Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

Abstract

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.

Figure 1.

Submitochondrial localization and structural organization of the paraplegin–AFG3L2 complex. (A) Submitochondrial localization of paraplegin and AFG3L2 in human primary fibroblasts. Disruption of the mitochondrial outer membrane by digitonin: pellet/mitoplast (lane 1) and supernatant fraction (lane 2); alkaline treatment of mitoplasts, isolating integral membrane proteins (lane 3) and soluble/peripheral membrane proteins (lane 4). (B) Coimmunoprecipitation of paraplegin and AFG3L2. HEK293 mitochondrial lysate was immunoprecipitated with AFG3L2 antibody (lane 1) and paraplegin antibody (lane 3). Precipitates were analyzed by SDS-PAGE and immunostained using paraplegin (lane 1) or AFG3L2 (lane 3) antibodies. Corresponding preimmune sera were used as controls for the specificity of the coimmunoprecipitation (lanes 2 and 4). (C) Gel filtration analysis of mitochondrial extracts from HEK293. Isolated mitochondria are solubilized and fractionated by Superose 6-gel chromatography. Fractions are analyzed by SDS-PAGE and immunostained by antibodies recognizing paraplegin or AFG3L2 (fractions are from two gels: top, fractions 18 and 19, and 20–33; bottom, fractions 18–26 and 27–33) . The peaks of elution for various proteins of known molecular mass are indicated by lines (900 kD, ferritin dimer; 450 kD, ferritin monomer; 232 kD, catalase). (D) Gel filtration analysis of mitochondrial extracts from control and HSP fibroblasts. Fractions are analyzed by SDS-PAGE and immunostained by AFG3L2 antibody (fractions 18–26 and 27–33 are from two gels).

Figure 2.

Mitochondrial depolarization of cells subjected to oxidative stress. Control and HSP fibroblasts are visualized by Rh123 (A) and JC-1 (B) staining.

Figure 3.

Hydrogen peroxide challenge and phenotype rescue. (A) ATP depletion after oxidative stress. Data represent means of six independent experiments. (B) MTT reduction assay, data are expressed as percentage between the absorbance (570 nm) of treated and untreated cells; values, in triplicates, represent means of four independent experiments. (C) HSP and control cells transfections by paraplegin expressing vector pSPG7. Data, in triplicates, are indicated as mean of three independent experiments. *, P < 0.0005; **, P < 0.000001. Bars represent ±SEM.

Figure 4.

Complex I deficiency in HSP fibroblasts. (A) Ratio of cell viability measured in galactose or glucose media; data, two replicates, represent means of three independent experiments. (B) ATP synthesis rates in permeabilized fibroblasts. 1, no substrate (basal activity); 2, pyruvate and l-malate (complexes I, II, III, IV, and V); 3, glutamate and l-malate (complexes I, II, III, IV, and V); 4, rotenone and succinate (complexes II, III, IV, and V); 5, antimycin, ascorbate, and TMPD (complexes IV and V). *, P < 0.05; **, P < 0.001; ***, P < 0.0001. Bars represent ±SEM.

Figure 5.

Blue native gel electrophoresis of mitochondrial complexes. (A) Coomassie brilliant blue staining of mitoplast preparations. (B) Complex I in situ activity staining by nitro blue tetrazolium reduction assay. (C) Rescue of complex I in situ activity in HSP cells after pSPG7 transfection. (D and E) Western blot analysis of complex I revealed by anti–39-kD antibody. Immunoblotting with HSP60 antibody was used to verify equal loading. Activity and protein amount of complex I were quantified by densitometric analysis. *, P < 0.05; **, P < 0.001. Bars represent ±SEM.

Figure 6.

Mitochondrial import and protein synthesis in HSP and control fibroblasts. (A) Mitochondrial protein import efficiency of the 39-kD protein in control and HSP mitochondria (p, precursor; m, mature). (B) Western blot analysis of the 39-kD protein in control and HSP mitoplasts. (C) Mitochondrial protein synthesis in control and HSP cells (CO1, cytochrome c oxidase subunit I; ND2, NADH dehydrogenase subunit 2; COIII, cytochrome c oxidase subunit III; COII, cytochrome c oxidase subunit II; A6, ATPase subunit 6; A8, ATPase subunit 8).

Figure 7.

Integrity of the proteasic site of paraplegin is not essential for complex I rescue. (A) Partial sequence alignment of human and yeast metalloproteases surrounding the divalent metal binding site (boxed area); the glutamic acid residue, E, replaced by glutamine, Q, is indicated. (B) Rescue of complex I in situ activity in HSP cells after pSPG7 and pSPG7^Q575 transfection. (C) Western blot analysis of complex I revealed by anti–39-kD antibody. Immunoblotting with HSP60 antibody was used to verify equal loading. Activity and protein amount of complex I were quantified by densitometric analysis. **, P < 0.001. Bars represent ±SEM.

Figure 8.

Complementation assays of Δyta10Δyta12 yeast cells with paraplegin–AFG3L2 complex. (A) Suppression of the respiratory deficiency of Δyta10Δyta12 yeast cells by paraplegin and AFG3L2 coexpression. Fivefold serial dilutions of yeast cells were spotted onto YP plates containing either 2% glucose (left, YPD) or 3% glycerol (right, YPG). (B) Western blot analysis of paraplegin–AFG3L2 complex on BN-PAGE: the human m-AAA protease has been revealed by c-Myc antibody, recognizing the AFG3L2 fusion protein; the same band was observed in an identical blot decorated by HA antibody, recognizing the paraplegin fusion protein (not depicted). (C) Proteolytic activity of paraplegin and AFG3L2 is required for respiratory competence of Δyta10Δyta12 cells; yeast cells harboring a proteolytic site mutation in paraplegin, AFG3L2, or both were cultured and spotted on YP plates containing 3% glycerol.