Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION

Abstract

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (>85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high.

Keywords: confocal microscopy; energy metabolism; live cell imaging; mitochondria; mitochondrial disease; mitochondrial dynamics; mitochondrial transport.

FIGURE 1.

Modeling the novel compound heterozygous mutation in human PiC using yeast. A, steady-state protein expression and growth analyses of PiC variants and humanized PiC in comparison with WT. Immunoblotting for Pic1p and the loading controls Tom70p and Atp2p was done using yeast whole cell extracts. B, 1:4 dilutions of the same set of strains were tested for functional growth using the indicated media. Plates were incubated at 30 or 37 °C for 3 days except for YPEG at 37 °C, which was cultured for 10 days. C, steady-state protein expression and growth analyses of WT PIC, PIC^L200W variant, and PIC^Hum in the presence or absence of co-expressed PIC^QIP as in A. D, 1:4 dilutions of the same set of strains were tested for functional growth using the indicated media at the designated temperature. Plates were incubated for 3 days (YPD at 30 °C), 5 days (YPD at 37 °C and YPEG at 30 °C), or 13 days (YPEG at 37 °C). E, quantification of PIC or PIC^L200W protein amounts at 37 °C in the presence or absence of PIC^QIP (n = 9). Data are presented as means; error bars represent S.E. n.s., differences not significant. EV, empty vector; YPD, 1% yeast extract, 2% peptone, 2% dextrose; YPEG, 1% yeast extract, 2% peptone, 1% ethanol, 3% glycerol.

FIGURE 2.

Lower PiC protein expression in primary fibroblasts harboring the compound heterozygous PiC mutation. A, mRNA, as assessed by quantitative PCR analysis, of isoforms A and B was unchanged by the PiC mutant. Ctrl data were obtained from fibroblasts from three normal individuals; PiC mutant data were obtained from two different passages of the fibroblasts expressing the compound SLC25A3 mutation. As controls for identification of isoform A and B mRNA, mRNA was isolated from primary human skeletal myoblasts and myotubes (upper panels); myotubes were expected to show up-regulated PiC-A compared with myoblasts. B, left panels, example of an immunoblot from PiC mutant fibroblasts and Ctrl fibroblasts from two different individuals. Arrow, band used to quantify PiC abundance. Right panel, quantification from Ctrl fibroblasts from four normal individuals (each tested at three different passages) and from five separate passages (less than passage 10) of the PiC mutant fibroblasts. For each genotype, band intensity was first normalized to that of α-tubulin. Data are presented as means, and error bars represent S.E. except for the skeletal muscle cells, which are each from a single sample. C-I, C-II, and C-III, complexes I, II, and III; Cyto-c and cyt c, cytochrome c.

FIGURE 3.

Slower proliferation rate in primary skin fibroblasts harboring the compound heterozygous PiC mutation. Cells tested in glucose plus glutamine had been growing in that substrate condition for two to three passages. Cells tested in β-hydroxybutyrate (BHB) plus glutamine (no glucose) were switched to that condition the day before cell counting was started. DMEM + 10% FBS was used as the base medium throughout. n = 3–6 growth curves/genotype derived from cells at different passages (less than passage 10). In the 25 mm glucose and no glucose conditions, Ctrl cells were from two different subjects (n = 3/subject). In the 5 mm glucose condition, Ctrl cells were from one subject. Data are presented as means; error bars represent S.E. Doubling time was significantly longer in PiC mutant cells; p values were calculated using an unpaired t test. fibro, fibroblasts.

FIGURE 4.

Suppressed bioenergetics in intact but not permeabilized PiC mutant skin fibroblasts. A, raw O₂ traces from a single experiment where a Ctrl line and the PiC mutant cells were studied in parallel. Cells were grown and the experiment was performed in 5 mm glucose + 4 mm glutamine as substrates. Values are means ± S.D. with six technical replicates each. Oligomycin (Oligo) was injected to inhibit ATP synthase to measure leak-dependent respiration. Capacity of the electron transport chain under the prevailing substrate condition was determined using the chemical uncoupler FCCP. Finally, antimycin was injected to inhibit the electron transport chain to reveal non-mitochondrial O₂ consumption. Note that protein abundance was similar for Ctrl and PiC mutant cells (∼10% higher for mutant cells). B and C, all values are mitochondrial O₂ consumption rates calculated by subtracting the O₂ consumption rate measured after antimycin. The basal rate is the rate measured in the absence of inhibitors or uncoupler. Cells were grown in the indicated substrate conditions for two to three passages before testing in those conditions. p values are as follows: ***, p < 0.01 from Tukey post hoc test after two-way ANOVA (p = 0.011 for genotype × JO₂ condition interaction); **, p = 0.03 from two-way ANOVA (genotype × JO₂ condition interaction not significant; p = 0.03 for genotype main effect; p < 0.001 for JO₂ condition main effect). C, cells were cultured in the indicated media and then preincubated in those media for 45 min in 0 CO₂ before being switched to intracellular medium containing 10 mm pyruvate, 2.5 mm malate, saturating ADP, and a permeabilizing reagent (see “Experimental Procedures” for further details). Permeabilization was confirmed by showing robust succinate-driven O₂ consumption; succinate does not cross the plasma membrane (not shown). Data were normalized to total cellular protein from parallel plates of intact cells. n = 3/genotype from cells at different passages (less than passage 10). In B and C, data are presented as means, and error bars represent S.E. For the 5 mm glucose condition, n = 4/genotype from cells at different passages (less than passage 10). For the 25 mm glucose condition, four separate Ctrl fibroblast lines were tested, each in triplicate (three different passages); triplicates were averaged. Data from PiC mutant cells were split into parallel cultures for the different controls and then tested at three different passages each. Thus, n = 4/genotype for 25 mm glucose.

FIGURE 5.

Acute PiC depletion in HeLa cells is still associated with substantial phosphorylating O₂ consumption. A, immunoblots from HeLa cells exposed for 72 h to one of two different siRNA duplexes targeting PiC or scrambled controls. For each genotype, bands were normalized to either succinate dehydrogenase subunit A (SDHA) or α-tubulin. n = 3/genotype. B, growth curves in HeLa cells exposed to siRNA for 72 h and grown in DMEM + 25 mm glucose + 4 mm glutamine. p values (doubling times) are from an unpaired t test. n = 3/genotype. C, mitochondrial bioenergetics measured in cells exposed to one of two different siRNA duplexes for 72 h, grown and tested in either DMEM + 25 mm glucose + 4 mm glutamine (Intact), or permeabilized and supplied with pyruvate and malate (Permeabilized). See “Experimental Procedures” and Fig. 4 for further details. n = 3–5/genotype. D, immunoblots showing lesser depletion of PiC in HeLa cells exposed to siRNA for 48 h. Cells were grown in DMEM + 25 mm glucose + 4 mm glutamine, and mitochondrial bioenergetics were measured in that medium (Intact) or subsequently permeabilized and supplied with pyruvate and malate (Permeabilized). See Fig. 4 for further details. n = 3–5/genotype. In all panels, data are presented as means, and error bars represent S.E. For the immunoblots, p values are as follows: ***, p < 0.01; **, p = 0.02; *, p < 0.046 (unpaired t test). For the JO₂ measurements, p values are as follows: ***, p < 0.001; *, p = 0.035 (Tukey post hoc tests following two-way ANOVA with significant genotype × JO₂ condition interaction). Two different siRNA duplexes were used (kd1 and kd2), and controls were run with each (Ctrl 1 and Ctrl 2). Oligo, oligomycin; C-I, C-II, and C-III, complexes I, II, and III; cyto-c, cytochrome c.

FIGURE 6.

Mitochondrial morphology in primary PiC mutant fibroblasts and PiC-deficient HeLa cells. Mitochondrial morphology was scored as follows: fragmented, mainly small and round; partly fragmented, intermediate, mixture of round and shorter tubulated; normal, tubulated, long and higher interconnectivity; elongated, very long, tubulated. The percentage of cells with the indicated mitochondrial morphologies was determined as a percentage of the total number of DsRed-transfected cells counted (≥18 cells per experiment; n = 4 independent experiments). A, representative figures of the different mitochondrial forms in Ctrl and PiC mutant fibroblasts. B, distribution of the different type of mitochondria in primary fibroblasts. C, distribution of the different types of mitochondria after PiC silencing in HeLa cells. Scr., scrambled siRNA; PiC kd1 and kd2, two different PiC-specific duplexes.

FIGURE 7.

Mitochondrial continuity and fusion activity in primary fibroblasts. A, image time series showing a representative fibroblast before and after 2P photoactivation of mtPA-GFP (white squares) at 0 s (first step of the imaging), 24 s (first step directly after photoactivation), 40 s (the mtPA-GFP diffusion within the first 16 s of photoactivation), and 500 s (further spreading after 8 min). A region of interest for each condition (dashed white line) is magnified and shown as PA-GFP only (green) images for each time point. B, the time course of the ratio of F_mtPA-GFP and F_mtDsRed for the RPA (above) and the decay of the fluorescence ratio in the RPA at 40 and 500 s (below). Asterisks indicate significant differences (p < 0.05). C, fusion event rate in primary fibroblasts. D, average fusion times. E, distribution of different fusion types (left) and orientation (right). F–I, HeLa cells treated with scrambled siRNA or kd1 or kd2 duplexes targeting PiC. F, the time course of the normalized F_mtPA-GFP for the region of photoactivation (RPA) (left) and the decay of the fluorescence ratio in the RPA at 500 s (right). Asterisks indicate significant differences (p < 0.05). Scr., scrambled siRNA-silenced cells; PiC kd1 and kd2, PiC siRNA-treated cells. G, fusion event rate in silenced HeLa cells. H, average fusion times in silenced HeLa cells. I, distribution of different fusion types (left) and orientation (right) in silenced HeLa cells. In B, C, D, F, G, and H, error bars represent S.E.

FIGURE 8.

Abundance of mitochondrial fusion and fission proteins in PiC-deficient primary fibroblasts and HeLa cells. A, Western blotting of the main fusion and fission proteins in the Ctrl and PiC-deficient fibroblasts. Left, representative blots; right, normalized protein abundance of the percentage of the average protein levels of control fibroblast. B, OPA1 isoforms. FCCP (5 μm) was used as a positive control to show the mitochondrial uncoupling-induced loss of the long form of OPA1. C, Western blotting of the main fusion and fission proteins in HeLa cells. Scr., scrambled siRNA-silenced cells; PiC kd, PiC siRNA-treated cells. Left, representative blots; right, normalized protein abundance of the percentage of the average protein levels of scrambled siRNA-silenced cells. In A and C, error bars represent S.E.