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. 2013 Feb 1;22(3):483-92.
doi: 10.1093/hmg/dds447. Epub 2012 Oct 24.

Seven functional classes of Barth syndrome mutation

Kevin Whited  1 , Matthew G Baile, Pamela Currier, Steven M Claypool
Affiliations

Seven functional classes of Barth syndrome mutation

Kevin Whited et al. Hum Mol Genet. .

Abstract

Patients with Barth syndrome (BTHS), a rare X-linked disease, suffer from skeletal and cardiomyopathy and bouts of cyclic neutropenia. The causative gene encodes tafazzin, a transacylase, which is the major determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, CL. In addition to numerous frame shift and splice-site mutations, 36 missense mutations have been associated with BTHS. Previously, we established a BTHS-mutant panel in the yeast Saccharomyces cerevisiae that successfully models 18/21 conserved pathogenic missense mutations and defined the loss-of-function mechanism associated with a subset of the mutant tafazzins. Here, we report the biochemical and cell biological characterization of the rest of the yeast BTHS-mutant panel and in so doing identify three additional modes of tafazzin dysfunction. The largest group of mutant tafazzins is catalytically null, two mutants encode hypomorphic alleles, and another two mutants are temperature sensitive. Additionally, we have expanded the defects associated with previously characterized matrix-mislocalized-mutant tafazzins to include the rapid degradation of aggregation-prone polypeptides that correctly localize to the mitochondrial IMS. In sum, our in-depth characterization of the yeast BTHS-mutant panel has identified seven functional classes of BTHS mutation.

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Figures

Figure 1.
Figure 1.
Every BTHS mutant still co-fractionates with mitochondria. Fractions were prepared from Δtaz1 yeast transformed with the indicated BTHS-mutant tafazzin through a series of differential centrifugations. Twenty-five micrograms of each fraction were separated by SDS-PAGE and analyzed by immunoblot using antisera specific for the indicated subcellular organelle.
Figure 2.
Figure 2.
The majority of BTHS missense mutations disrupt tafazzin transacylase activity. (A) The relative expression of each of the BTHS mutants was determined in isolated mitochondria (20 μg) by fluorescent immunoblotting for Taz1p (top panel); Porin, Aac2p and Aco1p served as loading controls (lower panels). (B) The quantified band intensities are expressed as the ratio of the Taz1p signal in Δtaz1 [WT] (mean ± SEM, n = 3). Asterisks indicate significantly lower expression relative to Δtaz1 [WT] (P ≤ 0.001). Lyso-PC → PC (C) and MLCL → CL (D) transacylase activities were determined for each of the BTHS mutants (mean ± SEM, n = 3). Unless indicated as not significant (NS), all differences are significant relative to Δtaz1 [WT] (P ≤ 0.001). In B–D, the dashed green line indicates the value observed in Δtaz1 [WT] and the dashed red line indicate that measured in WT [VA] (WT yeast transformed with vector alone). (E) Submitochondrial localization of WT and BTHS-mutant tafazzins. Intact mitochondria, mitochondria subjected to osmotic shock (mitoplasts) or mitochondria solubilized with 0.1% Triton X–100 were incubated in the presence of the indicated concentration of proteinase K, resolved by SDS-PAGE and immunoblotted as indicated. n = 3. The controls for every source of mitochondria are provided in Supplementary Material, Fig. S1. (F) 1.5% (wt/vol) digitonin extracts from mitochondria derived from the indicated strains were resolved by 2D blue native/SDS-PAGE and Taz1p-complexes detected by immunoblot. n = 3.
Figure 3.
Figure 3.
Aggregation-prone mutants with low-fidelity submitochondrial sorting. (A) Taz1p expression was determined in whole cell extracts derived from the indicated strains by immunoblotting for Taz1p, Yme1p, cytochrome c peroxidase (Ccp1p), and the loading control, Aac2p. n = 3 (B) Submitochondrial localization of WT and BTHS-mutant tafazzins in the presence and absence of Yme1p. n = 3. The controls for every source of mitochondria are provided in Supplementary Material, Fig. S2. (C and D) Solubility of Taz1p in detergents. Mitochondria isolated from the indicated strains were solubilized with digitonin and separated into a supernatant (S1) and pellet by centrifugation. Non-extracted material was solubilized with TX–100 and fractionated into a supernatant (S2) and pellet (P2) by centrifugation. (C) Fractions were resolved by SDS-PAGE and immunoblotted as indicated. (D) The percentage of Taz1p in TX–100 insoluble aggregates (light gray) and the percentage of Taz1p solubilized by digitonin (dark gray) (mean ± SEM, n = 3). Significant differences are indicated. (E) The mitochondrial MLCL:CL ratios following steady-state labeling with 32Pi (mean ± SEM, n = 6). Significant differences are indicated. (F and G) MLCL → CL transacylase assay. Lower panels in (F) show the expression of Taz1p in mitochondria (20 μg) by immunoblot. In (G), the calculated MLCL → CL activities were determined (mean ± SEM, n = 4) and significant differences are indicated.
Figure 4.
Figure 4.
Thermolabile BTHS alleles. Δtaz1 yeast transformed as indicated were grown at 30 or 37°C for 24 h. (A) Taz1p expression was determined in whole cell extracts by immunoblot; Pic1p and Tom70p are loading controls. The percentage of Taz1p at 37°C relative to 30°C is shown at the bottom (mean ± SEM, n = 3). (B) Mitochondrial phospholipids separated by thin-layer chromatography and revealed by phosphorimaging. (C) The derived MLCL:CL ratios (mean ± SEM n = 6). Significant differences are indicated. (D and E) MLCL → CL transacylase assay. Before initiating the reaction, mitochondria were pre-incubated at 4, 30 or 37°C for the indicated time. In (D), the calculated transacylase activities are expressed as a percentage of the maximal activity (mitochondria kept at 4°C) for a given source of mitochondria. (mean ± SEM, n = 3–4) The maximal activities were (pmols CL/min/mg protein): Δtaz1[WT] = 381.9 ± 45.7; Δtaz1[K65L] = 404.3 ± 25.8; Δtaz1[G261R] = 263.4 ± 10.7; and Δtaz1[K65L/G261R] = 119.3 ± 25.1. The significant differences relative to WT Taz1p treated the same are indicated. (E) The expression of Taz1p in the same samples (20 μg) by immunoblot; Porin, Aac2p, and Aco1p served as loading controls.
Figure 5.
Figure 5.
Model of yeast Taz1p. (A) Schematic representation of Taz1p with the position and functional class of each BTHS missense mutation indicated. Light blue boxes, predicted acyl transferase motifs A–E; blood red box, interfacial membrane anchor. (B and C) A homology model of yeast Taz1p was generated using the Phyre server, with the squash glycerol-3-phosphate acyltransferase as a template. (B) Taz1p in cartoon format colored from the N-terminus (blue) to the C-terminus (red). Surface representation is shown in grey. (C) The location of the class 4 mutant residues (green).
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