Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

Abstract

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.

Keywords: ATP synthase; Trypanosoma brucei; mitochondria; proteomics; protozoan.

Figure 1

Schematic depicting the structure and function of a typical mitochondrial F₁F_oATP synthase complex. A schematic showing ATP synthase subunit composition in yeast, with a comparison to the subunits found in the Euglena gracilis structure and their location. ATP synthesis in the F₁ catalytic head (blue) is coupled to proton translocation through the membrane-embedded F_o portion (red). The protons are translocated between subunit a and the c-ring, driving the rotation of the c-ring and the central rotor, made up of subunits γ, ε, and δ. Asymmetric interactions of subunit γ during this rotation force conformational changes in catalytic subunits α and β and thereby induce ATP synthesis. Futile rotation of the α/β headpiece is prevented by OSCP and the peripheral stalk, which holds the external parts of the F₁ moiety stationary. The subunits labeled on the scheme, along with proton-translocating subunit a, are found in all eukaryotic lineages, with the exception of p18, which is exclusively found in Euglenozoa. Schematic created with BioRender.com. IMS, intermembrane space; OSCP, oligomycin sensitivity–conferring protein.

Figure 2

Tb927.8.3070 is an integral mitochondrial inner membrane protein that interacts with the F₁F_oATP synthase complex.A, IF image of a procyclic Trypanosoma brucei cell line allowing tetracycline-inducible expression of Tb927.8.3070-myc. ATOM40 serves as a mitochondrial marker. DAPI marks both nuclear and mitochondrial DNA. The scale bar represents 10 μm. Cells were induced overnight. B, subcellular fractionation of cells expressing Tb927.8.3070-myc. Left panel, immunoblot analysis of whole cell lysates (WCLs) and digitonin-extracted mitochondria-enriched (P1) and soluble cytosolic (S1) fractions of cells expressing Tb927.8.3070-myc. The immunoblots were probed with anti-myc antibodies and antisera against VDAC and elongation factor 1-alpha (EF1a), which serve as markers for mitochondria and cytosol, respectively. Right panel, digitonin-extracted mitochondria-enriched fractions (P1) were subjected to alkaline carbonate extraction performed at pH 11.5 resulting in membrane-enriched pellet (P2) and soluble supernatant (S2) fractions. Subsequent immunoblots were probed with anti-myc and antisera against VDAC and cytochrome c (cyt c), which serve as markers for integral and peripheral membrane proteins, respectively. C, volcano plots of a SILAC–IP analysis of crude mitochondrial extracts from Tb927.8.3070-myc–expressing cells. Cells were induced with tetracycline for 1 day. Proteins were quantified in three independent biological replicates, with the mean log₂ of ratios (with/without Tet) plotted against the −log₂p value (two-sided t test). The bait is shown in red. The horizontal dashed line shows a significance level of p = 0.05. The vertical dotted lines mark specified enrichment factors. Left panel, the following groups of proteins are highlighted: mitochondrial IM proteins (IM, black), other proteins of the mitochondrial importome (Mito, black with a white center), and non mitochondrial proteins (non mito, gray). Right panel, the following groups of proteins are highlighted: ATP synthase F_o subunits (F_o, orange) and F₁ subunits (F_1,green). ATOM40, atypical translocase of the outer membrane 40; DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; IM, inner membrane; SILAC–IP, stable isotope labeling by amino acids in cell culture–immunoprecipitation; VDAC, voltage-dependent anion channel.

Figure 3

Tb927.8.3070 is an essential protein and comigrates with the F₁F_oATP synthase monomer.A, growth curve analysis of a procyclic Tb927.8.3070-RNAi cell line. Analysis was performed in triplicate, with error bars showing standard deviation (too small to be visible). Inset, Northern analysis after 2 days of induction of the level of Tb927.8.3070 mRNA. EtBr-stained rRNAs act as a loading control. B, left panel, BN-PAGE analysis of crude mitochondrial extracts of Tb927.8.3070-RNAi cells. ATP synthase complexes were visualized with a polyclonal antibody against ATP synthase F₁ subunit p18. A section of the Coomassie-stained gel serves as a loading control. Right panel, quantification of the BN-PAGE using ImageJ. Three or four biological replicates were analyzed. p Values were calculated using a one-way ANOVA with Dunnett’s multiple comparison post hoc test. C, growth curve analysis of the Tb927.8.3070-RNAi 3′ UTR cell line expressing Tb927.8.3070-myc. The RNAi construct in this cell line targets the 3′ UTR of Tb927.8.3070 and therefore allows reexpression of the protein in a different genomic context. Analysis was performed in triplicate, with error bars showing standard deviation. Inset, Northern analysis after 2 days of induction of the level of Tb927.8.3070 mRNA and the corresponding immunoblot analysis of the Tb927.8.3070-myc levels. EtBr-stained rRNA species and EF1a act as a loading control. D, BN-PAGE analysis of crude mitochondrial extracts from uninduced and induced cells used in C. Tb927.8.3070-myc and ATP synthase complexes were visualized either with anti-myc or anti-p18. A section of the Coomassie-stained gel serves as a loading control. BN-PAGE, blue native PAGE; EF1a, elongation factor 1-alpha.

Figure 4

Tb927.8.3070 shows similarities to F₁F_oATP synthase subunit b.A, a schematic showing a comparison of domains predicted in Tb927.8.3070 and the homologs of ATP synthase subunit b in spinach chloroplasts (atpF), yeast (ATP4), and Bacillus species (atpF). The regions of secondary structure homology as determined by HHpred are indicated by the colored lines. TMDs were predicted by HMMTOP and TMPred (86, 87). The yeast ATP4 mitochondrial targeting sequence (MTS) was previously defined experimentally (88). B, an alignment of the protein sequences of Tb927.8.3070 and Euglena gracilis subunit b using the EMBOSS Water Pairwise Sequence Alignment tool (89). TMD, transmembrane domain.

Figure 5

Tb927.8.3070 depletion selectively depletes F_oATP synthase subunits.A, volcano plots of a SILAC-based quantitative MS analysis of crude mitochondrial extracts of uninduced and induced Tb927.8.3070-RNAi cells. Cells grown in SILAC media were harvested after 4 days of induction. Proteins were quantified in three independent biological replicates, with the mean log₂ of normalized (norm.) ratios plotted against the −log₂p value (two-sided t test). Tb927.8.3070, the RNAi target, was not detected in this analysis. The horizontal dashed line indicates a significance level of p = 0.05. The vertical dotted lines mark proteins with a 1.5-fold change in abundance compared with control cells. The following groups of proteins are highlighted: ATP synthase F_o subunits (F_o, orange) and F₁ subunits (F_1,green). B, as in A, but proteins downregulated more than 1.5-fold are labeled with either their name or accession numbers. MS, mass spectrometry; SILAC, stable isotope labeling by amino acids in cell culture.

Figure 6

Tb927.8.3070 knockdown affects mitochondrial physiology.A, ATP production analysis of crude mitochondrial lysates from uninduced and 3 days induced Tb927.8.3070-RNAi cells. Oxidative phosphorylation or substrate-level phosphorylation was induced by the addition of either succinate, α-ketoglutarate (α-KG), or pyruvate plus succinate (pyruvate) as substrates. Atractyloside (ATR), an inhibitor of the ATP/ADP translocator, selectively prevents mitochondrial ATP production. Three biological replicates were analyzed. The p value was calculated by an unpaired t test. The mean of ATP production detected in crude mitochondrial lysates of uninduced cells treated with the respective substrate was set to 100%. B, measurement of ΔΨm in the uninduced and induced Tb927.8.3070-RNAi cell line. Analysis was performed by measuring TMRE fluorescence using flow cytometry. Three biological replicates were analyzed per time point. The p value was calculated using an unpaired t test. The average TMRE fluorescence of the uninduced cell line was set to 100%. Addition of FCCP, a ΔΨm uncoupler, acts as a negative control. C, measurement of mitochondrial superoxide production in uninduced and induced Tb927.8.3070-RNAi cell lines. Analysis was performed by measuring MitoSOX fluorescence using flow cytometry. Three biological replicates were analyzed per time point. The p value was calculated using an unpaired t test. The average MitoSOX fluorescence of the uninduced cell line was set to 100%. D, measurement of cellular reactive oxygen species production in the uninduced and induced Tb927.8.3070-RNAi cell line. Analysis was performed by measuring CM-H₂DCFDA fluorescence using flow cytometry. Three biological replicates were analyzed per time point. The p value was calculated using an unpaired t test. The average CM-H₂DCFDA fluorescence of the uninduced cell line was set to 100%. E, representative IF image of Tb927.8.3070-RNAi cells induced for 4 days. Top panel, DAPI marks both nuclear and mitochondrial DNA. Bottom panel, ATOM40 serves as a mitochondrial marker. The scale bar represents 10 μm. F, analysis of cell cycle progression upon depletion of Tb927.8.3070 by RNAi. Left panel, the visualization of DAPI-stained cells (n > 130 cells per time point). Right panel, the quantification of PI fluorescence using flow cytometry. The proportion of cells found in each cell cycle stage are shown as a percentage of the total population. Flow cytometry analysis was performed in three biological replicates. The average percent value here is presented with error bars showing standard deviation. ATOM40, atypical translocase of the outer membrane 40; CM-H₂DCFHDA, chloromethyl derivative of H₂DCFHDA; DAPI, 4′,6-diamidino-2-phenylindole; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; IF, immunofluorescence; PI, propidium iodide; TMRE, tetramethylrhodamine ethyl ester.