Two human patient mitochondrial pyruvate carrier mutations reveal distinct molecular mechanisms of dysfunction

Abstract

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.

Keywords: Carbohydrate metabolism; Cell Biology; Metabolism; Mitochondria; Transport.

Figure 1. Patient fibroblasts contain MPC1 mutations on highly conserved residues and express aberrant MPC1 and MPC2 levels.

(A) Schematic of MPC1 transcript indicating location of patient mutations. The c.C289T mutation produces 2 mRNAs shown as red arrows: a full-length transcript (R97W), and a truncated transcript (A58GfsX2). The c.T236A mutation coding for L79H is shown as a blue arrow. Sequence alignments show evolutionary conservation across multiple species. Two transmembrane regions were predicted using transmembrane helix prediction (TMHMM). MPC1 point mutations are marked by *, and truncated MPC1 mutant is indicated with #. (B) Representative MPC1, MPC2, and VDAC levels visualized by immunoblot of immortalized patient fibroblasts from Control, c.C289T, or c.T236A mutants (n = 3). (C and D) Quantification of relative MPC1 (C) and MPC2 (D) protein levels relative to VDAC in patient fibroblasts c.C289T and c.T236A as compared with WT (n = 3). (E and F) Relative MPC1 (E) and MPC2 (F) mRNA levels in patient fibroblasts c.C289T and c.T236A as compared with WT (n = 3). Data are presented as mean ± SEM. One-way ANOVA was performed for C–F (^##P ≤ 0.01, ^###P ≤ 0.001, ns = not significant; see also Supplemental Figure 1).

Figure 2. Generation and characterization of MPC1-knockout cell lines (ΔMpc1).

(A) Schematic displaying CRISPR sgRNA locations used to generate ΔMpc1 cell lines. (B) Representative MPC1, MPC2, and actin levels visualized by immunoblot in WT C2C12 following transduction with an empty vector (WT) or sgRNAs targeting Mpc1 (ΔMpc1) (n = 3). (C and D) Quantification of relative MPC1 (C) and MPC2 (D) protein levels relative to actin in WT and ΔMpc1 cells (n = 3). (E) [2-¹⁴C]-radiolabeled pyruvate uptake of mitochondria isolated from WT and ΔMpc1 cells (n = 4). (F and G) Respiration driven by 10 mM pyruvate (F) and 10mM glutamine (G) in WT and ΔMpc1 cells (n = 12, 3 clonal lines/genotype × 4 replicates/clonal line). Pyr, pyruvate; UK, UK5099; ROT, rotenone; FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. Data are presented as mean ± SEM. Two-tailed unpaired t test was performed for C–G (^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001; see also Supplemental Figure 2).

Figure 3. MPC1 C-terminal truncation causes MPC-complex instability and dysfunction.

(A) Representative MPC1, MPC2, and actin levels visualized by immunoblot in WT C2C12 transduced with a guideless Cas9 vector and subsequently with an empty vector (WT+EV) compared ΔMpc1 cells complemented with empty vector (EV), WT human MPC1 (WT), or MPC1 mutants C-terminally truncated by 3 (ΔC3), 9 (ΔC9), 12 (ΔC12), or 18 amino acids (ΔC18) (n = 3). (B and C) Quantification of relative MPC1 (B) and MPC2 (C) protein levels relative to actin, statistics versus ΔMpc1+WT (B) or WT+EV (C) (n = 3). (D) Respiration driven by 10 mM pyruvate of cell lines described in A. Letters represent significant differences in FCCP-stimulated respiration among ΔMpc1+EV, ΔMpc1+ΔC12, ΔMpc1+ΔC18 (a–c; P ≤ 0.001) versus WT+EV, and in UK5099-inhibited respiration among ΔMpc1+EV, ΔMpc1+WT, ΔMpc1+ΔC12, ΔMpc1+ΔC18 (d–g; P ≤ 0.001), and ΔMpc1+ΔC3, ΔMpc1+ΔC9 (h and i; P ≤ 0.01) versus WT+EV (n = 9; 3 clone lines/genotype × 3 technical replicates/clone line). (E) Quantification of FCCP-stimulated normalized to basal pyruvate-driven respiration by complemented ΔMpc1 cell lines as compared with WT+EV (n = 9; 3 clone lines/genotype × 3 technical replicates/clone line). Data are presented as mean ± SEM. One-way ANOVA was performed for B–E (^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001; see also Supplemental Figure 3).

Figure 4. MPC1 patient mutations display varied MPC complex stability and activity.

(A) Representative MPC1, MPC2, and VDAC with Actin levels visualized by immunoblot in WT C2C12 cells transduced with guideless Cas9 vector and subsequently with an empty vector (WT+EV) compared with ΔMpc1 cells complemented with an empty vector (EV), WT human MPC1 (WT), or MPC1 mutants (L79H, R97W, and A58G) (n = 3). (B and C) Quantification of MPC1 (B) and MPC2 (C) protein levels relative to Actin, statistics versus ΔMpc1+WT (B) or WT+EV (C) (n = 3). (D) Respiration driven by 10 mM pyruvate of cell lines described in A. Letters represent a significant difference in FCCP-stimulated respiration among ΔMpc1+EV, ΔMpc1+L79H, ΔMpc1+A58G (a–c; P ≤ 0.001) and ΔMpc1+WT, ΔMpc1+R97W (d and e, P ≤ 0.05) versus WT+EV, and in UK5099-inhibited respiration among ΔMpc1+EV and ΔMpc1+L79H, ΔMpc1+A58G (f–h; P ≤ 0.01) versus WT+EV (n = 12; 2 clone lines/genotype × 6 technical replicates/clone line). (E) Quantification of FCCP-stimulated normalized to basal pyruvate-driven respiration by complemented ΔMpc1 cell lines as compared with WT+EV (n = 12; 2 clone lines/genotype × 6 technical replicates/clone line). Data are presented as mean ± SEM. One-way ANOVA was performed for B–E (^#P ≤ 0.05, ^##P ≤ 0.01, ^###P ≤ 0.001; see also Supplemental Figures 4 and 5).

Figure 5. MPC1 patient mutations affect MPC activity.

(A) [2-¹⁴C]-radiolabeled pyruvate uptake of mitochondria isolated from WT (WT+EV) and ΔMpc1 cells complemented with an empty vector (EV), human MPC1 (WT), or MPC1-L79H mutant (L79H) (n = 4). (B) Respiration driven by 10 mM pyruvate in WT C2C12 cell lines complemented with an empty vector (EV), WT human MPC1 (WT), or MPC1 mutants (L79H, R97W, and A58G) (N = 6; parental polyclonal line × 6 technical replicates). (C) Quantification of FCCP-stimulated normalized to basal pyruvate-driven respiration by complemented WT cell lines (n = 6; parental polyclonal line × 6 technical replicates). (D) Quantification of MPC1 (R97W) and MPC1 (A58G) mRNA in patient fibroblasts harboring the c.C298T MPC1 mutation (n = 3). Data are presented as mean ± SEM. One-way ANOVA was performed for A–D (^#P ≤ 0.05, ^###P ≤ 0.001, ns = not significant; see also Supplemental Figures 6 and 7).

Figure 6. The MPC1(L79H)-MPC2 interaction is weaker than MPC1(WT)-MPC2.

(A) Representative MPC1, MPC2, FLAG, and HA levels visualized by immunoblot of input, immune-depleted (depleted), wash, and eluate fractions from coimmunoprecipitation of MPC1 proteins (L79H-FLAG or MPC1-HA) precipitated using FLAG or HA antibody in the ΔMpc1 cell line expressing both MPC1-HA and L79H-FLAG (n = 3). Quantification of relative MPC1 (B) and MPC2 (C) levels in eluates from L79H-FLAG and MPC1-HA pull-downs (n = 3). Data are presented as mean ± SEM. Two-tailed unpaired t test was performed for B and C (^#P ≤ 0.05, ns = not significant; see also Supplemental Figure 8).