The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity Heike Angerera, Michael Radermacherb, Michalina Mankowska a, Mirco Stegerc, Klaus Zwickerd, Heinrich Heidec, Ilka Wittigc,e, Ulrich Brandte,f, and Volker Zickermanna,e,1 aInstitute of Biochemistry II, Structural Bioenergetics Group, Medical School, Goethe University Frankfurt, 60438 Frankfurt, Germany; bDepartment of Molecular Physiology and Biophysics, College of Medicine, University of Vermont, Burlington, VT 05405; cFunctional Proteomics, SFB 815 Core Unit, and dInstitute of Biochemistry I, Medical School, Goethe University Frankfurt, 60590 Frankfurt, Germany; eCluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Germany; and fNijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, 6525 GA, Nijmegen, The Netherlands Edited by Wolfgang Baumeister, Max Planck Institute of Biochemistry, Martinsried, Germany, and approved February 25, 2014 (received for review December 4, 2013) Mitochondrial complex I is the largest and most complicated enzyme is identical to LYR motif containing protein 6 (LYRM6). Sever- of the oxidative phosphorylation system. It comprises a number al other LYRM proteins were shown to be associated with of so-called accessory subunits of largely unknown structure and the maintenance of mitochondrial homeostasis. LYRM4 (alterna- function. Here we studied subunit NB4M [NDUFA6, LYR motif tive designation ISD11, iron-sulfur protein biogenesis desulfurase- containing protein 6 (LYRM6)], a member of the LYRM family of interacting protein 11) plays an essential role in mitochondrial – proteins. Chromosomal deletion of the corresponding gene in iron sulfur cluster biogenesis, and it has been implied that the yeast Yarrowia lipolytica caused concomitant loss of the mito- it functions as an adaptor protein for anchoring frataxin to the chondrial acyl carrier protein subunit ACPM1 from the enzyme complex of the cysteine desulfurase Nfs1 and the iron-sulfur complex and paralyzed ubiquinone reductase activity. Exchanging protein assembly protein ISCU (8). LYRM7 and LYRM8 are assembly factors of respiratory complexes III and II, respectively the LYR motif and an associated conserved phenylalanine by ala- BIOCHEMISTRY nines in subunit NB4M also abolished the activity and binding of (5, 6). The function of LYRM6 (i.e., the NB4M/NDUFA6 subunit of complex I) is unknown, but it is interesting to note that it was subunit ACPM1. We show, by single-particle electron microscopy identified as a hot spot for peroxynitrite-induced tyrosine nitration and structural modeling, that subunits NB4M and ACPM1 form (9). Down-regulation of the NDUFA6 subunit has been linked a subdomain that protrudes from the peripheral arm in the vicinity with inactivation of complex I, leading to induction of apoptosis of central subunit domains known to be involved in controlling the and, ultimately, T-cell depletion in HIV infections (10). catalytic activity of complex I. The aerobic yeast Yarrowia lipolytica is a useful yeast genetic model to study eukaryotic complex I in health and disease (11). itochondrial complex I (proton pumping NADH:ubiqui- We have deleted the gene encoding the NDUFA6 ortholog Mnone oxidoreductase, EC 1.6.5.3) is a 1-MDa membrane NB4M in this organism and observed formation of a stable protein complex with a central function in cellular energy con- and nearly complete complex I subassembly. Proteomic analysis version (1). Redox-linked proton translocation by complex I revealed that the absence of NB4M caused detachment of the contributes to the electrochemical proton gradient across the accessory mitochondrial acyl carrier protein subunit ACPM1 inner mitochondrial membrane that drives ATP synthesis by from the enzyme complex, and EM of single particles permitted ATP synthase. Complex I dysfunction caused by mutations or assignment of subunits NB4M and ACPM1 to a specific domain toxins is associated with a number of neuromuscular and neu- rodegenerative human disorders, such as Parkinson’s disease. Significance Fourteen central subunits are conserved from bacteria to humans that harbor the core function of energy conversion. In eukary- Complex I is the largest membrane protein complex of the re- otes, a substantial fraction of the mass of the holoenzyme is spiratory chain. In addition to 14 central subunits conserved contributed by so-called accessory subunits (2). A broad range of from bacteria to man, the mitochondrial enzyme comprises functions from structural reinforcement to regulation of the some 30 so-called accessory subunits of largely unknown enzyme complex has been suggested for the accessory subunits, structure and function. We show that accessory subunit NB4M but in most cases, their specific role remains unresolved. Elec- [human ortholog NDUFA6, LYR motif containing protein 6 tron microscopy (EM) (3) and X-ray crystallographic analysis of (LYRM6)], a member of the LYR protein superfamily, anchors an mitochondrial complex I at 6.3 Å resolution (4) revealed the acyl carrier protein to complex I, thus forming a structurally well- arrangement of functional modules within the L-shaped complex defined subdomain essential for complex I activity. Our study of a highly hydrophobic membrane arm consisting of the proximal offers structural insights into the functional interplay of several and distal pump-modules (PP and PD modules) and a hydrophilic central and accessory subunits that seem to be specifically in- peripheral arm extruding into the mitochondrial matrix that com- volved in controlling the catalytic activity of mitochondrial prises the NADH oxidation and ubiquinone reduction modules complex I. This also provides the structural basis for human (N and Q modules). However, information on the structure and complex I inactivation in HIV-linked LYRM6 deficiency. position of individual accessory subunits is still very limited. In this study, we focused on accessory complex I subunit Author contributions: H.A., U.B., and V.Z. designed research; H.A., M.R., M.M., M.S., K.Z., NB4M (alternative designations NDUFA6 and B14). NB4M/ H.H., I.W., U.B., and V.Z. performed research; H.A., M.R., K.Z., I.W., U.B., and V.Z. analyzed NDUFA6 belongs to the Complex1_LYR family of LYRM data; and H.A., U.B., and V.Z. wrote the paper. proteins (5–7) that is characterized by a motif comprising an The authors declare no conflict of interest. N-terminal leucine-tyrosine-arginine sequence located upstream This article is a PNAS Direct Submission. of several conserved arginines and an invariant phenylalanine. 1To whom correspondence should be addressed. E-mail: [email protected]. The human genome contains at least 11 proteins of the LYRM This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. superfamily, and the mammalian complex I subunit NDUFA6 1073/pnas.1322438111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322438111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 of the peripheral arm of complex I. Remarkably, despite Taken together, our data clearly show that subunit ACPM1 complete assembly of all central subunits, ubiquinone reductase was the only additional subunit not present in complex I from activity of complex I was abolished in the NB4M deletion strain. strain nb4mΔ. This indicated that the binding of this protein depended on the presence of subunit NB4M. The combined Results mass of the two missing subunits is 24 kDa. This is less than 3% Complex I from Strain nb4mΔ Lacks Two Accessory Subunits. Blue of the total mass of complex I and explains why neither via native (BN)-PAGE analysis of mitochondrial membranes pre- BN-PAGE nor in the elution profile from the size exclusion pared from Y. lipolytica strain nb4mΔ, in which we had deleted column was a significant difference between complex I from the the entire NB4M ORF by homologous recombination, indicated parental strain and strain nb4mΔ observed. a somewhat reduced amount of complex I compared with the Because the position of subunit NB4M had remained ambig- parental strain (Fig. 1A). To obtain a comprehensive picture of uous in a topological model for the arrangement of the subunits the migration profiles of the complex I subunits in a native gel in different functional modules of complex I (15), we used and the composition of possible subassemblies, we applied our controlled dissociation of purified complex I with the detergent recently developed Complexome Profiling approach (Fig. S1) lauryldimethylamine-oxide to biochemically prepare subcomplex (12). With the notable exception of subunit ST1, which is known Iλ that corresponds essentially to the peripheral arm. dSDS- to dissociate from complex I during BN-PAGE (13) and was PAGE (Fig. S3) and mass spectrometric analysis (Table S1)of indeed found exclusively as free subunit, in both strains, the the subcomplex unambigously revealed that subunits NB4M and major fraction of all detected subunits was present in monomeric ACPM1 were associated with the peripheral arm of complex I. complex I and in the supercomplexes formed by its association with complexes III and IV (14). Of several complex I subunits, All Iron–Sulfur Clusters Are Present in Complex I from Strain nb4mΔ. a small fraction also migrated as free proteins, and some sub- EPR spectroscopy of purified complex I from strain nb4mΔ at 12 K – complexes corresponding to the membrane arm and its PD showed that the signals