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. 2011 Feb 7;192(3):447-62.
doi: 10.1083/jcb.201008177.

Barth syndrome mutations that cause tafazzin complex lability

Steven M Claypool  1 , Kevin Whited, Santi Srijumnong, Xianlin Han, Carla M Koehler
Affiliations

Barth syndrome mutations that cause tafazzin complex lability

Steven M Claypool et al. J Cell Biol. .

Abstract

Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.

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Figures

Figure 1.
Figure 1.
ClustalW alignment of exon 5–deleted human and mouse Taz1p and full-length yeast Taz1p. BTHS mutations that occur at identical and conserved amino acids are indicated. Truncation mutations are indicated with an X above the last amino acid translated in the mutant protein. Gray boxes highlight the conserved acyltransferase motifs A–E; the black box reveals the determined location of the integral interfacial membrane anchor of Taz1p. Asterisks indicate identical residues. Single dots indicate lesser conserved residues, and double dots indicate highly conserved residues.
Figure 2.
Figure 2.
18/21 BTHS mutations result in dysfunctional yeast Taz1p. (A) The relative expression of each of the BTHS mutants was determined from whole-cell extracts by immunoblotting for Taz1p (bottom) with α-ketoglutarate dehydrogenase (KDH) serving as a loading control (top). (B) The relative abundance of MLCL was determined for each strain and is expressed as a percentage of the total phospholipid in each strain (means ± SEM; n = 3). The dashed red line indicates the highest level of MLCL detected in Δtaz1 (WT Taz1). Asterisks indicate significant accumulations of MLCL relative to Δtaz1 (WT Taz1; P < 0.001) as determined by one-way analysis of variance (ANOVA) with Holm–Sidak pairwise comparisons.
Figure 3.
Figure 3.
The BTHS mutant tafazzins localize to and within mitochondria normally. (A) Immunoprecipitation (IP) of Taz1p from the indicated metabolically labeled yeast extracts. The asterisk highlights a nonspecific band. (B) Subcellular fractions were prepared from the indicated yeast strains through a series of differential centrifugations. 25 µg of each fraction was separated by SDS-PAGE and analyzed by immunoblotting using antisera specific for the indicated subcellular organelle. (C) Submitochondrial localization of wt and BTHS mutant tafazzins. Intact mitochondria, mitochondria subjected to osmotic shock (mitoplasts), or mitochondria solubilized with 0.1% TX-100 were incubated alone or in the presence of the indicated concentration of proteinase K. 50 µg/lane wt Taz1p and 100 µg/lane BTHS mutants were resolved by SDS-PAGE and immunoblotted as indicated. For simplicity, only one set of control immunoblots is shown. The controls for every source of mitochondria are provided in Fig. S2. The four BTHS mutants being characterized in the present study are shown in red. The previously characterized matrix-mislocalized BTHS mutant tafazzin is shown in purple. KDH, α-ketoglutarate dehydrogenase. Mito, mitochondria. (A–C) n = 3.
Figure 4.
Figure 4.
The BTHS mutant tafazzins are degraded by the i-AAA protease. (A and B) Steady-state expression was determined from whole-cell extracts derived from the indicated strains by immunoblotting for Taz1p, Yme1p, cytochrome c peroxidase (CCPO), and the loading control, porin. n = 3. (C) Increased half-life of the BTHS mutants in the absence of Yme1p. Whole-cell extracts were harvested after incubation with cycloheximide (CHX) for the indicated times, and the Taz1p remaining was determined by immunoblotting. Yme1p and porin are mitochondrial controls, and hexokinase (Hxk1p) is a cytosolic control. The four BTHS mutants being characterized in the present study are shown in red. Relative molecular masses are shown on the left.
Figure 5.
Figure 5.
The BTHS mutant tafazzins assemble in normal complexes. (A) 1.5% (wt/vol) digitonin extracts from mitochondria derived from the indicated strains were resolved by 2D BN/SDS-PAGE, and Taz1p complexes were detected by immunoblotting. 150 µg Δtaz1 (WT Taz1) and 250 µg Δtaz1 yeast transformed with empty vector or BTHS mutant tafazzin were analyzed. The red arrows highlight a complex only detected in mutant tafazzin extracts. (B) Digitonin extracts from mitochondria derived from the indicated strains were separated by glycerol density gradient centrifugation, and collected fractions (1 = top and 20 = bottom) were immunoblotted for F1-β (top) and Taz1p (bottom). 100% of each BTHS mutant fraction was analyzed versus 40% of Δtaz1 (WT Taz1) fractions. The numbers above the arrowheads indicate the molecular weights of the high molecular weight standards (Stds). n = 3. (C) Endogenous AAC2 was immunoprecipitated from digitonin extracts from the indicated mitochondria. 0.5% of the starting material and final flow through versus 100% of the material remaining attached to the immunoprecipitation beads after washing were immunoblotted for the indicated mitochondrial proteins. exp, exposure. The four BTHS mutants being characterized in the present study are shown in red. (A and C) n = 5.
Figure 6.
Figure 6.
i-AAA protease mutants bind the A88E BTHS mutant, but not wt, tafazzin. (A) Steady-state expression was determined from whole-cell extracts derived from Δtaz1Δyme1 yeast transformed with the indicated Taz1p and Yme1p variants by immunoblotting for Taz1p, Yme1p, and the loading control, porin. (B) Digitonin extracts from mitochondria derived from the indicated strains were subjected to Ni2+ nitrilotriacetic acid (NiNTA) chromatography. The indicated amount of total, TCA-precipitated flow through, and bound material was resolved by SDS-PAGE and immunoblotted as indicated. The asterisk highlights the cross-reaction with porin of the AAC2 antiserum. (C) Digitonin extracts from mitochondria derived from the indicated strains were resolved by 2D BN/SDS-PAGE, and Taz1p complexes were detected by immunoblotting. 150 µg Δtaz1Δyme1 yeast transformed with empty vector or Yme1 mutants and 250 µg Δtaz1Δyme1 (Wt Yme1) were analyzed. The four BTHS mutants being characterized in the present study are shown in red. Stds, molecular weight standards. (A–C) n = 3.
Figure 7.
Figure 7.
Increased function of the A88E and L148H BTHS mutants in the absence of the i-AAA protease. (A and B) The relative abundance of MLCL (A) and CL (B) was determined for each strain and is expressed as a percentage of the total phospholipid in each strain (means ± SEM; n = 5). Student t tests were performed for each construct as expressed in Δtaz1 and Δtaz1Δyme1 yeast strains with significant differences indicated. (C) Comparison of CL in Δtaz1 and Δtaz1Δyme1 yeast strains transformed as indicated. The mass content of each molecular form of CL (acyl chain composition indicated) was determined by multidimensional mass spectrometric array analyses by comparison of the peak intensity of each individual ion to that of the internal standard (means ± SEM; n = 3). The four BTHS mutants being characterized in the present study are shown in red. m/z, mass to charge ratio.
Figure 8.
Figure 8.
Increased assembly, but with protein aggregation, of the A88E and L148H BTHS mutants in the absence of the i-AAA protease. (A) Fractions were prepared from the indicated yeast strains through a series of differential centrifugations. 25 µg of each fraction was separated by SDS-PAGE and analyzed by immunoblotting for the indicated subcellular organelle. (B) Digitonin extracts from mitochondria derived from the indicated strains were resolved by 2D BN/SDS-PAGE, and Taz1p complexes were detected by immunoblotting. The red arrows highlight a complex only detected in mutant tafazzin extracts. 250 µg (Δtaz1 transformed with [A88R], [A88E], [S140R], or [L148H]) and 150 µg (all the rest) were analyzed. Stds, molecular weight standards. (A and B) n = 3. (C) Mitochondria derived from the indicated strains were treated exactly as described in Fig. 3 C. 50 µg/lane of each sample was resolved by SDS-PAGE and immunoblotted as indicated. (D) Solubility of Taz1p in detergents. Mitochondria isolated from the indicated strains were solubilized with digitonin and separated into a supernatant (S1) and pellet (P1) by centrifugation. Nonextracted material (P1) was solubilized with TX-100 and fractionated into a supernatant (S2) and pellet (P2) by centrifugation. Fractions were resolved by SDS-PAGE and immunoblotted as indicated. The asterisk highlights the cross-reaction with porin of the AAC2 antiserum. The four BTHS mutants being characterized in the present study are shown in red. Mito, mitochondria. (B and D) n = 4.
Figure 9.
Figure 9.
The A88R/E, S140R, and L148H BTHS mutants are recognized as defective by the i-AAA protease quickly after import. (A and B) In vitro import of 35S-radiolabeled wt Taz1 or the BTHS mutant Taz1 precursor into wt (A) or Δyme1 (B) mitochondria. The nonimported precursor was removed with trypsin, mitochondria were reisolated, and the OM was ruptured by osmotic shock in the absence or presence of proteinase K (Prot. K). C, control (2.5% of precursor proteins + 100 µg mitochondria). The samples were resolved by SDS-PAGE, the bottom half of the gel was analyzed by phosphoimaging, and the top half was immunoblotted for markers of the OM (Tom70p), membranes facing the IMS (Dld1p), and the matrix (α-ketoglutarate dehydrogenase [KDH]). The percentage of each precursor imported at each time point was determined after phosphoimaging. Significant differences were determined by one-way ANOVA with Holm–Sidak pairwise comparisons and are indicated. Relative molecular masses are shown on the left. (C) Comparison of import into wt versus Δyme1 mitochondria. Significant differences between the percentage of each precursor imported into wt versus Δyme1 mitochondria were determined at each time point by Student t tests, with significant differences indicated. The four BTHS mutants being characterized in the present study are shown in red. Values are means ± SEM (n = 5–7 for import into wt mitochondria, and n = 4 for import into Δyme1 mitochondria).
Figure 10.
Figure 10.
BTHS mutant tafazzin complexes are unstable, and the mutant polypeptide accumulates in aggregates. Mitochondria were harvested from the indicated strains after incubation with CHX for the indicated times. (A) Solubility of Taz1p in detergents was determined as described in Fig. 8 D. The asterisk highlights the cross-reaction with porin of the AAC2 antiserum. For Taz1p immunoblots, 50 µg (Δtaz1 [A88E]) and 20 µg (all the rest) were analyzed. For immunoblots of control proteins (Tom70p, F1-α/β, and AAC2), 20 µg was analyzed. (B) Versadoc-captured images were quantified using the affiliated Quantity One software, and the percentage of Taz1p in TX-100–insoluble aggregates and the percentage of Taz1p solubilized by digitonin were determined (means ± SEM; n = 5). Significant differences were determined by one-way ANOVA with Holm–Sidak pairwise comparisons and are indicated. (C) Digitonin extracts from mitochondria derived from the indicated strains were resolved by 1D BN-PAGE, and immunoblotting was performed for Taz1p, the ATP synthase/complex V (F1-β), and respiratory supercomplexes (Cox2p). The migrations of the Vdimer, Vmonomer, III2IV2, and III2IV supercomplexes are indicated where appropriate. 250 µg (Δtaz1[A88E]) and 150 µg (all the rest) were analyzed. 25 µg mitochondrial protein was additionally resolved by SDS-PAGE and immunoblotted as indicated. n = 3. (D) wt Taz1p assembles with partner proteins (generically denoted A) into final Taz1p complexes that are very stable. In contrast, the characterized BTHS mutant tafazzins have a retarded rate of assembly into final Taz1p complexes that quickly disassemble. The i-AAA protease recognizes both the delayed rate of BTHS mutant Taz1p assembly and the unregulated disassembly of Taz1p complexes. In the absence of the i-AAA protease, disassembled BTHS Taz1 forms aggregates. The BTHS mutants being characterized in the present study are shown in red.
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