Figure 1.
ADP-stimulated oxygen consumption and inhibitor insensitivity of ATPase activity in T. thermophila mitochondria.
(A) Succinate-dependent oxygen consumption in digitonin-permeabilized T. thermophila cells is stimulated upon addition of ADP, consistent with oxidative phosphorylation by a mitochondrial electron transport chain and ATP synthase. Additions to the oxygen electrode reaction chamber are indicated by arrows. Final concentrations were 0.3 µg/µl cell protein, 5 mM succinate, 17 µM ADP. Numbers below the trace give the rate of oxygen consumption in nmoles O2/min/mg protein). (B) and (C) show the concentration dependence of the inhibition of the ATPase activity of isolated T. thermophila mitochondria by oligomycin and sodium azide, respectively. Error bars indicate standard deviation (n = 3). Yeast mitochondrial ATPase activity was inhibited 50% by 1 µM oligomycin and 91% by 1 mM sodium azide (not shown) under these assay conditions.
Figure 2.
BN-PAGE of solubilized T. thermophila mitochondrial membranes.
(A) BN gel (3%–10%) run with T. thermophila mitochondria (1 mg) solubilized with digitonin (5 µg of detergent/µg of protein), and stained with colloidal Coomassie blue. (B) In-gel ATPase staining of a BN gel strip of digitonin-solubilized T. thermophila mitochondria. The gel was incubated overnight (8–12 h) and was briefly (2 min) washed with 10% acetic acid to remove excess lead precipitate on the surface. (C) 2-D BN/BN-PAGE. The first dimension was completed as in (A) and a strip was excised and briefly soaked in cathode buffer containing 0.03% docecyl maltoside (the strip shown here above the 2-D gel is a second strip cut from the same 1-D BN-PAGE that was stained with Coomassie blue; the image of the strip was cropped below the position of band 3). The second dimension was a 4%–12% gradient BN-PAGE run with 0.03% dodecyl maltoside in the cathode buffer (see Materials and Methods). The band 1 (V2, I+III2, II2) separated into two spots designated as spot 1 and 2. The band 2 (V2, I+III2) separated into two spots designated as spot 3 and 4, while band 3 (III2) ran as a single spot, labeled as spot 5. The image of the 2-D gel was cropped on the right side so that most of the material running below band 3/spot 5 is not shown.
Figure 3.
2-D projection maps of dimeric ATP synthase from Tetrahymena thermophila.
(A–E) represent the side view, (F) top view, and (G) intermediate view. The Complexes were extracted either with digitonin (F, G), dodecyl maltoside (A, D, E), or the mixed dataset was used for image analysis (B, C). (E) Dimeric ATP synthase with an additional density next to the c subunit rotor of the left monomer (sum of 64 projections). (H) Interpretation of the projection (B, average of 3,254 projections) with the help of X-ray structure of yeast ATP synthase (PDB accession number 1QO1, [34]). Blue arrowheads (A) mark additional subunits on the extreme left and right positions of the c subunit rotors; green arrowheads (B and C), OSCP subunits; yellow arrowheads (B and D) point to an apparent connection between the domains seen at the extremities of the c rotor (cf., blue arrowheads in (A) and the F1 headpiece); the orange arrowhead (C), to a connection between the F1 part and the matrix exposed domain; and the dark blue arrowhead (E) points to an unknown large extra mass attached to Fo. The bar represents 10 nm and applies to all frames.
Table 1.
Assigned proteins detected in BN-PAGE Spot 1 by LC/MS/MS.
Table 2.
Unassigned proteins detected in BN-PAGE spot 1 by LC/MS/MS.
Figure 4.
Position of arginines in putative TM6 of Ymf66 and possible alignment with the region of ATP synthase subunits a that contains the conserved Arg.
(A) Prediction of transmembrane helices and topology by TMHMM (similar results are obtained with other algorithms). The predicted extent of transmembrane segments is indicated by red bars above the plotted probability scores; the sixth transmembrane segment containing two Arg residues is colored dark red with a star at the position of the second Arg. (B) An alignment of Ymf66 transmembrane helix 6 with the region of ATP synthase subunits a that contains an essential conserved Arg. Residues that are identical to or chemically similar to the consensus amino acid are shown with reverse green coloration; the conserved Arg is shown in red and highlighted with yellow background, and the second partially conserved Arg is shown in dark red with yellow-green background. The extent of known (E. coli) and predicted (Ymf66) transmembrane helices is indicated by dark red bars. Sequence data: E. coli, gi:16131606; B. taurus, gi:60101830; A. thaliana, gi:6851018, R. americana, gi:2258385; T. brucei, gi:343544; T. thermophila, gi:15027631; P. Aurelia, gi:8928578.
Figure 5.
Phylogenetic trees inferred for subunits γ and c and alignment of the interface regions of subunits δ and c.
(A and B) show trees inferred by Bayesian analysis (MrBayes [76], see Methods) for subunit γ and subunit c, respectively. Numbers near branch nodes indicate Bayesian posterior probabilities/maximum likelihood bootstrap support (200 replicates) (maximum likelihood analysis employed PhyML [80] [see Methods] indicates a maximum likelihood support of less than 50%; dt indicates a different branch topology was supported by the maximum likelihood analysis). Branches with less than 0.5 posterior probability have been collapsed to a common node. Sequences from α-proteobacterial spp. were included to provide a root, but turned out not to compose the most divergent clade in each analysis; the trees are nevertheless shown as rooted by the bacterial clade. Major taxonomic groups are indicated by color shading (it should be kept in mind, however, that gene product trees can differ from species trees via a number of biological mechanisms, as well as methodological and statistical error). The bar at the lower left provides the scale of substitutions per site. (C) compares the interface regions of subunits δ (denoted ε in prokaryotes) and c from selected prokaryotic and eukaryotic spp. Amino acid residues determined to be critical for interaction by site directed mutagenesis [49] are shown in dark red with a yellow background. Highly conserved residues are dark red on a light green background; those that are identical to or chemically similar to the consensus amino acid are shown with reverse coloration on a green background. (Consensus residues were calculated in Jalview 2.4 [96] using alignments of representative eukaryotic spp. from a broad range of taxa, but omitting ciliates. Consensus is not indicated for two positions of subunit δ that have a very low degree of conservation). Residues of subunit ε that were shown to be in proximity to the loop region of subunit c by cross-linking in the E. coli complex [48],[97] are enclosed in boxes. Basic (positive) residues are colored red, and acidic (negative) residues blue. Positions of secondary structural elements as found in high resolution structures of bacterial subunits [98],[99] are indicated above the alignment. Ciliate species names are shown with red-shaded lettering and P. falciparum, another alveolate, with blue lettering.