Ultrastructural analysis of human fibroblasts from HuAtp12p(W94R) patient. Transmission electron micrographs of Epon 60-nm-thick sections of fibroblasts cultured from the HuAtp12p(W94R) patient (panels A and B) and control source (panels C and D). m, mitochondria. Bars, 0.5 μm.

Western blot analysis of wild type and mutant Atp12p in mitochondria. Mitochondria were prepared from yeast cells grown in GAL media as described previously (53) and suspended to 10 mg/ml in 0.3 ml of 20 mm Tris-HCl, pH 8.0, and sodium deoxycholate was added to 0.1% to permeabilize the membranes. An aliquot of total mitochondria (M) was removed, and the rest of the sample was centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant (S) was collected, and the pellet (P) was resuspended to the original volume in the same buffer. Samples of mitochondria (50 μg) and equivalent volumes of the supernatant and pellet fractions were resolved on a 12% SDS-polyacrylamide gel, and following the transfer of proteins to nitrocellulose membranes, Western analysis was done using polyclonal antibody against ScAtp12p (see “Experimental Procedures”). Panel A, isogenic wild type (W303) and ATP12::LEU2 disruption strain (Δatp12). Panel B, Δatp12 bearing single (CEN) or multicopy (2μ) plasmid for wild type or W103R ScAtp12p. Panel C, Δatp12 bearing multicopy plasmid for wild type or W94-substituted HuAtp12p. The migration of Atp12p is shown by the arrowhead. The positions of molecular mass standards are shown on the right.

Western blot analysis of wild type and mutant ScAtp12p in Δatp12Δfmc1. The double mutant Δatp12Δfmc1 was the recipient of plasmids for ScAtp12p. All other aspects of this figure are as described in Fig. 3, panel B. M, mitochondria; S, supernatant; P, pellet.

MD simulations of wild type (WT) and W94R mutant HuAtp12p. Cα-r.m.s.d. (RMSD) values (Å) of each frame at time t with respect to the frame at time t = 0 were calculated for the wrist (residues 33–119) and the palm (residues 120–277) domains. The contribution from the wrist domain was further broken down into residues 33–49, where the largest conformational differences between wild type and W94R occur, and residues 50–119.

HMM Logo for Atp12p. The HMM logo for the Atp12p Pfam domain consists of alternating stacks for match (white) and insert (red) states for all positions in the sequence. The stack height (information content) indicates the sequence conservation at that position, whereas the height of the letters within the stack indicate the relative frequency of each amino acid at that position. The stack width (hitting probability) indicates the frequency of having an amino acid at that position; narrow white stacks denote high probability of amino acid deletion, and wide red stacks denote high probability of amino acid insertion. The stack corresponding to Trp-94 of HuAtp12p is annotated with gray shading.

Behavior of recombinant HuAtp12(W94R) in bacterial cell fractions. Panel A, small scale expressions from pPROEX/Atp12ph(W94R) in bacteria. Transformed cells (JM109 and BL21) were cultured overnight at 30 °C in 2× YT (1.6% bacto tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0), supplemented with ampicillin (50 μg/ml), diluted into 10 ml of fresh media the next morning, and grown to mid-log phase in 50-ml flasks. A sample (1 ml) was removed before induction (BI), after which the cultures were brought to 1 mm isopropyl β-d-thiogalactopyranoside and, where indicated, 2% ethanol, and incubated under the conditions for time and temperature specified for each experiment. Cells were collected by centrifugation and suspended in 1 ml of 20 mm Tris-HCl, pH 7.5; a sample (20 μl) of whole cells (W) was removed, and the remainder was exposed to sonic irradiation to break the cells. The sonicated material was centrifuged at 13,000 × g for 2 min; the supernatant (S) was collected, and the pellet (P) was suspended in 20 mm Tris-HCl, pH 7.5, to the original volume. Aliquots of the cells before induction, and post-induction whole cell, supernatant, and pellet samples were brought to 1× gel loading buffer and applied (10 μl) to 12% SDS-polyacrylamide gels. Gels were stained with Coomassie Blue R-250. The migration of HuAtp12p(W94R) (arrowhead) relative to molecular weight standards (right) is shown. Panel B, large scale expression from pPROEX/Atp12ph(W94R) in bacteria. Transformed JM109 cells were induced with 1 mm isopropyl β-d-thiogalactopyranoside in 2 liters of 2× YT + ampicillin for 8 h at 15 °C. The cells were then collected by centrifugation at 5000 × g for 5 min at 4 °C, suspended in 25 ml of breaking buffer (20 mm Tris-HCl, pH 7.5, 0.2 mg/ml lysozyme, 0.01% phenylmethylsulfonyl fluoride), and broken by passing through a French press three times. Streptomycin sulfate was added (1%) to precipitate DNA, and the broken cells were centrifuged at 100,000 × g for 1 h. The supernatant was filtered through a 0.2-μm membrane, diluted to 80 ml, and applied to a 10-ml TALON® cobalt-chelate resin column (Clontech). The column was washed with 120 ml of 20 mm Tris-HCl, pH 7.5, and eluted with 20 ml of the buffer supplemented with 0.2 m imidazole. Samples of cells before induction (BI) and after breaking (whole cell (W), pellet (P), and supernatant (S)) and the flow-through (FT) and eluate (E) from the cobalt column were analyzed in SDS gels as described for panel A above.

Growth of S. cerevisiae mutants on fermentable and nonfermentable carbons. Δatp12Δfmc1, both untransformed and transformed with either a multicopy or a single copy plasmid for wild type or mutant ScAtp12p (pSc12^2μ, pSc12^CEN, pSc12(W103R)^2μ, pSc12(W103R)^CEN), was tested for growth on glucose (YPD) and nonfermentable ethanol glycerol (EG) plates at 30 or 37 °C. Following overnight growth in liquid YPD, cell cultures were adjusted to A₆₀₀ = 1.0, and then serially diluted by a factor of 2. Five μl of each dilution were applied to plates that were incubated at the indicated temperature for 48 h.

Homology models of wild type (WT) and W94R mutant HuAtp12p. Proteins are colored according to the electrostatic potentials (+1 kT/e, blue; −1 kT/e, red) at the solvent-accessible surface.

Representative conformations of wild type (WT) and W94R mutant HuAtp12p observed in the equilibrium phase (20–40 ns) of an MD simulation at 300 K. Wild type and W94R mutant proteins are shown, respectively, in the upper and lower panels. The leftmost panels show a superposition of the three most representative conformations. The three conformations of residues 33–49 are shown in blue, magenta, and cyan. The other panels show the solvent-accessible surface of the wild type and W94R protein in the three conformations. The surface of a hydrophobic groove in the wrist domain is shown in red. The surface corresponding to Trp-94 or Arg-94 is shown in yellow. The relative contributions of the three conformations to the ensemble in solution in indicated above the surfaces.