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. 2014 Jun 1;23(11):2901-13.
doi: 10.1093/hmg/ddu003. Epub 2014 Jan 8.

Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase

Myriam Bourens  1 , Aren Boulet, Scot C Leary, Antoni Barrientos
Affiliations

Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase

"VSports手机版" Myriam Bourens et al. Hum Mol Genet. .

Abstract

Cytochrome c oxidase (CIV) deficiency is one of the most common respiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleus-encoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here, we have used small interference RNA and transcription activator-like effector nucleases (TALENs) technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2 VSports手机版. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into early CIV subassemblies. Immunoprecipitation assays using a stable COX20 knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction between COX20 and newly synthesized COX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps of COX2 maturation, stabilizing the newly synthesized protein and presenting COX2 to its metallochaperone module, which in turn facilitates the incorporation of mature COX2 into the CIV assembly line. .

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Figures

Figure 1.
Figure 1.
COX20 knockdown causes an isolated CIV deficiency by destabilizing newly synthesized COX2. (A) Schematic representation of the COX20 mRNA regions that are recognized by siRNA duplex oligonucleotides #1 and #2. (B) and (C) COX20 mRNA abundance in HEK293T cells transfected with 10 nm of the indicated COX20 siRNA duplex, estimated by qPCR. COX20 mRNA levels were normalized to those of HPRT1 (hypoxanthine phosphoribosyltransferase 1) and expressed as a percentage of the abundance at Day 1. (D) Immunoblot analysis of COX20 protein levels in extracts from mock-transfected cells and cells transfected with 10 nm of siRNA #1. (E) Endogenous cell respiration following silencing of COX20 expression for 10 days with 10 nm of siRNA #1. Data are expressed as percentage of the respiratory rate of mock-transfected cells. (F) Steady-state levels of representative subunits of the oxidative phosphorylation complexes (indicated in parentheses) in whole cells extracts from mock-treated HEK293T cells (−) and cells transfected with 10 nm of siRNA #1 or #2 for 6 days. Tubulin was used as a loading control. (G) and (H) BN-PAGE analysis of mock-transfected cells and cells transfected with 10 nm of siRNA for 6 days. The assembly intermediates with sizes compatible with subassemblies S1 (COX1), S2 (COX1-COX4-COX5A) and S3 are indicated. (I) BN-PAGE analysis of HEK293T that were mock-transfected or transfected with 10 nm siRNA #2 for 6 days, and of immortalized fibroblasts from patients carrying mutations in either SCO1 or SURF1. (J) In vivo mitochondrial protein synthesis. The left panel shows the incorporation of [35S]methionine into mitochondrial proteins in siRNA transfected cells (10 nm, 6 days) and mock-treated cells after a 30 min pulse. The right panel highlights the stability of newly synthesized proteins 2, 3 and 4 h after washing off residual [35S]methionine following the 30 min pulse.
Figure 2.
Figure 2.
A KO-COX20 cell line generated using TALENs technology exhibits an isolated CIV deficiency that is complemented by stable expression of COX20-FLAG. (A) Schematic representation of the first exon of the COX20 (FAM36A) gene and the sequence recognition sites of both TALEs. (B) Immunoblot analysis of the steady-state levels of COX20 in mitochondria isolated from HEK293 T (WT) and HEK293T KO-COX20 cell lines. VDAC was used as a loading control. (C) Endogenous cell respiration and (D) mitochondrial enzyme activities of HEK293T (WT) and HEK293T KO-COX20 cell lines. CS, citrate synthase; NCCR, NADH-cytochrome c reductase (complex I + III). (E) Immunoblot analysis of the steady-state levels of CIV subunits in mitochondria isolated from HEK293T (WT) and HEK293T KO-COX20 cell lines. (F) and (I) Steady-state levels of oxidative phosphorylation complexes were analyzed by BN-PAGE and detected by immunoblotting with the indicated antibodies. (G) BN-PAGE and immunoblot analysis of CIV subcomplexes, highlighting the accumulation of early assembly intermediates. An antibody against the h70 kDa complex II subunit was used as a loading control. (H) Immunoblot analysis of the steady-state levels of CIV subunits in KO-COX20 cells alone and stably expressing COX20-FLAG compared with the parental line (WT). (J) Analysis of respiratory chain supercomplexes in HEK293T (WT) and the KO-COX20 cell line, extracted with digitonin and separated by BN-PAGE. The left most panel shows an in-gel CI activity staining. The remaining three panels show immunoblots probed with the indicated antibodies.
Figure 3.
Figure 3.
ScCox20 and HsCOX20 do not functionally substitute for each other in the heterologous systems. (A) Alignment of HsCOX20 and ScCox20. The localization of the two predicted transmembrane domains is indicated. (B) Serial dilutions of wild-type (WT) and Δcox20 W303 yeast strains on fermentable (glucose) and respiratory (ethanol + glycerol) media. H. sapiens COX20 (HsCOX20), S. cerevisiae COX20 (ScCOX20) and a S. cerevisiae COX20 lacking the first 58 first amino acids (NΔ58ScCOX20) were expressed from either a multicopy (YEp) or an integrative (YIp) plasmid under the control of a TEF1 promoter. (C) Steady-state levels of Cox20-HA and Cox2 in mitochondria isolated from the indicated yeasts strains. The two panels presented for the anti-HA reaction are two different exposure times. In the overexposed panel, the asterisks indicate faint bands of ScCOX20-HA and NΔ58ScCOX20-HA when expressed from an integrative plasmid. Porin was used as a loading control. (D) Radiolabeling of mitochondrial proteins synthesized during a 15-min pulse followed by a 1-hour chase. Cox2p, Cox2 precursor; Cox2 m, Cox2 mature. (E) BN-PAGE of HEK293T KO-COX20 cell lines stably expressing HsCOX20-FLAG, HsCOX20-HA, ScCox20-HA or NΔ58ScCox20-HA. (F) Immunoblot analysis of COX20 protein levels in mitochondria purified from HEK293T (WT) stably expressing HsCOX20-HA or ScCox20-HA. The asterisk represent a non-specific band that crossreacts with the HA antibody.
Figure 4.
Figure 4.
COX20 interacts with and stabilizes newly synthesized COX2. (A) and (B) Mitochondrial translation products were pulse-labeled with [35S]methionine for 30 min in HEK293T (WT), KO-COX20 and KO-COX20 + COX20-FLAG cells in the presence of emetine to inhibit cytoplasmic protein synthesis, followed by the indicated chase time. Cellular protein extracts were run on an SDS–PAGE gel, transferred to nitrocellulose and exposed to film. (C) Mitochondrial translation products were pulse-labeled in HEK293T (WT) and KO-COX20 + COX20-FLAG cells for 30 min. Whole cell extracts were then used for COX20-FLAG immunoprecipitation with anti-FLAG-conjugated agarose beads. E, Extract; Un, Unbound; W, Wash and IP, immunoprecipitate. (D) Immunoprecipitation of COX20-FLAG from mitochondrial extracts prepared from a KO-COX20 + COX20-FLAG stable cell line using FLAG-conjugated agarose beads. Resultant membranes were immunoblotted with COX2 and FLAG antibodies. HEK293T (WT) mitochondrial extracts were used as a negative control.
Figure 5.
Figure 5.
COX20 forms a stable complex of ∼ 90 kDa independent of COX2 synthesis. (A) BN-PAGE of whole cell extracts from HEK293 T (WT) and KO-COX20 + COX20-FLAG cell lines, with the resultant immunoblot decorated with FLAG antibody. (B) Sucrose gradient analyses of KO-COX20 + COX20-FLAG mitochondrial extracts prepared from cells grown overnight in the absence (upper panel) or presence of 15 µg/ml of doxycycline to inhibit mitochondrial protein synthesis. The gradient was calibrated with hemoglobin (67 kDa) and lactate dehydrogenase (130 kDa). Ex, Extract. (C) Steady-state levels of proteins in mitochondria isolated from untreated and doxycycline-treated cells.
Figure 6.
Figure 6.
Transient interactions between COX20 and SCO1 and SCO2 depend on COX2 and are required for maturation of the newly synthesized polypeptide. (A) Immunoprecipitation of COX20-FLAG from mitochondria. E, Extract; Un, Unbound; W, Wash and IP, immunoprecipitate. The resultant blot was probed with FLAG, SCO1 and OXA1L antibodies. (B) Immunoprecipitation of SCO1 from KO-COX20 + COX20-FLAG mitochondria. An anti-SCO1 antibody was conjugated with protein A Dynabeads (Invitrogen) and incubated with mitochondrial extracts. The blot was probed with SCO1 and FLAG antibodies. (C) Steady-state levels of COX20, COX2, SCO1 and SCO2 in purified mitochondria isolated from cells grown overnight in the absence or presence of 15 µg/ml of doxycycline. (D) Immunoprecipitation of COX20-FLAG using FLAG antibody-conjugated beads from mitochondrial extract of HEK293T, KO-COX20 and KO-COX20 + COX20-FLAG cells grown overnight in the absence or presence of doxycycline. (E) Sucrose gradient analyses of KO-COX20 + COX20-FLAG, HEK293 T and KO-COX20 mitochondrial extracts. The gradient was calibrated with hemoglobin (67 kDa) and lactate dehydrogenase (130 kDa). Ex, Extract. (F) Control, SCO1 (SCO1-1, SCO1-2) and SCO2 patient fibroblasts were grown for 9 days in basal media (−) or transfected on Days 0, 3 and 6 with one of two COX20 siRNAs (siRNA1 and siRNA2) or a scrambled siRNA (Alexa). Digitonized mitoplasts were prepared from these fibroblasts, extracted in PBS containing 1.5% lauryl maltoside and a complete protease inhibitor cocktail and equal amounts of protein (15 μg/lane) were separated by SDS–PAGE and blotted with the indicated antibodies. The asterisk indicates a non-specific band that crossreacts with the COX20 antibody.
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