “nucleoid orphan,” the respiratory complex I assembly factor NDUFS6 (Kmita et al, 2015), western blotting revealed its presence in crosslinked nucleoids enriched by anti-V5 pull-down (from HEK 293T cells expressing TFAM-V5; Figure 2G). A second nucleoid orphan, C7ORF55, was analyzed by fluorescence imaging.…”

Section: Resultsmentioning

confidence: 99%

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

Han

Udeshi

Deerinck

et al. 2017

Cell Chemical Biology

113

120

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SUMMARY A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.

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Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I

Cited by 72 publications

References 42 publications

Mitochondrial genomes are retained by selective constraints on protein targeting

Mitochondrial genomes are retained by selective constraints on protein targeting

Structure of subcomplex Iβ of mammalian respiratory complex I leads to new supernumerary subunit assignments

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

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