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Rcf1 Modulates Cytochrome c Oxidase Activity Especially Under Energy-Demanding Conditions

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Rcf1 Modulates Cytochrome c Oxidase Activity Especially Under Energy-Demanding Conditions

Hannah Dawitz et al. Front Physiol.

Abstract

The mitochondrial respiratory chain is assembled into supercomplexes. Previously, two respiratory supercomplex-associated proteins, Rcf1 and Rcf2, were identified in Saccharomyces cerevisiae, which were initially suggested to mediate supercomplex formation. Recent evidence suggests that these factors instead are involved in cytochrome c oxidase biogenesis. We demonstrate here that Rcf1 mediates proper function of cytochrome c oxidase, while binding of Rcf2 results in a decrease of cytochrome c oxidase activity. Chemical crosslink experiments demonstrate that the conserved Hig-domain as well as the fungi specific C-terminus of Rcf1 are involved in molecular interactions with the cytochrome c oxidase subunit Cox3. We propose that Rcf1 modulates cytochrome c oxidase activity by direct binding to the oxidase to trigger changes in subunit Cox1, which harbors the catalytic site. Additionally, Rcf1 interaction with cytochrome c oxidase in the supercomplexes increases under respiratory conditions. These observations indicate that Rcf1 could enable the tuning of the respiratory chain depending on metabolic needs or repair damages at the catalytic site.

Keywords: Rcf1; Rcf2; Saccharomyces cerevisiae; bc1 complex; cytochrome c oxidase; interaction partners; respiratory supercomplex.

Figures

FIGURE 1
FIGURE 1
Rcf1 and Rcf2 affect respiratory growth but influence supercomplex assembly only slightly. (A) A 10-fold serial dilution growth assay on fermentable and non-fermentable medium at different temperatures showed that absence of Rcf1 decreases respiratory ability of the strains. The absence of Rcf2 showed only in combination with loss of Rcf1 a strong decrease in growth. (B) Mitochondria were purified from cells grown to logarithmic phase in YPGal (30°C). Supercomplex assembly of mitochondria solubilized in 2% digitonin was analyzed by BN-PAGE followed by subsequent Western blot and immuno decoration showing supercomplex assembly in the absence of Rcf1 and Rcf2. The antibody against Rcf2 shows unspecific binding in the lower molecular weight range. (C) Respiratory chain complex assembly of mitochondria purified from cells grown in YPGal (logarithmic phase, 30°C) was analyzed. Mitochondria were solubilized in 2% digitonin, separated by BN-PAGE followed by separation on 2D polyacrylamide gels and Western blot analysis. Rcf1 and Rcf2 comigrate with supercomplexes. Upon loss of Rcf1, Rcf2 shifts mostly out of the supercomplexes. Still Rcf1 and Rcf2 can associate with supercomplexes in the absence of the respective other protein. Supercomplexes can still assemble in the absence of Rcf1, Rcf2 or both proteins combined although in a lower amount. T, total; III2IV2, III2IV, forms of supercomplexes; III2, dimer of bc1 complex; IV, cytochrome c oxidase.
FIGURE 2
FIGURE 2
Rcf1 and Rcf2 affect cytochrome c oxidase activity without affecting its subunit amount. (A) Steady state protein levels of cells grown to logarithmic or stationary phase showed that the loss of Rcf1 or Rcf2 did not change the protein levels of bc1 complex (Cor1, Rip1) or cytochrome c oxidase subunits (Cox2). As control aconitase (Aco1) levels and total protein amount (ponceau) were used. (B) Cytochrome c reduction measurements showed that only in absence of both Rcf1 and Rcf2 bc1 complex (Complex III) activity was decreased. Oxygen consumption measurements demonstrated that cytochrome c oxidase (Complex IV) activity was decreased upon loss of either Rcf1 or both Rcf1 and Rcf2 while loss of Rcf2 alone led to an increase in activity. The coupled activity of the supercomplexes (Coupled) was not affected by the absence of either Rcf1 or Rcf2 [the standard errors were calculated from a sample size of n = 3 (complex III and complex IV) or n = 4 (coupled)]. n.s., P > 0.05; P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001. (C) Supercomplexes were purified from mitochondria (cells grown YPG, 30°C, to logarithmic phase) via a FLAG-tag on Rcf1 (Rcf1F), Rcf2 (Rcf2F) or the cytochrome c oxidase subunit Cox6 (Cox6F). All three proteins co-purified supercomplexes but to different amounts.
FIGURE 3
FIGURE 3
Rcf1 and Rcf2 are no stoichiometric subunits of cytochrome c oxidase. (A) and (B) Steady-state protein levels of His-tagged Rcf1, Rcf2 and the cytochrome c oxidase subunit Cox4. Decoration against the epitope of the His-tag showed 30% lower protein levels of Rcf1 and Rcf2 compared to Cox4. The proteins were extracted from mitochondria purified from cells grown in YPG, 30°C. The Western blot signal was quantified using the software ImageJ (the standard errors were calculated from a sample size of n = 3). (C) BN-PAGE analysis followed by 2D SDS-PAGE, Western blot and immuno decoration of wild-type mitochondria solubilized in 2% DDM showed that Cox4 solely comigrated with cytochrome c oxidase, while Rcf1 and Rcf2 comigrated additionally with other complexes. T, total; III2, dimer of bc1 complex; IVa and IVb, forms of cytochrome c oxidase. n.s., P > 0.05; P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001.
FIGURE 4
FIGURE 4
Rcf1 and Rcf2 associate with supercomplexes depending on working load of respiratory chain. (A) Mitochondria isolated from cells grown in fermentable (YPD) or non-fermentable medium (YPG) were solubilized in 2% digitonin, separated using BN-PAGE followed by 2D SDS-PAGE, Western blot and immuno decoration. (B) Based on the 2D analysis, the amount of the respective subunit of cytochrome c oxidase or the amount of the proteins Rcf1 and Rcf2 residing in the supercomplexes was calculated in respect to the total subunit amount. While the cytochrome c oxidase subunits slightly shift into the supercomplexes (fold change of ∼1.4), Rcf1 and Rcf2 shift strongly into supercomplexes (∼2.5 and ∼3.7, respectively). The Western blot signals were analyzed using the software OriginPro (the standard errors were calculated from a sample size of n = 3). (C) Model of protein distribution due to different carbon sources. Under fermentable conditions (YPD) cytochrome c oxidase subunits (Cox2, Cox12, and Cox13) are shifted to the monomeric complex or a free form while under respiratory conditions (YPG) cytochrome c oxidase shifts into supercomplexes. Rcf1 and Rcf2 follow the trend but in a higher amount. T, total; SC, supercomplexes; I, intensity of developed western blot.
FIGURE 5
FIGURE 5
The conserved Hig-domain of Rcf1 fulfills Rcf1 function. (A) Schematic view of truncation constructs tagged with a FLAG-tag: full-length Rcf1 (Rcf1), Rcf1ΔN (C-terminus of Rcf1 fused to Cyb2 membrane domain) and Rcf1ΔC (Hig-domain). (B) Steady state protein levels of strains expressing full-length Rcf1FLAG (Rcf1; 25 kDa), Rcf1ΔN (23 kDa), Rcf1ΔC (20 kDa) or no Rcf1 (rcf1Δ). Mitochondria were solubilized and analyzed using SDS-PAGE followed by Western blot and immunodecoration against Rcf1 and FLAG-peptide. Decoration against Rcf1 shows degradation products which are not visible by decorating against the FLAG-peptide indicating degradation of the FLAG-tag and not the Rcf1-protein itself. (C) A 10-fold dilution series of strains grown in YPD to logarithmic phase was spotted on YPD and YPG plates and incubated at 30°C and 37°C. Loss of the Hig-domain (Rcf1ΔN) induces a similar growth defect as loss of the entire protein while loss of the fungi specific C-terminus (Rcf1ΔC) does not affect growth. This growth phenotypes indicate that the conserved Hig-domain (present in Rcf1ΔC) is sufficient to support respiratory growth. (D) Oxygen consumption measurements to determine activity of cytochrome c oxidase activity in truncated mutants. As expected from the growth assay, loss of the Hig-domain (Rcf1ΔN) led to almost 40% decrease in activity, similar to rcf1Δ, while loss of the C-terminus (Rcf1ΔC) showed no effect on the complex activity. (E) Truncated mutants were analyzed with BN-PAGE and subsequent 2D SDS-PAGE followed by Western blot and immuno decoration against Cor2, Cox2, and Rcf1. While the C-terminus (Rcf1ΔN) can only comigrate weakly with cytochrome c oxidase (green arrow) but not with the supercomplexes (red arrows), the Hig-domain (Rcf1ΔC) retains the function to interact with cytochrome c oxidase as well as supercomplexes (green arrows). T, total; III2IV2, III2IV, forms of supercomplexes; III2, dimer of bc1 complex; IV, cytochrome c oxidase. n.s., P > 0.05; P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001; ∗∗∗∗P ≤ 0.0001.
FIGURE 6
FIGURE 6
Interaction partners and interaction sites of Rcf1. (A) Proteins in purified mitochondria were chemically crosslink followed by protein extraction, SDS-PAGE, Western blotting and immuno decoration against Rcf1. Use of different crosslink reagents (differences in reactive group and length) showed that Rcf1 has multiple interaction partners. The controls (-, DMSO) show cross reactions of the Rcf1 antiserum, which was considered during analysis in this and the following blots. (B) Chemical crosslink studies were performed as described in (A) with the crosslink reagent SMPB in the presence and absence of either Rcf1 or Rcf2 showed that the crosslink products were independent of Rcf2. (C) Chemical crosslink studies of Rcf1 or Rcf1FLAG with SMPB and BMH showed that all crosslink products of Rcf1 shift only 6 kDa (green lines), indicating that none of the crosslink products are self-crosslinks of a Rcf1 dimer. (D) Crosslinked (SMPB “+”) and non-crosslinked (SMPB “–“) mitochondria were solubilized in 2% digitonin and separated using BN-PAGE. The pattern of the supercomplexes was not affected by the crosslink. (E) As indicated in (D) gel samples containing either supercomplexes (SC) or cytochrome c oxidase (CIV) as well as total mitochondria fraction (T) were loaded on a polyacrylamide gel followed by transfer to a nitrocellulose membrane and immuno decoration. The crosslink pattern shows the same crosslink products in supercomplexes as in cytochrome c oxidase (green arrows). (F) Chemical crosslink studies with SMPB (DMSO as control) using the truncation mutants show interactions of both N- and C-termini with Cox3. The antibody against the FLAG-peptide showed unspecific binding in the control (DMSO), which was considered during analysis. (G) Model of Rcf1 binding to Cox3 of cytochrome c oxidase (adapted from PDB ID: 6HU9). We propose that Rcf1 either directly modulates the active site in Cox1, which is adjacent to Cox3, or Rcf1 modulates the lipid cleft and, thus, the active site through interaction with TM helices of Cox3.
FIGURE 7
FIGURE 7
Model of Rcf1 and Rcf2 modulating cytochrome c oxidase upon metabolic changes. Rcf1 positively modulates cytochrome c oxidase activity while Rcf2 has a negative effect. Upon a high working load of the respiratory chain under non-fermentable conditions (YPG) Rcf1 and Rcf2 shift overproportional into supercomplexes compared to a slight shift of cytochrome c oxidase subunits. Therefore, we speculate that the respiratory chain must be tightly regulated or has higher need of repair under a high working load. Structures are adapted from PDB ID: 6HU9.

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