Journal of Molecular Biology
C1orf163/RESA1 Is a Novel Mitochondrial Intermembrane Space Protein Connected to Respiratory Chain Assembly
Graphical Abstract
Introduction
Mitochondria are organelles of bacterial origin with a role in several important cellular processes, including energy production. Energy production in mitochondria takes place at the inner mitochondrial membrane (IMM) during the process known as oxidative phosphorylation (OXPHOS). This process, performed by four OXPHOS complexes (NADH:ubiquinone oxidoreductase or complex I, succinate:ubiquinone oxidoreductase or complex II, ubiquinol:ferricytochrome c oxidoreductase or complex III, and cytochrome c oxidoreductase or complex IV), creates a proton gradient across the IMM, which is then later used by F1FO ATPase (complex V) to capture energy in the form of ATP [1]. In mammalian mitochondria, OXPHOS complexes contain only 13 subunits encoded by the mitochondrial genome, whereas the majority of the subunits are nuclear encoded. The assembly of the OXPHOS complexes is a complicated process, involving many assembly factors [1], [2], [3]. For example, yeast complex IV contains three mitochondrial encoded subunits, Cox1, Cox2, and Cox3, the translation of which is controlled by at least seven translational regulators. Cox1 first forms an intermediate with Cox5 and Cox6, to assemble in later steps with the remaining two mitochondrial encoded subunits, and then with Cox4, Cox7, Cox8, and Cox9, and finally with Cox12 and Cox13. This process is supervised by more than 20 different proteins, which control membrane insertion, heme a synthesis, copper transport, and insertion and act as assembly chaperones [4]. Complexes I, III, and IV also combine to form supercomplexes, which are of crucial importance for the OXPHOS [5].
Since most of the mitochondrial proteins, including the components of the OXPHOS complexes, are encoded by the cell nucleus and translated on cytosolic ribosomes, mitochondria have developed import machineries that take up mitochondria-targeted proteins, import them, and sort them to their proper location. The translocase of the outer membrane (TOM) complex serves as an entry point for practically all mitochondrial proteins. The sorting and assembly machinery (SAM/TOB complex), with its central component Sam50, assembles β-barrel proteins into the outer mitochondrial membrane (OMM). Two translocases of the IMM (TIM23 and TIM22 complexes) perform the import into the matrix and/or integration of proteins into the IMM [6]. Intermembrane space (IMS) proteins can follow several different import routes. IMS proteins with specific cysteine-rich motifs are imported with the help of the mitochondrial IMS import and assembly pathway machinery (MIA complex) [6], [7].
Recently, a novel complex was described in the yeast IMM and termed MINOS, for mitochondrial inner membrane organizing system [8]; MITOS, for mitochondrial organizing structure [9]; or MICOS, for mitochondrial contact site [10]. This protein complex is crucial for the maintenance of the cristae structure. In mammalian mitochondria, the components of the MINOS complex interact with the SAM complex in the OMM, forming a large mitochondrial intermembrane space bridging (MIB) complex [11], [12], [13]. We have observed that the depletion of the MIB complex components, primarily Sam50 and mitofilin, affects the steady-state levels of practically all OXPHOS complexes, the most affected being complexes I and IV. Furthermore, the assembly of newly imported subunits of complexes I and IV was impaired in mitochondria depleted of Sam50 or mitofilin [11].
To better understand how the knockdowns of Sam50 and mitofilin affect mitochondria and OXPHOS, we have performed stable isotope labeling with amino acids in cell culture (SILAC) combined with quantitative mass spectrometry to determine which proteins are reduced after Sam50 or mitofilin depletion [11]. One of the proteins identified in this screen was a conserved, uncharacterized protein, C1orf163. This protein belongs to the hcp beta-lactamase family and is also known as SEL1 repeat containing 1 protein (SELRC1). In this work, we show that C1orf163 is a soluble protein localized to the mitochondrial IMS, present in two complexes of around 60 kDa and 150 kDa. The steady-state levels as well as the import and assembly of C1orf163 into these protein complexes are diminished after Sam50 depletion. Knockdown of C1orf163 leads to a strong reduction in the assembly and activity of complex IV, although all other OXPHOS complexes except complex II are affected to a lesser extent. Whereas the relation of C1orf163 to Sam50 and mitofilin remains unclear, our results indicate that C1orf163 is a novel IMS factor related to the OXPHOS complex assembly, possibly playing a role in the assembly of complex IV. We propose, therefore, to rename C1orf163 into RESA1 (for RESpiratory chain Assembly 1).
Section snippets
C1orf163 is a soluble mitochondrial protein located in the IMS
Our work so far showed that the depletion of a mitochondrial protein usually leads to reduction in levels of its interaction partners or substrates. For example, Tom40 depletion leads to reduction in the levels of the TOM complex components, such as Tom20 or Tom22, whereas other proteins are not affected. Likewise, depletion of Sam50 influences the levels of the SAM complex components Metaxin 1 and Metaxin 2 and the SAM complex substrates such as VDAC, but most other proteins remain unreduced
Discussion
In this study, we analyzed an uncharacterized protein, C1orf163/SELRC1. We identified this protein as one of the proteins reduced after the knockdown of Sam50 [11]. We show that C1orf163 is a soluble mitochondrial protein localized in the IMS with a putative function in the assembly of the OXPHOS complex, presumably complex IV.
The C1orf163 gene localizes to the short arm of chromosome 1, to the 1p32.3 region. C1orf163 belongs to the SLR family of proteins and its homologues are found from
Cell culture and isolation of mitochondria
HeLa cells with an inducible shRNA-mediated knockdown were generated as described elsewhere [14], [22]. The sequence of C1orf163kd-5 shRNA is 5′-GATGGTGTTGATAAGGATGA-3′. sam50kd-2 and mflkd-2 cells were previously generated [11], [14]. Cells were grown in RPMI 1640 or Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal calf serum (Biochrom) and penicillin/streptomycin. Expression of shRNAs was induced by adding 1 μg/ml doxycycline (BD Biosciences) to the growth medium for 7
Acknowledgements
This work was supported by Deutsche Forschungsgemeinschaft (Grant KO3882/1-1 to V.K.-P. and RU 631/7-1 to T.R.). We thank G. Krohne for help with electron microscopy and J. A. Enríques for useful discussion. We thank P. Rehling and E. Fernández-Vizarra for sharing their protocols.
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