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Research ArticleResearch Article
Open Access

A Mitochondrial LYR Protein Is Required for Complex I Assembly

Aneta Ivanova, Mabel Gill-Hille, Shaobai Huang, Rui M. Branca, Beata Kmiec, Pedro F. Teixeira, Janne Lehtiö, James Whelan, Monika W. Murcha
Aneta Ivanova
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Perth 6009, AustraliaThe Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, Australia
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Mabel Gill-Hille
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Perth 6009, AustraliaThe Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, Australia
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Shaobai Huang
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Perth 6009, AustraliaThe Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, Australia
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Rui M. Branca
Clinical Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory and Karolinska Institutet, Stockholm 171 77, Sweden
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Beata Kmiec
Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm SE-106 91, Sweden
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Pedro F. Teixeira
Department of Biochemistry and Biophysics, Stockholm University, Arrhenius Laboratories for Natural Sciences, Stockholm SE-106 91, Sweden
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Janne Lehtiö
Clinical Proteomics Mass Spectrometry, Department of Oncology-Pathology, Science for Life Laboratory and Karolinska Institutet, Stockholm 171 77, Sweden
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James Whelan
Department of Animal, Plant and Soil Science, School of Life Science, The ARC Centre of Excellence in Plant Energy Biology, La Trobe University, Bundoora 3086, Australia
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Monika W. Murcha
School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Perth 6009, AustraliaThe Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth 6009, Australia
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  • For correspondence: monika.murcha@uwa.edu.au

Published December 2019. DOI: https://doi.org/10.1104/pp.19.00822

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Abstract

Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.

Mitochondrial Complex I (CI; also known as NADH:ubiquinone oxidoreductase), the first protein complex of the respiratory chain, is involved in the oxidation of NADH and the initiation of electron transfer across the mitochondrial inner membrane into the mitochondrial electron transport chain. CI is a large L shaped complex consisting of a hydrophilic peripheral arm that protrudes into the matrix and a hydrophobic membrane arm embedded in the inner membrane. The matrix arm transfers electrons to ubiquinone across a chain of seven Fe-S clusters while the membrane arm pumps protons from the matrix to the intermembrane space providing up to 40% of the protons necessary for ATP production (Braun et al., 2014). The membrane and matrix arms can be further separated into functional modules, whereby the peripheral matrix domain consists of the N module involved in NADH oxidation, and the Q module involved in ubiquinone reduction. The membrane arm can also be divided into distinct modules, PP and PD, for proximal and distal ends of the membrane arm with respect to the peripheral arm (Hunte et al., 2010). In Arabidopsis (Arabidopsis thaliana), CI is composed of 49 individual subunits, consisting of 14 core subunits, which are highly conserved in animal, plants, and fungi and over 30 additional accessory subunits (Meyer, 2012; Subrahmanian et al., 2016; Senkler et al., 2017a). Nine CI subunits, located within the membrane arm, are encoded in the mitochondrial genome named Nad (NADH dehydrogenase) subunits. The remaining subunits are encoded in the nucleus and are required to be imported from the cytosol across both mitochondrial membranes, processed, inserted, and assembled within the complex at the inner membrane (Meyer, 2012; Subrahmanian et al., 2016; Senkler et al., 2017a).

CI assembly is an intricate process, involving coordination of both nuclear and mitochondrial gene expression, protein synthesis, cofactor incorporation, and, finally, the assembly of the individual subunits. Through the analysis of CI mutants, 15N labeling, BN-PAGE, and complexome profiling, it has been determined that CI is assembled in a sequential manner with distinct intermediates and modules generated to produce the final holocomplex resolving at 1000 kD (Klodmann et al., 2010, 2011; Kühn et al., 2011; Meyer et al., 2011; Schertl et al., 2012; Li et al., 2013; Ligas et al., 2018).

The first intermediate of CI assembly contains the plant-specific carbonic anhydrase (CA) domain, observed to resolve at 200 kD on a BN-PAGE gel (Li et al., 2013). The function of this plant-specific domain is unknown but because this domain is not present in animal or fungal CI, it has been suggested to be involved in photorespiration (Soto et al., 2015; Fromm et al., 2016a). Nevertheless, the assembly of this domain, containing 3 CA subunits, CA1/2/3, and CAL1/2, serves as the initiator of the PP module and subsequent CI assembly (Klodmann et al., 2010; Klodmann et al., 2011; Meyer et al., 2011; Li et al., 2013; Ligas et al., 2018). Further investigation identified that this intermediate initially forms as an 85 kD complex with subsequent association of the subunits Nad2, P2, and 20.9 kD subunits to form the 200 kD intermediate (Ligas et al., 2018). The next 400–450 kD intermediate contains additional membrane bound subunits to form the fully assembled Pp module (Meyer et al., 2011; Fromm et al., 2016b; Ligas et al., 2018). Interestingly, the assembly of the matrix arm domain is carried out independently whereby the Q and N submodules (120 and 170 kD) are first assembled in the matrix (120 kD) to combine and form a 350-kD NQ subcomplex (Ligas et al., 2018). This subcomplex is further attached to the 450-kD inner membrane complex intermediate to form the 800-kD intermediate (Ligas et al., 2018). The final maturation of CI involves the addition of the PD module to the peripheral membrane end of CI forming the 1000-kD holocomplex (Schertl et al., 2012; Ligas et al., 2018). CI assembly involves a range of assembly factors, usually defined as proteins that are found to associate with CI subunits or intermediates, but that are not part of the final 1000-kD holocomplex. In mammals, 14 CI assembly factors have been identified. Commonly annotated as NADH-dehydrogenase α-subcomplex assembly factor (NDUFAF), these factors appear to have varied functions and many operate via yet undefined mechanisms (Vogel et al., 2005; Vogel et al., 2007; Andrews et al., 2013; Guarani et al., 2014; Zurita Rendón et al., 2014; Formosa et al., 2015). Additionally, some assembly factors are not involved in intermediate assembly but rather in CI subunit biogenesis, for example, NDUFAF6 plays a role in maintaining the stability of individual CI subunits before import into mitochondria (Zhang et al., 2013), or IND1, which has a role in Fe-S cluster biogenesis of CI subunits (Bych et al., 2008; Sheftel et al., 2009).

In plants, only two CI assembly factors have been identified: l-galactono-1,4-lactone dehyrodrogenase (GLDH; At3g47930), an enzyme also involved in the ascorbate biosynthesis pathway (Leferink et al., 2008), and INDH (iron-sulfur protein required for NADH dehydrogenase), the putative plant ortholog for Yarrowia lipolytica IND1 (Wydro et al., 2013). GLDH was initially identified as a CI subunit (Heazlewood et al., 2003). However, subsequent studies showed it is instead a subunit that associates with the 200-, 470-, and 800-kD intermediates (Senkler et al., 2017b). As GLDH is peripherally attached to the inner membrane within the intermembrane space side, it was proposed that GLDH is involved in the assembly of the CI membrane arm domain subunits at multiple intermediate stages (Schertl et al., 2012; Schimmeyer et al., 2016). The second plant CI assembly factor, INDH, was identified due to its sequence homology to the Y. lipolytica CI assembly factor IND1 (Bych et al., 2008). INDH was determined to be a mitochondrial matrix protein, and the indh mutant line in Arabidopsis exhibited a disrupted CI, with a stalled assembly intermediate at 650 kD. It was proposed that INDH plays a role in the translation of mitochondrial-encoded CI subunits (Wydro et al., 2013).

Mitochondrial LYR proteins (LYRM) are an exclusively eukaryotic family of proteins that do not exhibit specific amino acid homology, but are instead defined by a Leu, Tyr, and Arg motif at the N terminus (that can be followed by either a Ile/Leu and a Phe). LYRM proteins are typically small proteins ranging in size from 10 to 22 kD, positively charged, and mitochondrial located (Angerer, 2013). In yeast (Saccharomyces cerevisiae), humans (Homo sapiens), and Trypanosoma brucei, thirteen LYRM proteins have been identified with a range of diverse roles (Table 1; Angerer, 2013). LYRM proteins were classified following the characterization of LYRM4, or ISD11, a protein involved in the stability of the Cys desulfarase (NFS1) Fe-S biogenesis complex in the mitochondrial matrix (Pfam database classification https://pfam.xfam.org/clan/CL0491; Lim et al., 2013; Yan et al., 2016; Boniecki et al., 2017). LYRM4/ISD11 mutations in a human patient resulted in CI, CII, and CIII deficiencies (Lim et al., 2013), most likely due to the fact that many subunits of the respiratory chain require Fe-S cluster insertion. Several other LYRM proteins have been found to be either respiratory chain complex subunits or respiratory chain complex assembly factors. For example, LYRM7/mzm1 has been implicated in either the insertion of Fe-S clusters and maintaining the stability of the CIII subunit Rieske Fe-S protein (RISP) in yeast (Ghezzi et al., 2009; Atkinson et al., 2011; Cui et al., 2012; Sánchez et al., 2013). LYRM8/SDHAF1 has been implicated in CII assembly in humans, yeast, and Drosophila melanogaster (Ghezzi et al., 2009; Na et al., 2014), whereby the LYR motif could act as a signal for engaging HSC20-mediated chaperone transfer of Fe-S clusters to recipient proteins (Sánchez et al., 2013; Maio et al., 2014). Additionally, the LYRM protein FMC1 interacts with ATP12, a CV assembly factor (Lefebvre-Legendre et al., 2001). LYRM5 may also be a CI assembly factor as it was shown to interact with the CI assembly factor NDUFAB1 and also shown to be involved in removing FAD from the electron transfer flavoprotein required for electron transfer to CI in humans. Furthermore, deletion of LYRM5 resulted in a moderate reduction in CI activity (Pagliarini et al., 2008; Floyd et al., 2016).

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Table 1. Arabidopsis LYRM proteins belonging to the Complex I_LYR-Like superfamily

A table listing all Arabidopsis LYRM proteins, size, known or predicted localization (as determined by the Arabidopsis Complexome map [Senkler et al., 2017b] or SUBA4 [Heazlewood et al., 2007]), closest nonplant putative homolog as determined by phylogeny (SF3), and known function.

In Arabidopsis, two LYRM proteins have been previously identified both as CI subunits, LYRM3, also named NDUFB9/B22, and LYRM6, also named NDUFA6/B14 (Sunderhaus et al., 2006; Meyer et al., 2008; Ligas et al., 2018). NDUFB9/B22 is an integral CI subunit found within the PD membrane arm domain (Ligas et al., 2018). Deletion mutants of B22 in Arabidopsis (named CIB22) display the characteristic CI defective phenotype of being small and developmentally delayed (Meyer et al., 2008; Han et al., 2010). Although the function of B22 has not yet been determined in plants, in Y. lipolytica and mammals, B22 anchors an acyl carrier protein (ACP) to CI (Zhu et al., 2015; Angerer et al., 2017) and maintains complex oligomerization (Wu et al., 2016).

In Y. lipolytica, LYRM6/B14 was shown to be located within the matrix arm domain, and although the B14 mutant contained a complete CI with regards to size, its activity was affected and it lacked the acyl carrier protein subunit M1 (ACPM1; Angerer et al., 2014). In Arabidopsis, B14 was also identified as a subunit of the matrix-facing NQ module (Ligas et al., 2018), although the consequences of deleting this subunit in plants have yet to be determined.

In this study we identify and characterize a new LYRM protein from Arabidopsis named CIAF1, encoded by At1g76060. The physiological implications of deleting this protein are investigated with regards to CI assembly, particularly its role in the assembly of the matrix arm domain of the 1000-kD holoenzyme. A lack of CI results in an increased abundance of many proteins involved in mitochondrial biogenesis, revealing a mitochondrial response to decreased or limited respiratory chain activity.

RESULTS

At1g76060 Encodes for a Mitochondrial LYR Protein

A CI deficiency was previously identified in the T-DNA insertion line SALK_143656 (Wang et al., 2012). This line was annotated to contain a T-DNA insertion in the Tim23-2 gene (At1g72750), which was experimentally confirmed by PCR genotyping and Sanger sequencing following multiple rounds of backcrossing to Col-0 (Wang et al., 2012). TAIL PCR (Liu and Whittier, 1995) on SALK_143656 reveals an additional T-DNA insertion at the At1g76060 locus, an intronless gene, 60 nucleotides after the start ATG codon (Supplemental Fig. S1). At1g76069 is now annotated as COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), and SALK_143656 is thus named ciaf1-1. To unambiguously define the function of this protein, an independent second allele was obtained (SALK_122338), containing a T-DNA insertion about 260 nucleotides after the ATG codon (Fig. 1A) and named ciaf1-2. Both lines exhibit similar developmental delay compared with Col-0 (Fig. 1Bi), as previously observed with SALK_143656 (Wang et al., 2012). To confirm that the observed phenotype was attributed to the T-DNA insertion within At1g76060, ciaf1-1 and ciaf1-2 were cross-pollinated with no restoration of the phenotype in the first filial generation (F1). This indicates that ciaf1-1 and ciaf1-2 are allelic (Fig. 1Bi). Complementation of the T-DNA lines was carried out using CIAF1 cloned under the CaMV 35S promoter, which resulted in a restoration of growth phenotype comparable with wild type (Col-0; Fig. 1Bii). Thus, the severely reduced growth phenotype could be unambiguously attributed to inactivation of At1g76060. Both lines were confirmed as knock-down mutants by reverse transcription-quantitative PCR showing a dramatic reduction in At1g76060 transcript abundance (Fig. 1C).

Figure 1.
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Figure 1.

SALK_143656 and At1g76060 T-mutants. A, SALK_143656, previously identified as containing a T-DNA insertion within the promoter of Tim23-2 (At1g72750) was found to also contain an additional T-DNA insertion within At1g76060. A second T-DNA insertion line for At1g76060 (CIAF1) was obtained and confirmed by PCR, SALK_122338. B, Both lines exhibit the same developmental delays that cannot be restored upon cross-pollination (i). Complementation of SALK_143656 with CIAF1 resulted in a restoration of the developmental and growth delays (ii). C, Both lines were tested for CIAF1 (At1g76060) transcript abundance by reverse transcriptase PCR.

Phenotypic characterization of the two T-DNA insertion lines (ciaf1-1 and ciaf1-2) shows a similar developmental profile to what was previously observed with ciaf1-1 (SALK_143656; Wang et al., 2012). On medium containing 1.5% (w/v) Suc, radical emergence (stage 0.5) was observed nearly 2 d after Col-0, two rosette leaves of >1 mm (stage 1.02) were observed 6 and 5 d following Col-0, and the development of four rosette leaves (stage 1.04) was also observed 6 and 5 d following Col-0 in ciaf1-1 and ciaf1-2, respectively (Supplemental Fig. S2A). When grown on soil for 2 to 8 weeks, both lines displayed developmental delays. Four rosette leaves of >1 mm (stage 1.04) were observed 5 d after Col-0 for both lines, ten rosette leaves of >1 mm stage (1.10) were observed 7 d after Col-0 (Supplemental Fig. S2Bi), whereas stage 5.10 (first flower buds visible) took place 11 and 12 d after Col-0 in ciaf1-1 and ciaf1-2, respectively (Supplemental Fig. S2Biii). Both mutant lines exhibited stunted growth with regard to growth parameters such as maximum rosette radius and plant height after 40 d, being twice and three times less for ciaf1-1 and ciaf1-2, respectively, compared with Col-0 (Supplemental Fig. S2B). The phenotypic aberrations in the mutant lines were restored upon complementation with CIAF1 (Supplemental Fig. S2, A and B).

At1g76060 was initially annotated as EMB1793, due to nonlethal embryo defective phenotype observed at the cotyledon stage in the At1g76060 T-DNA insertion line SALK_122338 (http://seedgenes.org/NomarskiImages_alleleSymbol_emb_1793-2.html; Meinke et al., 2008). The predicted protein encoded by At1g76060 contains a conserved Complex I LYR (Leu/Tyr/Arg) motif (PFAM PF05347), characteristic of all mitochondrial LYR (LYRM) proteins (Fig. 2A). At1g76060 encodes a protein of 157 residues with the LYR motif beginning at residue 72 and the conserved Phe at residue 126, characteristic of all LYRM proteins from S. cerevisiae (Fig. 2A). Keyword searches in The Arabidopsis Information Resource (TAIR) identified 10 LYRM proteins in Arabidopsis, including the confirmed CI subunits B14 and B22 (Ligas et al., 2018; Table 1). Several of these are putative Arabidopsis orthologs to previously identified LYRM proteins from Y. lipolytica, T. brucei, and H. sapiens (Angerer, 2013), whereas an additional three do not have direct orthologs including CIAF1 (Table 1). The protein characteristics, such as domains, localization, and known functions, are listed in Table 1. Phylogenetic analysis of LYRM proteins from Arabidopsis, Y. lipolytica, T. brucei, and H. sapiens reveals that CIAF1 branches in a distinct clade to all other LYRM proteins. This includes the CI subunits B14 and B22, and the LYRM protein shown to be involved in Fe-S cluster biogenesis, LYRM4/ISD11 (Fig. 2B; Supplemental Fig. S3). Sequence homology searches show that CIAF1 has putative orthologs in the unicellular green alga Chlamydomonas reinhardtii, early land plants Physcomitrella patens and Selaginella moellendorffii, and is present in a wide variety of land plants (Supplemental Fig. S3). No direct orthologs to CIAF1 could be identified in any nonplant model species with the exception of Dictyostelium discoideum (XP_641605.1).

Figure 2.
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Figure 2.

CIAF1 encodes for a new mitochondrial LYR protein. A, CIAF1 (At1g76060) encodes a protein of 156 residues that contains the LYR motif domain (pfam PF05347) at residues 76–78 conserved for mitochondrial LYR proteins across a range of eukaryotes. Protein sequence alignment of At1g76060 with all LYRM proteins identified in yeast, with the conserved LYR (Leu,Tyr,Arg), AF (Ala, Phe), and F (Phe) residues highlighted, is shown. B, Phylogenetic analysis of all putative LYRM orthologs from Saccharomyces cerevisiae, Yarrowia lipolytica, Trypanosoma brucei, Homo sapiens, and Arabidopsis. The phylogenetic tree was analyzed and generated using MEGA7 using the maximum likelihood tree method and the Jones-Thornton-Taylor model after 1,000 replications.

Arabidopsis CIAF1 Is a Mitochondrial Matrix Protein

CIAF1 has previously been identified as a mitochondrial protein, resolving as a soluble protein under <100 kD in a mitochondrial complexome map (Senkler et al., 2017b). To confirm its mitochondrial localization, GFP targeting and in vitro import assays were carried out (Fig. 3). The predicted protein coding sequences of CIAF1 was cloned upstream of GFP and biolistically transformed into Arabidopsis cell cultures (Fig. 3A). GFP fluorescence was observed exclusively in mitochondria as determined by the colocalization with a mitochondrial RFP marker (mt-RFP). To confirm its mitochondrial targeting ability and to determine its submitochondrial localization, in vitro imports of radiolabeled CIAF1 were carried out into isolated mitochondria (Fig. 3B). The protein was imported and processed to a mature protein of 14 kD (Fig. 3B, lanes 2 and 3), dependent on the presence of a membrane potential as determined by the addition of valinomycin (Fig. 3B, lanes 4 and 5). To determine the submitochondrial localization, the outer membrane was ruptured by osmotic shock and treated with Protease K (PK) following import (Fig. 3B, lanes 6–9). The presence of a 14-kD mature protein band following PK treatment suggests that CIAF1 is protected from protease digestion and therefore targeted to the matrix. A similar import profile is observed for the soluble matrix protein Gly Decarboxylase (GDC-H) and the matrix facing inner membrane bound alternative oxidase (AOX; Fig. 3B, ii and iii). The import, rupture, and subsequent PK treatment of the integral inner membrane protein Translocase of the Inner Membrane 23 (Tim23-2) results in the production of a smaller band representing the inner membrane PK-protected portion of Tim23-2, confirming that the mitochondria are intact before osmotic shock (Fig. 3B, iv, lane 7). Carbonate extractions carried out following the import of radiolabeled protein revealed that radiolabeled CIAF1 is present in the soluble fraction, similar to GDC-H. Combined, these data suggest that CIAF1 is a soluble mitochondrial matrix protein (Fig. 3C).

Figure 3.
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Figure 3.

CIAF1 is a soluble mitochondrial matrix protein. A, Biolistic transformation of CIAF1-GFP shows colocalization with the mitochondrial RFP marker (AOX-RFP). B, In vitro translated and radiolabeled CIAF1 protein was incubated with isolated mitochondria under conditions that support uptake of proteins. Lane 1, Precursor protein alone showing a product with an apparent molecular mass of 16 kD. Lane 2 shows incubation of precursor protein with mitochondria under conditions that support import. Lane 3, as in lane 2 with Proteinase K (PK) added to 0.4 µg/ml to digest all protein still exposed on the outer membrane. Lane 4, as in lane 2 with the addition of valinomycin before the commencement of the import uptake assay. Lane 5, as in lane 4 with PK added following the import uptake assay. Lanes 6 and 7, as in lanes 2 and 3 except that before the addition of PK the outer mitochondrial membrane was ruptured (Mit*OM). Lanes 8 and 9, as in lanes 6 and 7 with the addition of valinomycin before the commencement of the import uptake assay. The mitochondrial proteins AOX and the GDC-H were used as import controls. Tim23-2 was used to test mitochondrial import ability and successful rupture of the outer membrane as evidenced by the generation of a 14-kD inner membrane located, PK protected band. C, Carbonate extractions following the import of radiolabeled CIAF1, AOX, GDC-H, and Tim23-2 into isolated mitochondria.

CIAF1 Is Required for Complex I Activity

The T-DNA insertional mutant line SALK_143656 (ciaf1-1) was previously characterized as a CI-defective mutant lacking CI abundance and activity (Wang et al., 2012). To confirm that this is due to a nonfunctional At1g76060 gene, a second independent line ciaf1-2 (SALK_122338) was analyzed for CI abundance and activity. Mitochondria were isolated from 10-d-old seedlings and resolved by BN-PAGE for Coomassie staining, CI activity, and immunodetection of respiratory chain complexes (Fig. 4). BN-PAGE analysis of mitochondria isolated from ciaf1-1 and ciaf1-2 shows that both lines have a substantial decrease in the abundance of monomeric CI and the Supercomplex I+III, as evidenced by reduced Coomassie staining and CI activity staining (Fig. 4). Immunodetection with antibodies against the CI subunit B14.7 show a clear lack of the 1000-kD and 1500-kD bands that can be observed in Col-0 (Fig. 4). Instead, a band with an apparent molecular mass of ∼650 kD is observed in mitochondria isolated from both ciaf1-1 and ciaf1-2. Immunodetection with antibodies raised against respiratory chain subunits of Complex III (RISP), Complex II (SDH1-1), Complex IV (COX II), and Complex V (β-ATP synthase) show an equal or higher abundance of these respiratory chain complexes in mitochondria isolated from both SALK lines compared with Col-0. Furthermore, the mobility of the respiratory chain complexes on the BN-PAGE is comparable between Col-0 and mutants (Fig. 4). Enzymatic activity assays specific for Complex I, II, III, IV, and V were carried out on isolated mitochondria from Col-0, ciaf1-1, ciaf1-2, and the complemented line ciaf1-1::CIAF1. A significant reduction in activity could only be observed in mitochondria isolated ciaf1-1 and ciaf1-2 with regard to the deamino-NADH:Q reductase, an indicator for CI activity (Fig. 5A). This assay primarily relies on the 51-kD subunit containing FMN for the initial electron transfer from NADH. No significant change in activity could be observed for Complex III (CIII), Complex IV (CIV), or Complex V (CV), whereas Succinate Dehydrogenase (CII) showed a significant increase in activity in ciaf1-1 mitochondria (P < 0.05; Fig. 5, B–E). Furthermore, CI activity was restored back to Col-0 levels in mitochondria isolated from ciaf1-1::CIAF1 and BN-PAGE analysis confirmed a restoration of the 1000-kD monomeric CI and the 1500-kD Supercomplex I+III (Supplemental Fig. S4). This confirms that CIAF1 is required for a functional CI. In order to determine if there were any other changes in mitochondrial protein abundance high resolution isoelectric focusing (HiRIEF) liquid chromatography–mass spectrometry (LC-MS) analysis (Branca et al., 2014; Kmiec et al., 2018) was carried out on Col-0 and ciaf1-1 total rosette tissue (Fig. 6A; Supplemental Table S1). Mapman representation of fold changes to mitochondrial proteins reveals an up-regulation of many mitochondrial proteins, except for CI subunits (Fig. 6A). Consistent with what was previously reported at the transcript level with regard to ciaf1-1 (Wang et al., 2012) and in the mitochondrial proteome of the ca1ca2 CI mutant (Fromm et al., 2016a), we also observed up-regulation of many proteins involved in mitochondrial biogenesis (Fig. 6A). CIAF1 abundance in the ciaf1-1 mutant line was shown to be around 30% compared with Col-0 (Supplemental Table S1).

Figure 4.
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Figure 4.

Deletion of CIAF1 results in CI defects. Mitochondrial isolation and BN-PAGE analysis of Col-0, ciaf1-1, and ciaf1-2 shows a substantial decrease in the monomeric form of CI and the Supercomplex I+III. CI activity staining confirm a lack of CI activity, and immunodetection against the CI subunit shows a lack of the 1000 kD monomeric Complex I and the 1500 kD Supercomplex I+III in mitochondria isolated from ciaf1-1 and ciaf1-2. Instead, a smaller complex band at ∼650 kD (indicated with a #) is detected. Immunodetection with antibodies specific for Complex II (160 kD), III (500 kD), IV (210 kD), and V (650 kD) subunits shows the holocomplex of Complex II-V resolving at their expected size and abundances comparable or greater than Col-0. The location of the protein marker (NativeMark) is indicated on the right.

Figure 5.
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Figure 5.

Enzymatic activities of mitochondrial respiration complexes measured using isolated mitochondria from Col-0, ciaf1-1, ciaf1-2, and the complemented ciaf1-1::CIAF1. A, Complex I (CI) enzymatic activity (n = 3). B, complex II (CII) enzymatic activity (n = 3). C, Complex III (CIII) enzymatic activity (n = 4). D, Complex IV (CIV) enzymatic activity (n = 3). E, Complex V (CV) enzymatic activity (n = 4). Error bars = se, analysis was carried out using the Student’s t test; * P < 0.05; ** P < 0.01.

Figure 6.
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Figure 6.

HiRIEF LC-MS proteomics to investigate whole plant proteomic changes in ciaf1-1. HiRIEF LC-MS proteomics was carried out on Col-0 and ciaf1-1 total rosette tissue to investigate whole proteomic changes. A, Mapman visualization of fold changes of mitochondrial proteins. B, Fold changes to Complex I subunits. The fold changes and detailed classification of the proteins are shown in Supplemental Table S1.

Analysis of the protein abundance changes for individual CI subunits shows three patterns: (1) The majority of the matrix arm subunits decrease in abundance ranging from 1.25- to 3-fold (Fig. 6B), (2) CI membrane arm constituents and the matrix facing carbonic anhydrase (CA) arm levels remain unchanged compared with Col-0, and, (3) the abundance of many mitochondrial encoded ND subunits increase from 1.25- to 2.5-fold (Fig. 6B). The latter change is consistent with the increased capacity for mitochondrial transcription and translation observed at both the transcript (Wang et al., 2012) and protein levels (Fig. 4B).

CIAF1 Is Required for the Assembly of the 1000-kD Complex I Holoenzyme

Immunodetection of CI subunit B14.7 in ciaf1-1 and ciaf1-2 mitochondria suggested that the assembly of CI is stalled at around 650 kD (Fig. 4). Coomassie staining of mitochondria following BN-PAGE failed to visualize an accumulated assembly intermediate but both ciaf1-1 and ciaf1e cod-2 are observed to contain the mixed intermediate complex (MIC) previously identified in ciaf1-1 and shown to consist of various CI and Complex III subunits (Wang et al., 2012; Fig. 7A, indicated by *).

Figure 7.
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Figure 7.

Deletion of CIAF1 results in defective Complex I assembly. A, Coomassie stain of Col-0 and ciaf1-2 mitochondria resolved by BN-PAGE. The apparent molecular weights are indicated on the side. The MIC is indicated by the *, and the 650- and 800-kD assembly intermediates are indicated by the “i” and “i2”. B, Import of radiolabeled CI subunits into isolated mitochondria and analyzed by BN-PAGE. CA1 (i); CAL (ii); B16.6/GRIM19 (iii); B14.7 (iv); and B13 (v). The apparent molecular weights are indicated on the side. C, Import of CIAF1, CIII, and CV subunits into isolated mitochondria and analyzed by BN-PAGE β-ATP synthase subunit of CV (i), α-MPP subunit of CIII (ii), and CIAF1 (iii).

To test if the import and assembly of radiolabeled CI subunits are compromised in ciaf1-2 mitochondria, known constituents of the various assembly intermediates were translated and imported into isolated mitochondria. Subsequent analysis by BN-PAGE allows the visualization of assembly intermediates as the radiolabeled protein is incorporated and CI assembled further in a step-wise manner (Duncan et al., 2015). Import of the matrix-facing peripheral Carbonic Anhydrase subunits CA1 and CAL1 shows incorporation into the initial 200- and 400-kD CI assembly intermediates (Fig. 7B, i and ii), as previously observed (Li et al., 2013). CA1 and CAL1 can also be observed labeling the holocomplex at 1000- and 1500-kD Supercomplex I+III (Fig. 7B, i and ii) in Col-0 mitochondria, but when imported into mitochondria isolated from ciaf1-2, only the 650-kD intermediate appears to be labeled (Fig. 7B, i, indicated with an “i”); CA1 can also be observed weakly in the MIC (Fig. 7B, i and ii, indicated by *). B16.6, a known constituent of the 450-kD subcomplex (Meyer et al., 2011; Schertl et al., 2012), was observed to weakly incorporate into the 450-kD intermediate (Fig. 7B, iii) and to strongly incorporate into the 650-kD intermediate in mitochondria isolated from both Col-0 and ciaf1-2. This is possibly due to the fact that the 450-kD intermediate is rapidly matured to the 650-kD intermediate. B16.6 did, however, strongly label the 800-kD subcomplex in mitochondria isolated from ciaf1-2, but not in Col-0, suggesting that the import and assembly of B16.6 was stalled and accumulated at 650 and 800 kD (Fig. 7B, iii, indicated with a “i” and “i2”). Import of B14.7, an integral membrane arm component known to be a constituent of the 650-kD intermediate (Ligas et al., 2018), was also tested. Strong labeling of B14.7 was observed within both 650-kD and 800-kD intermediates in mitochondrial isolated from ciaf1-2 (Fig. 7B, vi, indicated as i and ii, respectively), while following import into Col-0 mitochondria, strong labeling was observed within the monomeric CI at 1000-kD and the 1500-kD Supercomplex I+III (Fig. 7B, vi). Some weak incorporation of B14.7 could also be observed within the MIC (Fig. 7B, vi, indicated with a *). B13, a subunit located within the matrix Q arm module of CI and recently identified as one of the first subunits assembled within the Q module in the matrix (Ligas et al., 2018), showed clear labeling of the mature 1000-kD CI and 1500-kD supercomplex I and III2, without any labeling of smaller intermediate products in Col-0 (Fig. 7B, vii). A similar labeling profile was also observed in ciaf1-2 mitochondria, but with much lower intensity (Fig. 7B, vii), suggesting that B13 may have the ability to be incorporated into CI independently of the known assembly intermediates.

These data indicate that the import and assembly of CI subunits into ciaf1 (except B13) is stalled at the 650- and 800-kD intermediates, confirming a defect in CI assembly. The import of the β-subunit of ATP synthase (CV) and the mitochondrial processing peptidase/Bc1 (MPP; CIII) was also tested (Fig. 7C, i and ii) and was observed to be incorporated into the respective mature complexes of the expected sizes (Fig. 7C, i and ii). MPP could also be seen to incorporate into the 1500-kD Supercomplex I+III in Col-0 mitochondria. The observed weak incorporation into the ciaf1-2 mitochondria suggests that CIAF1 does not play a role in Supercomplex I and III2 assembly. Furthermore, as with other CI subunits, MPP was also observed to incorporate within the MIC (Fig. 7C, i and ii, indicated with a *). To investigate the possibility that CIAF1 is itself a subunit of complex I, the import and assembly of CIAF1 was also carried out and analyzed by BN-PAGE (Fig. 7C, iii). No labeling could be detected at the expected size of CI or any of its intermediates (Fig. 7C, iii). A more comprehensive time course import of CIAF1 and B14.7 was also carried out (Supplemental Fig. S5), and no incorporation of CIAF1 could be observed into any mitochondrial complex. This finding is consistent with several independent complexome studies that did not identify CIAF1 within CI or any of its intermediates (Meyer et al., 2008; Meyer et al., 2011; Schertl et al., 2012; Schimmeyer et al., 2016; Senkler et al., 2017b; Ligas et al., 2018).

CIAF1 Interacts with the 23 kD Subunit and Can Complement a Yeast Deletion Mutant Involved in Fe-S Cluster Biogenesis

The role of CIAF1 in CI assembly was further investigated by yeast-2-hybrid assays. The ability of CIAF1 to interact with 17 CI subunits, spanning the matrix, membrane, and carbonic anhydrase domains, was tested (Fig. 8). CIAF1 was observed to interact strongly with the 23-kD subunit, also known as TYKY (Fig. 8A). Furthermore, CIAF1 was observed to interact with itself, suggesting an ability to self-oligomerize (Fig. 8A). Because the 23 kD/TYKY-1 subunit is predicted to contain 4 Fe-S clusters, a possible role in Fe-S cluster assembly was investigated (Fig. 8B). Yeast ISD11 has been previously shown to be involved in Fe-S cluster assembly, and a yeast deletion strain (GAL-ISD11) was previously generated that exhibits suppressed growth on Glc media due to a defect in the assembly of Fe-S centers (Adam et al., 2006; Fig. 8Bi). The ability of the Arabidopsis homolog to ISD11 (AtISD11, At5g61220) and CIAF1 to complement this deletion strain was tested (Fig. 8B). Both genes showed an ability to restore growth on Glc (Fig. 8B, ii and iii). Growth rates indicated that AtISD11 could restore yeast viability back to wild-type levels, whereas CIAF1 could only partially restore growth rates during the midlog phase, with both wild type and AtISD11 reaching the late log phase earlier than CIAF1 (Fig. 8B, iv and v). This suggests that CIAF1 may have a similar function to ISD11 and may be involved in Fe-S assembly.

Figure 8.
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Figure 8.

CIAF1 interacts with the 23 kD/TYK1 subunit of Complex I and has the ability to complement yeast ISD11. Ai, Yeast-2-Hybrid interaction assays of CIAF1 with a range of CI subunits. CIAF1 was cloned into the cg-pGBK and transformed in AH109. Complex I subunits were cloned into either cg-pGAD and transformed in Y187. Successful diploid mating was determined by growth on Double Drop-Out media (DDO, SD-leu-trp) whereas positive interactions were determined by growth on Quadruple Drop-Out media (QDO, SD-leu-trp-ade-his) with serial dilutions shown. No interactions were observed with empty vector, whereas SV40 and Lam were used as positive and negative controls. Aii, Representation of CI interaction data; subunits colored red indicate a positive interaction, and subunits colored green indicate a negative interaction. Subunits not tested are indicated in gray. B, CIAF1 has the ability to complement yeast ISD11. The yeast ISD11 deletion mutant (GAL10-ISD11) shows limited growth ability on Glc media compared with wild type (YPH499; i). Transformation with the Arabidopsis ISD11 homolog (pAG423_ISD11) and CIAF1 (pAG423_CIAF1) restores its ability to grow on Glc media (ii) unlike the empty vector alone (pAG423; iii). Growth rates of complemented lines in Glc (iv) and Gal (v) media show AtISD11 can fully restore the growth defect of GAL10-ISD11, whereas CIAF1 can partially restore the growth defect of GAL10-ISD11. Error bars = SE; analysis was carried out using the Student’s t test; *P < 0.05; **P < 0.01. Error bars = SE, analysis was carried out using the Student’s t test; *P < 0.05; **P < 0.01.

Mitochondrial Biogenesis Is Upregulated in CIAF1 Deletion Mutants

Previous characterization of ciaf1-1 showed a general up-regulation of mitochondrial biogenesis (Wang et al., 2012). We therefore tested mitochondrial translation rates in mitochondria isolated from ciaf1-1 and ciaf1-2 mutant lines (Supplemental Fig. S6). As previously observed with ciaf1-1, mitochondrial translation rates (determined by in organelle labeling) were increased in the mutant lines compared with Col-0 (Wang et al., 2012). This increase in mitochondrial translation rates corresponds to the observed increase in all mitochondrial transcripts in ciaf1-1 and the observed increase in mitochondrial encoded Nad subunits (Fig. 6B; Supplemental Table S1). To confirm that the protein import ability of CI subunits was not compromised, protein uptake assays were carried out using mitochondria isolated from Col-0, ciaf1-1, and ciaf1-2 (Supplemental Fig. S7). Import of radiolabeled 20-kD and 24-kD CI subunits was tested, and the band intensity of the mature processed proteins appeared stronger in mitochondria isolated from ciaf1-1 and ciaf1-2, suggesting that protein import ability was greater in the mutants compared with Col-0. Import of the AOX, an inner membrane protein involved in alternative mitochondrial respiration, also showed increased import uptake rates in the mutants, as documented previously (Wang et al., 2012). This indicates that CIAF1 does not play a role in mitochondrial translation or in the import of nuclear-encoded proteins and, therefore, the CI defect cannot be attributed to these mechanisms.

DISCUSSION

The results presented here show that the gene At1g76060 encodes a mitochondrial matrix LYR protein involved in the assembly of CI. As such, the previously annotated embryo defective EMB1793 protein has been named COMPLEX I ASSEMBLY FACTOR 1 (CIAF1). CIAF1 has been defined as a CI assembly factor due to the fact that the loss of CIAF1 results in a loss of the CI holocomplex. Moreover, we have shown that CIAF1 has the ability to interact with the 23-kD subunit of Complex I and its role is possibly attributed to Fe-S cluster insertion.

CI assembly in plants can be divided into the sequential assembly of various modules and intermediates. The most recent model of CI assembly proposed by Ligas et al. determined that the N and Q modules are initially assembled as individual intermediates along with yet unidentified assembly factors that are subsequently released (Ligas et al., 2018). The N and Q modules are then brought together to form a 350-kD intermediate complex that is assembled to form an 800-kD intermediate. The final stages of assembly involve the addition of the remaining membrane domain subunits at the peripheral domain known as the PD (Ligas et al., 2018) to form the 1000-kD holocomplex. Thus, the matrix domain modules are fully assembled before attachment to the peripheral membrane arm (Ligas et al., 2018). This assembly mechanism is unique to plants; in contrast in humans, the Q and N modules are anchored to the membrane domain sequentially (Guerrero-Castillo et al., 2017). Deletion of CIAF1 results in the generation of aberrant CI intermediates at 650 and 800 kD (Fig. 7) and the apparent Mrs of these intermediates correspond to a CI holocomplex lacking the NQ module (350 kD; Fig. 9). Whole proteomic analysis confirms a >2- to 3-fold decrease in the matrix domain P and Q subunits, and because the import of these subunits is maintained at high levels, it is likely that the NQ submodule or its individual subunits are degraded, due to either defective assembly or subunit instability. Yeast-2-hybrid assays show that CIAF1 interacts with the 23-kD subunit of the Q module (Fig. 8). This subunit is located on the peripheral end of the Q module between the N module interface and may be essential for the maintaining the structural integrity of the NQ submodule (Fig. 9). Additionally, the 23-kD apoprotein requires 4 Fe-S cluster insertions for its final maturation and function. Our yeast complementation data shows CIAF1 to partially complement the yeast ISD11 deletion strain, known to be involved in maintaining the stability of the mitochondrial matrix iron sulfur cluster insertion complex (Adam et al., 2006). Thus, CIAF1 likely plays a role in Fe-S cluster insertion into the 23-kD subunit. This hypothesis is probable as several studies implicate LYRM proteins to have a chaperone function for Fe-S cluster insertion across respiratory complexes and species. For example, in yeast and mammals, Mzm1 stabilizes RISP before CIII insertion and recruits a Fe-S cluster insertion complex necessary for its Fe-S cluster insertion (Atkinson et al., 2011; Cui et al., 2012; Sánchez et al., 2013). In Complex II assembly, SDHAF1/LYRM8 also plays a chaperoning/stabilizing role required for Fe-S cluster insertion within the SDH2 subunit (Ghezzi et al., 2009; Maio et al., 2014; Maio et al., 2016). Interestingly another LYRM protein, SDHAF3, was also shown to be involved in complex stability, specifically to shield Fe-S clusters within SDH2 from deleterious antioxidants (Na et al., 2014; Dwight et al., 2017). It appears that the LYR motif is crucial for proteins to bind the Fe-S cluster transfer apparatus and that their mode of action is very much conserved. It has been suggested that the LYRM protein acts as a guide to allow targeted delivery of Fe-S clusters to recipient proteins (Lane et al., 2014). Therefore, it is likely that CIAF1 is involved in Fe-S cluster delivery to the 23-kD subunit. Whether insertion occurs into the monomeric protein, the submodules, or the fully assembled complex remains to be determined (Fig. 9). In the ciaf1 mutants, defective maturation of the matrix arm domain results in defective assembly and/or turnover resulting in the accumulation of an intermediate CI containing the membrane and PD module subunits only (Fig. 9).

Figure 9.
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Figure 9.

Model for the proposed role of CIAF1 in the assembly of the matrix modules in Complex I. A model for the proposed role of CIAF1 in the assembly of the matrix domain modules of Complex I in Col-0 and the ciaf1 mutants. The decreased abundance of the CIAF1 and Complex I subunits are indicated by opaque subunits. The 650–800 kD Complex I intermediates that are observed in mitochondria isolated from ciaf1 is likely to be a composition of the Pp and PD modules. Assembly intermediates and Complex I module composition are defined as described by Ligas et al. (2018). The nomenclature of Complex I subunits used as listed in Subrahmanian et al. (2016). The N module is indicated in yellow, the Q module is indicated in pink, the Pp module is indicated in purple, and the PD module is indicated in green.

Both ciaf1 knock-out lines do display a small amount of mature 1000-kD holocomplex and 1500-kD Supercomplex I and III. Our proteomic data reveals that CIAF1 has been reduced to 30% of Col-0; this shows that these lines are functionally capable of assembling a mature CI either by the actions of the small amount of CIAF1 present or by an alternative LYRM. Phylogenetic analysis shows an additional two plant-specific LYRM proteins with an unknown function that may be involved (Table 1).

The changes in the mitochondrial proteome associated with the large decrease in CI add to the growing body of evidence that perturbation of CI function initiates a mitochondrial retrograde signaling pathway. The increase in abundance of mitochondrial proteins involved in RNA transcription and modification, translation, assembly, and protein import points to an up-regulation of mitochondrial biogenesis (Fig. 6A). This is consistent with the increase in protein import capacity, in organelle translation rates, and transcript abundance for these components in ciaf1-1 that was reported previously (Wang et al., 2012). Significantly increased rates of protein import have also been observed for other CI mutant lines, such as rug3 and ndusf4 (Wang et al., 2012), suggesting this is a common response to CI perturbation. It has also been observed that there are cellular-wide changes in the proteome in response to Complex I dysfunction (Fromm et al., 2016a), including increases in proteins involved in glycolysis, the TCA cycle, and antioxidant defense. CI-perturbed plants have a characteristic slow growth phenotype that is hard to explain by loss of a single coupling site alone, especially as plants are often grown under nonrestrictive growth conditions. Thus, the growth phenotype may be associated with the cellular-wide changes in the proteome and metabolome (Meyer et al., 2009) associated with CI dysfunction that prioritize defense over growth, which position mitochondria as a sensor and initiator of stress response signaling pathways (Wang et al., 2018). Although the molecular mechanism of CI retrograde signaling are not known, it has been reported that a protein of the mitochondrial twin Cys family, At12Cys, typically found in the intermembrane space, is induced and found in mitochondria, chloroplasts, and the cytosol in the absence of functional CI (Wang et al., 2016). This protein associates with Supercomplex I+III, and thus the loss of CI may release this protein to initiate a signaling pathway resulting in changes in the proteome that occur in mitochondria, chloroplasts, and the cytosol in CI mutants (Fromm et al., 2016a).

CONCLUSION

This research provides further insight into the mechanisms of CI assembly by identifying CIAF1 as a novel CI factor involved in the assembly of the matrix arm domain subunits.

MATERIALS AND METHODS

Phylogenetic Analysis

Protein sequences for all mitochondrial LYR motif-containing proteins (LYRM; PFAM PF05347) were retrieved from TAIR for Arabidopsis (Arabidopsis thaliana; https://www.arabidopsis.org/) and the Saccharomyces Genome Database for Saccharomyces cerevisiae (https://www.yeastgenome.org/). LYRM protein sequences from Yarrowia lipolytica, Trypanosoma brucei, and Homo sapiens, as previously listed (Angerer, 2013), were retrieved from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/;Supplemental Table S2). Putative orthologs to all Arabidopsis LYRM proteins were retrieved from Phytozome 12.0 (Goodstein et al., 2012). Protein sequences were aligned using ClustalW, and phylogenetic trees were created using MEGA7 (Kumar et al., 2016) using the Jones-Taylor-Thornton (JTT) model and 1000 replicates. The tree was drawn using iTol: Interactive Tree of Life (Letunic and Bork, 2016).

Plant Material and Growth

T-DNA mutants and Col-0 seeds were germinated on half-strength MS salt medium containing 1.5% (w/v) Suc and grown at 22°C with a light intensity of 80 μmol quanta m−2 s−1 in a 16-h photoperiod. The SALK T-DNA insertion lines (SALK_ 143656 and SALK_122338) were obtained from the Arabidopsis Biological Resource Center (The Ohio State University, Columbus) seed stock center (Alonso et al., 2003). Both lines were PCR screened for homozygosity of the T-DNA insert with the primers listed in Supplemental Table S3 according to Salk Institute T_DNA Arabidopsis mapping tool suggestions. Plant phenotyping was performed according to the parameters as described previously (Boyes et al., 2001). Allelism between ciaf1-1 and ciaf-2 was confirmed by examination of the phenotype following cross-pollination between of the two lines.

TAIL PCR

Identification of the additional T-DNA insertion within SALK_ 143656 line was carried out by Thermal Asymmetric Interlaced (TAIL) PCR as described by Liu and Whittier (Liu and Whittier, 1995). Three nested primers (pROK2-LB1,2 and 3; Supplemental Table S3) specific to the sequence of the Left Border of pROK2 vector (Salk Institute Genomic Analysis Lab), together with an arbitrary degenerate primer (AD2), were used in three consecutive PCR cycles to amplify the DNA adjacent to the T-DNA insertion. The final PCR product was sequenced using Sanger sequencing with the LB3 primer and mapped to the genome using the BLAST tool in TAIR.

Reverse Transcription-Quantitative PCR

Total RNA from 10-d-old seedlings from Col-0, ciaf1-1, and ciaf1-2 was extracted using FavorPrep Plant Total RNA Purification Mini Kit according to the manufacturer’s instructions (Favorgen). RNA was converted to complementary DNA (cDNA) using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s instructions. CIAF1 and Actin2 were amplified with primers listed in Supplemental Table S3.

PCR and cDNA Clones

Genomic DNA was isolated from 14-d-old plants, and PCR was performed using the MyTag HS Mix (Bioline) according to the manufacturer’s instructions. Cloning from cDNA was performed as described below using primers listed in Supplemental Table S3. Full-length cDNA was amplified using gene-specific primers flanked by Gateway recombination cassettes and cloned into pDONR201. LR reactions were carried out into C-terminal GFP fusion vectors for GFP localization (Carrie et al., 2009). pDEST14 was used for in vitro transcription and translation, pB2GW7 (35S CaMV promoter) and pGWB1 (endogenous promoter) for Agrobacterium tumefaciens-mediated transformation (Karimi et al., 2002), and cg-pGBK and cg-pGAD for yeast two-hybrid analysis (Stellberger et al., 2010).

GFP Targeting and Microscopy

Biolistic transformation of CIAF1 cloned with a C-terminal GFP and mtCherry fusion vector (Nelson et al., 2007) was carried out on 5-d-old Arabidopsis (Ler) cell suspensions as described previously (Duncan et al., 2015). Plasmid (5 µg) was coprecipitated onto gold particles and bombarded using the PDS-1000/He biolistic transformation system (Bio-Rad). GFP and mtCherry expression was visualized and captured at 100× magnification using the Olympus BX61 microscope at 460/480 nm (GFP) and 570/625 nm (mtCherry).

Mitochondrial Isolation, In Vitro Import, and Carbonate Extractions

Mitochondria were isolated from 10-d-old seedlings grown in water pots as described previously (Duncan et al., 2015) for all subsequent assays. [35S] Met-labeled proteins were synthesized using the rabbit reticulocyte TNT in vitro transcription translation lysate (Promega). AOX (X68702; Whelan et al., 1995) and Tim23-2 (Murcha et al., 2003) cDNA clones were used as described previously. CIAF1 (At1g76060) and GDC-H (At2g35370) were cloned into pDEST14 as described above, with the inclusion of 4 additional methionines for CIAF1 (At1g76060). In vitro import reactions were carried out as described previously (Duncan et al., 2015). Carbonate extractions were carried out following import assays accordingly; mitochondrial pellets were resuspended in 0.1 m Na2CO3 with 1 mm phenylmethylsulfonyl fluoride and incubated on ice for 20 min. An aliquot was removed, and the remaining samples were centrifuged at 200,000 g for 1 h at 20°C. The pellet was resuspended in loading buffer and taken as the membrane fraction, whereas the supernatant was taken as the soluble fraction. For BN-PAGE import assays, CA1 (At1g19580), CAL1 (At1g63510), B16.6 (At2g33220), and B13 (At3g03070) were cloned into pDEST14 and translated as described above. B14.7 (At2g42210), β-ATP synthase (At5g08670), and α-MPP/bc1 (At3g16480) were used as described previously (Wang et al., 2012). Radiolabeled protein was incubated with 30 μg of freshly isolated mitochondria for 40 min. Mitochondria were pelleted by centrifugation and processed as described below.

Blue-Native PAGE and Activity Staining

BN-PAGE was carried out as described previously (Eubel et al., 2005) using 5% (w/v) digitonin and precast 4-16% Bis-Tris Gels (NOVEX). Gels were transferred to PVDF membrane and immunodetected using anti-B14.7 (Wang et al., 2012), anti-RISP (Carrie et al., 2010), anti-COXII (Agrisera), anti-ATP synthase (Agrisera), and anti-SDH1-1 (Peters et al., 2012) using standard procedures. In gel CI activity staining was carried out as described by Schertl and Braun (2015).

In Organello Translation

In organello translation assays were carried out on freshly isolated mitochondria as described previously (Giegé et al., 2005). Briefly, 30 μg of mitochondria was incubated in buffer with added [35S] Met for the specified amount of time. The reaction was stopped by the addition of cold Met and samples resolved by SDS-PAGE and processed as described above.

Measurements of Enzymatic Activities of Complex I, II, III, IV, and V

All enzymatic assays were performed at 25°C using a spectrophotometer (SHIMADZU UV-1800). Complex I, II, and IV enzymatic assays were carried out as described in Huang et al. (2015). Deamino-NADH:Q reductase (Complex I) specific activity was measured by the following reaction. The reaction solution contained Tris-HCl, 50 mm (pH 7.2); NaCl, 50 mm; FeCN, 1 mm; deamino-NADH, 0.2 mm. The decrease of A420 was recorded after adding 10 µg mitochondrial protein in 1 mL reaction solution. The extinction coefficient of FeCN at 420 nm is 1.03 mm−1 cm−1. Succinate dehydrogenase (Complex II) specific activity was measured using a phenazine methosulfate 2,6-dichlorophenolindophenol coupled method. The reaction solution contained K phosphate buffer, 50 mm (pH 7.4); KCN, 10 mm; ETDA-Na2, 10 mm; bovine serum albumin, 0.1% (w/v); phenazine methosulfate, 1.6 mm; 2,6-dichlorophenolindophenol, 0.1 mm; Succinate, 10 mm. The decrease of A600 was recorded after adding 20 µg mitochondrial protein in 1 mL reaction solution. The extinction coefficient of DCPIP at 600 nm is 21 mm−1 cm−1. Ubiquinol-cytochrome c reductase (Complex III) specific activity was measured by the reduction of cytochrome c (Luo et al., 2008). The reaction solution contained Tris-HCl, 50 mm (pH 7.4); NaN3, 4 mm; decylubiquinol, 0.05 mm; cytochrome c, 0.05 mm. The increase of A550 was recorded after adding 10 µg mitochondrial protein in 1 mL reaction solution. The extinction coefficient of cytochrome c at 550 nm is 19.6 mm−1 cm−1. Cytochrome c oxidase (Complex IV) specific activity was measured by the oxidation of the reduced cytochrome c. The reaction solution contained Tris-HCl, 50 mm (pH 7.4); NaN3, 4 mm; decylubiquinol, 0.05 mm; reduced cytochrome c, 0.05 mm. The increase of A550 was recorded after adding 3 µg mitochondrial protein in 1 mL reaction solution. The extinction coefficient of cytochrome c at 550 nm is 19.6 mm−1 cm−1. ATP synthase (Complex V) activity was measured according to published methods (Catterall and Pedersen, 1971). The reaction solution contained TES-KOH, 10 mm (pH 7.2); NaCl, 10 mm; MgSO4, 2 mm; bovine serum albumin, 0.1% (w/v); lactate dehydrogenase, 2 units; pyruvate kinase, 4 units; KCN, mm; NADH, 0.2 mm; n-propyl-gallate, 50 µm; carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, 5 µm; ATP, 1.5 mm; and 50 µg isolated mitochondria subjected to three freeze/thaw cycles in liquid nitrogen. Phosphoenol pyruvate at 1 mm was then added to start the reaction and recorded at 340 nm. The extinction coefficient of NADH at 340 nm is 6220 m-1 cm−1.

Yeast-2-Hybrid Interactions

Bait constructs were cloned into cg-pGBK or pGBKT7 and transformed into Y187, whereas prey constructs were cloned into cg-pGADCg or pGADT7 and transformed into AH109 (Stellberger et al., 2010). Matings were carried out in flat-bottomed 96-well plates as outlined in the Matchmaker GAL4 Two Hybrid System (Clontech) manufacturer’s instructions and plated on double dropout (synthetic defined) SD_Leu-Trp and quadruple dropout SD-Leu-Trp-Ade-His media. Positive interactions were identified after 4 d of growth at 30°C and only regarded as positive interactors when growth was observed in three biological replicates. Interacting strains were serially diluted and plated onto double dropout and quadruple dropout media.

Yeast Complementation

Arabidopsis ISD11 (At5g16620) and CIAF1 (At1g76060) were cloned into pAG423GPB [pAG423GPB-ccdB was a gift from Susan Lindquist (Addgene plasmid # 14149; http://n2t.net/addgene:14149; RRID:Addgene_14149)] and transformed into yeast ISD11 depletion (GAL10-ISD11) and wild-type (YPH499) strains (Adam et al., 2006). Transformed yeast were transferred from Gal media 5% (w/v) lacking His (SD-his) to SD-his Glc media 5% (w/v) for two rounds. Growth rates were determined during the early, mid, and late log phases via absorbance measurements following dilution to OD600 = 0.3.

HiRiEF LC-MS Proteomics

Extraction

All steps in the HiRiEF LC-MS proteomics profiling were performed on two independent biological replicates. Leaves 8 to 10 of the mature rosette (stage 1.12) were ground in buffer (25 mm HEPES-KOH [pH 8.2], 4% [w/v] SDS, 1 mm dithiothreitol [DTT]). Typically, 15 leaves from Col-0 and ciaf1-1 were harvested. Extracts were heated to 95°C for 5 min with agitation and then sonicated 2 × 15 s. Samples were centrifuged at 13000 g, the supernatant was kept, and protein concentration measured using the BioRad DC assay (similar to the Lowry method).

Digestion, alkylation, and peptide labeling with TMT10plex

Total protein (300 µg) in lysis buffer (25 mm HEPES [pH 8.2], 4% [w/v] SDS, 1 mm DTT) was processed according to the filter aided sample preparation (Wiśniewski et al., 2009) protocol with minor modifications. Shortly, the filter units (Nanosep Centrifugal Devices with Omega Membrane, 10K, blue, P/N OD010C34, Pall Corporation) were washed with 200 µL of Milli-Q water (Millipore Corporation) by centrifugation at 14000 g for 15 min. The sample was diluted in 200 µL of urea buffer (8 m urea, 1 mm DTT, in 25 mm HEPES [pH 7.6]) and loaded onto the filter units that were then centrifuged and washed with 200 µl of urea buffer. The buffer containing urea and 25 mm iodoacetamide was added, and the filter units were incubated for 10 min at room temperature with agitation. After centrifugation, the filters were washed 2× with 200 µL of diluted urea buffer (4 m urea in 25 mM HEPES [pH 7.6]). Upon discarding the flow-through, 100 µL of Lys-C buffer (0.5 m urea, 50 mm HEPES, Lys-C protease from Thermo Pierce, 1:50 enzyme to protein mass ratio) was added and the filter units were incubated with mild agitation at 37°C for 16 h. Trypsin buffer (100 µL; 50 mm HEPES, trypsin 1:50 enzyme to protein ratio, Thermo Pierce) was added on to the filter units and incubation continued for 16 h at 37°C. The DC-protein assay (BioRad) was used to estimate peptide concentration. Peptides (100 µg) from each sample were labeled with TMT10plex (Thermo Fisher Scientific) according to the manufacturer's instructions.

HiRIEF separation

The samples for in each TMT set were pooled together, and each set was cleaned by strong cation exchange solid phase extraction (SCX-SPE, Phenomenex Strata-X-C, P/N 8B-S029-TAK). After SpeedVac drying (Thermo SPD111V with refrigerated vapor trap RVT400), 250 µg of peptides of each TMT set was dissolved in 250 µL of 8 m urea, 1% (w/v) pharmalyte (broad range pH 3-10, GE Healthcare, P/N 17-0456-01), and the immobilized pH gradient drystrip (pH 3–10, 24 cm, GE Healthcare, P/N 17-6002-44) rehydrated overnight with the solution. Isoelectric focusing was performed on an Ettan IPGphor 3 system (GE Healthcare) ramping up the voltage to 500 V in 1 h, then to 2000 V in 2 h, and finally to 8000 V in 6 h, after which voltage was held at 8000 V for additional 20 h or until 150 kVh were reached. Upon completion, a well-former with 72 wells was applied onto each strip, and liquid-handling robotics (modified Ettan Digester from GE Healthcare) added 50 µL of solvent to each well and transferred the 72 fractions into a microtiter plate (96 wells, polypropylene, V-bottom, Greiner P/N 651201), which was then dried in a SpeedVac. The solvents used were (1) milliQ water, (2) 35% (v/v) acetonitrile, and (3) 35% (v/v) acetonitrile, 0.1% (v/v) formic acid.

LC-MS analysis

In each LC-MS run of a HiRIEF fraction, the automatic sampler (Ultimate 3000 RSLC system, Thermo Scientific Dionex) dispensed 20 µL of mobile phase A (95% water, 5% [v/v] dimethyl sulfoxide, 0.1% [v/v] formic acid) into the designated well of a microtiter plate, mixed through aspirating/dispensing 10 µL (10 times), and after that injected 10 µL onto a C18 guard desalting column (Acclaim pepmap 100, 75 µm × 2 cm, nanoViper, Thermo). After 5 min of flow at 5 µL/min with the loading pump, the 10-port valve changed to analysis mode in which the NC pump achieved a flow of 250 nL/min through the guard column. The curved gradient (corresponding to curve 6 in the Chromeleon software) proceeded from a 3% mobile phase B (90% [v/v] acetonitrile, 5% [v/v] dimethyl sulfoxide, 5% [v/v] water, 0.1% [v/v] formic acid) to a mobile phase with 45% (v/v) B in 50 min followed by wash at 99% (v/v) B and subsequent re-equilibration. The LC-MS total run time was 74 min. We used a nano EASY-Spray column (pepmap RSLC, C18, 2 µm bead size, 100Å, 75 µm internal diameter, 50 cm long, Thermo) on the nano electrospray ionization EASY-Spray source (Thermo) at 60°C. Online LC-MS was run using a hybrid Q-Exactive HF mass spectrometer (Thermo Scientific). FTMS master scans with 60,000 resolution (and mass range 300–1600 under m/z) were followed by data-dependent MS/MS (30,000 resolution) on the top 5 ions using higher energy collision dissociation (HCD) at 30% normalized collision energy. Precursors were isolated with a 2 m/z window and 0.5 m/z offset. Automatic gain control targets were 1e6 for MS1 and 1e5 for MS2. Maximum injection times were 100 ms for MS1 and MS2. The entire duty cycle lasted <1 s. Dynamic exclusion was used with 60-s duration. Precursors with unassigned charge state or charge state 1 was excluded. An underfill ratio of 0.1% was used.

Proteomics database search

All MS/MS spectra were searched by MSGF+/Percolator under the Galaxy platform (https://usegalaxy.org) using a target-decoy strategy. The reference database used was Araport11 (40782 protein entries, 2016-06). We used a precursor mass tolerance of 10 ppm and high-resolution setting on MS2 level. Only peptides with fully tryptic termini was allowed. Carbamidomethylation on Cys and TMT-10plex on Lys and N terminus was considered as fixed modifications, and Met oxidation as variable modification. The quantification of TMT-10plex reporter ions was performed by using an integration window tolerance of 10 ppm. Peptide spectral matches and peptides were filtered at 1% false discovery rate (FDR; peptide level) and proteins were filtered additionally at 1% FDR (protein level) using the “picked” protein FDR method (Savitski et al., 2015).

Statistical Analysis

Statistical analyze were conducted using student’s t test with biological replicate numbers as indicated in figure legends.

Accession Numbers

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD011795. Sequence data from this article can be found in the Arabidopsis Genome Initiative under the following accession numbers: AT3g18780 (Actin2), At1g72750 (Tim23-2), At1g76060 (CIAF1), At2g35370 (GDC-H), At2g42210 (B14.7), At5g13430 (RISP), AT5G66760 (SDH1.1), At3g16480 (MPPα), At5G08670 (ATPβ), AT3g03070 (13 kD), AT5g67590 (18 kD [NDUFS4]), AT4g02580 (24 kD), AT5g08530 (51 kD), AT5g37510 (75 kD), AT5g47890 (B8), AT5g08060 (B14.5), AT3g03100 (B17.2), AT5g11770 (20 kD [PSST]), AT1g79010 (TYKY1 [23 kD]), AT1g16700 (TYKY2), AT5g63510 (CAL1), AT3g48680 (CAL2), AT4g16450 (MNLL), AT5g66510 (CA3), AT1g67350 (P1), AT5g52640 (B13), AT3g27380 (SDH2.1), At3g16480 (MPPα), and AT5g56090 (COX15).

Supplemental Data

The following supplemental materials are available.

  • Supplemental Figure S1. TAIL PCR identifies an additional T-DNA insertion within At1g76060 in the T-DNA insertion line SALK_143656.

  • Supplemental Figure S2. Phenotypic analysis of Col-0, CIAF1 deletion and complemented lines.

  • Supplemental Figure S3. Phylogenetic analysis of all LYRM proteins from plant and nonplant species.

  • Supplemental Figure S4. Complementation of the T-DNA insertion line ciaf1-2 with CIAF1 restores Complex I and the Supercomplex I+III.

  • Supplemental Figure S5. CIAF1 does not integrate within CI or any of its known intermediates.

  • Supplemental Figure S6. Deletion of CIAF1 results in an increase in mitochondrial protein translation ability.

  • Supplemental Figure S7. Deletion of CIAF1 results in an increase in mitochondrial protein import ability.

  • Supplemental Table S1. Fold changes (> ±1.25) for all mitochondrial proteins from ciaf1-1 plants a and b, compared to Col-0. Mapman bincodes used for Figure 6 are indicated in column F. Ciaf1 (At1g76060) is indicated in blue. Complex I subunits are indicated in pink.

  • Supplemental Table S2. FASTA sequences used for phylogenetic analysis.

  • Supplemental Table S3. Primers used in this study.

ACKNOWLEDGMENTS

We thank Kai Hell for the yeast strains (GAL10-ISD11 and YPH499) and Reena Narsai for helpful comments.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.19.00822

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Monika W. Murcha (monika.murcha{at}uwa.edu.au).

  • A.I. and M.W.M. designed the research; A.I., M.G.-H., S.H., R.M.B., B.K., P.F.T., and M.W.M. performed the research; A.I., R.M.B., J.L., J.W., and M.W.M. analyzed the data; all authors contributed to writing the manuscript.

  • ↵1 This work was supported by Australian Research Council Future Fellowships FT130100112 and FT130101338 (to M.W.M. and S.H.) and the Australian Research Council Centre of Excellence in Plant Energy Biology (CE140100008 to J.W.).

  • ↵3 Senior author.

  • ↵[OPEN] Articles can be viewed without a subscription.

  • Received July 10, 2019.
  • Accepted September 25, 2019.
  • Published October 10, 2019.

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A Mitochondrial LYR Protein Is Required for Complex I Assembly
Aneta Ivanova, Mabel Gill-Hille, Shaobai Huang, Rui M. Branca, Beata Kmiec, Pedro F. Teixeira, Janne Lehtiö, James Whelan, Monika W. Murcha
Plant Physiology Dec 2019, 181 (4) 1632-1650; DOI: 10.1104/pp.19.00822

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A Mitochondrial LYR Protein Is Required for Complex I Assembly
Aneta Ivanova, Mabel Gill-Hille, Shaobai Huang, Rui M. Branca, Beata Kmiec, Pedro F. Teixeira, Janne Lehtiö, James Whelan, Monika W. Murcha
Plant Physiology Dec 2019, 181 (4) 1632-1650; DOI: 10.1104/pp.19.00822
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Plant Physiology: 181 (4)
Plant Physiology
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