Identification of a protein mediating respiratory supercomplex stability

Abstract

The complexes of the electron transport chain associate into large macromolecular assemblies, which are believed to facilitate efficient electron flow. We have identified a conserved mitochondrial protein, named respiratory supercomplex factor 1 (Rcf1-Yml030w), that is required for the normal assembly of respiratory supercomplexes. We demonstrate that Rcf1 stably and independently associates with both Complex III and Complex IV of the electron transport chain. Deletion of the RCF1 gene caused impaired respiration, probably as a result of destabilization of respiratory supercomplexes. Consistent with the hypothetical function of these respiratory assemblies, loss of RCF1 caused elevated mitochondrial oxidative stress and damage. Finally, we show that knockdown of HIG2A, a mammalian homolog of RCF1, causes impaired supercomplex formation. We suggest that Rcf1 is a member of an evolutionarily conserved protein family that acts to promote respiratory supercomplex assembly and activity.

Figure 1. Rcf1 is a mitochondrial inner membrane protein important for respiration. (Also see Figure S1)

(A) The rcf1Δ mutant expressing Rcf1-GFP and MtRFP grown to log phase in SD medium was imaged by fluorescence microscopy. (B) Intact mitochondria, hypotonic-swollen mitoplasts, and TritonX-100–solubilized mitochondria of a strain expressing Rcf1-His₆/HA₃ were treated either with (+) or without (−) Proteinase K and analyzed by immunoblot with the whole cell lysate (WCL) and post mitochondrial supernatant (PMS). Cox2, Mge1, Cyb2, and Fzo1 are inner membrane, matrix, intermembrane space, and outer membrane proteins, respectively. (C) Soluble (S) and pellet (P) fractions from alkali and high salt extracted mitochondria expressing Rcf1-His₆/HA₃ were analyzed by immunoblot. Cyb2 is a soluble intermembrane space protein; Cox4 is an inner membrane-associated protein; Cox2 is an integral inner membrane protein; and Por1 is an integral outer membrane protein. (D) Five-fold serial dilutions of the indicated strains harboring an empty vector (EV) or a plasmid expressing Rcf1-His₆/HA₃ were spotted on plates and incubated at 30°C and 37°C. (E) Wild-type and rcf1Δ mutant strains of the BY4741 background were grown in glucose, lactate or glycerol to log phase and the rate of oxygen consumption was measured. Data shown is representative of three independent experiments and mean ± SD is shown. *, P<0.05; **, P<0.01

Figure 2. Rcf1 physically interacts with respiratory Complex III and IV

(A) Mitochondria from an EV-containing wild-type strain or an rcf1Δ mutant strain expressing Rcf1-His₆/HA₃ were subjected to two-step purification. The final eluate was analyzed by silver staining. Discrete bands were excised and the protein identity was determined by mass spectrometry as indicated in the text. (B) Digitonin-solubilized mitochondria extracted from yeast strains either expressing Rcf1-His₆/HA₃ (+) or not (−) were immunoprecipitated using anti-HA antibody. One percent of the mitochondrial lysate and the final eluate were immunoblotted using anti-Cor1&Qcr2, Cyt1, Cox1, Cox2, Cox3, Cox4 and HA antibodies. * indicates IgG light chain. (C) Rcf1-His₆/HA₃ was purified from digitonin and DDM-solubilized mitochondria, respectively. The final eluate was visualized by silver stain and protein identities were determined by mass spectrometry. Cox3 is indicated based on SDS-PAGE/Western blot as it was not detected by mass spectrometry. (D) Mitochondria extracted from a strain harboring a plasmid expressing Rcf1 or Rcf1-His₆/HA₃ were solubilized by digitonin or DDM and subjected to BN-PAGE/Western blot. Rcf1, Complex III and Complex IV were immunoblotted by anti-HA, Cor1&Qcr 2 and Cox2 antibodies, respectively.

Figure 3. Rcf1 interacts with Complex III and Complex IV independently. (Also see Figure S2)

(A) Rcf1 was immunoprecipitated using anti-HA antibodies from the digitonin-solubilized mitochondria of the indicated strains harboring either with (+) or without (−) a plasmid expressing Rcf1-His₆/HA₃. Both 1% crude lysate and final eluate were immunoblotted using anti-Cor1&Qcr2, Cyt1, Cox1, Cox2, Cox3, Cox4, HA and Por1 antibodies, respectively. Asterisk on the anti-Por1 blot indicates the Cox2 signal from a previous exposure and arrowhead on the anti-Cox3 blot indicates IgG light chain. (B) Mitochondria from the indicated strains were analyzed as in (A).

Figure 4. Respiratory supercomplexes are impaired in the absence of RCF1. (Also see Figure S3)

(A) Mitochondria from wild-type and the rcf1Δ mutant grown to log phase in synthetic raffinose medium were analyzed by BN-PAGE/Western blot. Complex III and Complex IV were immunoblotted by anti-Cor1&Qcr2 and Cox3 antibodies, respectively. Porin serves as a control. (B) The mitochondria from (A) were subjected to SDS-PAGE and immunoblotted using Cox1, Cox2, Cox3, Cor1&Qcr2, Cyt1, Rip1,and Sdh2 antibodies. Por1 and Mge1 are loading controls. (C) Enzymatic activities of Complex III, Complex IV and Complex V were measured in lysates of the wild-type and rcf1Δ mutant grown in synthetic raffinose media to log phase. Each was measured in three independent cultures and data shown is mean + SD. *, P<0.05 (D) Mitochondria from the wild-type strain with or without a 2µ-based plasmid overexpressing Rcf1 grown in raffinose medium to log phase were analyzed by BN-PAGE/Western blot. Complex III, Complex IV and porin complex were immunoblotted by anti-Cor1&Qcr2, Cox3 and Por1 antibodies, respectively.

Figure 5. RCF1 genetically interacts with AAC2 and COX13 to stabilize respiratory supercomplexes. (Also see Figure S4)

The indicated strains were grown in YPAD media to log or stationary phase as indicated were spotted on YPAD plates and incubated at 30°C (A) or 37°C (B). (C) Mitochondria from the indicated strains grown in raffinose medium to log phase were analyzed by BN-PAGE/Western blot. Complex III, Complex IV and porin complex were immunoblotted by anti-Cor1&Qcr2, Cox3 and Por1 antibodies, respectively. III₂* indicates a Complex III intermediate. (D) The indicated strains were spotted on SD and SGlycerol/ethanol plates and incubated at 30°C. (E) Mitochondria from the indicated strains grown in 1% glucose medium for 1 day were analyzed by BN-PAGE/Western blot. Complex III, Complex IV and Complex V were immunoblotted by anti-Cor1&Qcr2, Cox2 and Atp2 antibodies, respectively. III₂* indicates a Complex III intermediate.

Figure 6. Increased mitochondrial oxidative stress in the absence of RCF1. (Also see Figure S5)

(A) Cell lysates of the indicated strains grown in SD media for 0.5 and 2.5 days were subjected to aconitase activity assay and immunoblotted with anti-Aco1 antibody. Aconitase activity was determined specifically by normalizing NADP reduction rate to Aco1 protein band intensity. sod2Δ was used as a control. Mean ± SD of three independent cultures is shown. **, P<0.01; ***, P<0.0005 (B) The wild-type strain harboring either EV or a 2µ-based plasmid overexpressing Rcf1 were grown for 1 day in 1% glucose and analyzed as in (A). Mean ± SD of three independent cultures is shown. *, P<0.05 (C) The indicated strains were harvested after being grown in SD media for 0.5 day and analyzed as in (A). Mean ± SD of three independent cultures is shown. *, P<0.05; **, P<0.01 (D) Log phase cultures (0.5 day) of the wild-type and rcf1Δ mutant were pre-treated with H₂O₂ for 2 hours, spotted on synthetic glucose medium and incubated at 30°C. (E) Log phase cultures (0.5 day) of the indicated strains were harvested and immunoblotted for Sod2, which was normalized to Por1 intensity. Mean ± SD of six independent cultures is shown. **, P<0.01 (F) Wild-type and the rcf1Δ mutant strains of the BY4741 background were grown to log phase in glucose media and equal number of cells were plated on media containing the indicated concentration of CCCP. Number of colonies was counted after 48 hours. Mean ± SD of three independent cultures is shown. *, P<0.05

Figure 7. Mammalian HIG2A is critical for normal respiratory supercomplex organization. (Also see Figure S6)

(A) Mitochondria extracted from C2C12 cells transfected with either scrambled (scr) or HIG2A-targeted siRNA (si1) were solubilized in 2% digitonin and resolved by BN-PAGE, followed by immunoblotting with the indicated antibodies. Respiratory complex species are indicated following the convention of (Acin-Perez et al., 2008), specifically: 1 (I+III+V); 2 (I+II+III+IV); 3 (I+II+III+IV); 4 (I+III+IV); 5 (I+III+V); 6 (III+IV); 7 (III+IV)*. (B) HIG2A mRNA level normalized to ACTIN was measured from cells in (A) using quantitative RT-PCR. (C) Mitochondria extracted from C2C12 cells transfected with scrambled (scr) or one of three unique HIG2A-targeted siRNA (si2, 3, 4) were treated as in (A). A longer exposure of the anti-Cox1 immunoblot is shown to enable visualization of the supercomplexes #1, 2 and 4. Note that si4 is not efficacious in HIG2A knockdown. (D) HIG2A mRNA level normalized to ACTIN was measured from cells in (C) using quantitative RT-PCR.