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. 2016 May 1;25(9):1754-70.
doi: 10.1093/hmg/ddw046. Epub 2016 Feb 16.

Defining functional classes of Barth syndrome mutation in humans

Ya-Wen Lu  1 , Laura Galbraith  2 , Jenny D Herndon  3 , Ya-Lin Lu  4 , Mia Pras-Raves  5 , Martin Vervaart  5 , Antoine Van Kampen  6 , Angela Luyf  6 , Carla M Koehler  3 , J Michael McCaffery  7 , Eyal Gottlieb  2 , Frederic M Vaz  5 , Steven M Claypool  8
Affiliations

Defining functional classes of Barth syndrome mutation in humans

Ya-Wen Lu et al. Hum Mol Genet. .

Abstract

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ.

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Figures

Figure 1.
Figure 1.
One isoform of TAZ is expressed and localized to mitochondria in mammalian cells and tissues. (A) Whole cell extracts (50 μg) from healthy control (C109) and BTHS patient fibroblasts (TAZ001 and TAZ003) were immunoblotted with three different mAbs generated against recombinant human TAZ. (B) Subcellular fractions (50 μg) derived from C109 cells immunoblotted as indicated. (C) Mitochondrial extracts (50 μg) from the indicated source immunoblotted with the TAZ mAbs. (D) The epitope recognized by each TAZ mAb was determined by peptide array. The exon that harbors each epitope is indicated. (E) TAZ was immunoprecipitated with 3D7F11 from 1.5 mg of the indicated cell extracts. Fifty micrograms of starting material and non-binding flow through and 100% of bound material (Preclear and IP) were immunoblotted using 2C2C9. (F) Whole cell extracts from WT or taz1Δ yeast transformed as indicated and mitochondrial extracts from the designated source immunoblotted for TAZ. EV, empty vector. *nonspecific band.
Figure 2.
Figure 2.
Generation of a BTHS cell culture model using TALEN-mediated genome editing. (A) TALEN-mediated disruption of TAZ in 293 Flp-In cells introduces nucleotide deletions that result in an in-frame deletion in tazTALEN.2 and a missense and in-frame deletion in tazTALEN.19. (B) Whole cell extracts from the indicated cells grown in dextrose- or galactose-based media were resolved by SDS–PAGE and immunoblotted. (C) WT 293 Flp-In and tazTALEN cells were incubated for 4 h with 20 μm of the proteasome inhibitor, MG132. 40 μg of whole cell lysates were resolved by SDS–PAGE and immunoblotted for the indicated proteins. Cyclin D1, a target of the ubiquitin–proteasome pathway (35), is used as a positive control. (D) Relative mRNA level of TAZ in WT (293 Flp-In) and each tazTALEN cell. Data were analyzed by the comparative CT (ΔΔCT) method, represented as mean ΔΔCT ± SEM n = 8 relative to WT expression level, which was set to 0. (E) Mitochondria (50 μg) from the designated source were immunoblotted as listed. (F) Protein expression in (E) was quantified by densitometry and normalized to WT levels (mean ± SEM; n = 3). Significant differences are indicated. (G) In organello import of radiolabeled [35S]-Su9-DHFR into WT 293 Flp-In and tazTALEN mitochondria in the presence or absence of a membrane potential. Following import, mitochondria were osmotically ruptured, treated with trypsin, resolved by SDS–PAGE, and bands detected by phosphoimaging. P, precursor (5% of precursor protein + 100 μg mitochondria); p, precursor; m, mature. (H) The percentage of precursor imported at each time point was calculated (mean ± SEM; n = 3). See also Supplementary Material, Figure S1 and Table S1.
Figure 3.
Figure 3.
tazTALEN cells have altered mitochondrial CL and MLCL profiles as determined by high-performance liquid chromatography-mass spectrometry. The relative amounts of CL (A), MLCL (B) and PG (D) was determined by high-performance liquid chromatography-mass spectrometry. (C) The ratio of MLCL:CL as determined from (A and B). The abundance of CL (E) and MLCL (L) of a given acyl chain length (E and L) or containing the indicated number of double bonds (F and M) was determined as a percentage of the total amount of CL or MLCL. The distribution of double bonds per acyl chain length for CL (G–K) and MLCL (N and O) expressed as a percent of the sum of all species of the indicated length. All data are the mean ± SEM (n = 3). Significant differences are indicated. See also Supplementary Material, Figures S2–S4.
Figure 4.
Figure 4.
Membrane association and assembly of TAZ. (A) Mitochondria were sonicated and equal volumes of the pellet (P) and TCA-precipitated supernatant (S) fractions were immunoblotted as indicated. SM, starting material. (B) Mitochondria were incubated in 0.1 m Na2CO3 of the listed pH. Integral proteins (P) were separated from released proteins (S) by ultracentrifugation and equal volumes of each were immunoblotted as indicated. (C) Quantified band intensities of the P and S fractions were plotted as the percentage of total protein released into the supernatant for each pH (mean ± SEM; n = 3 (C109) or 4 (293)). (D) Mitochondria from control and BTHS fibroblasts (450 μg), 293 Flp-In cells (200 μg) and mouse heart and liver (200 μg) were solubilized with digitonin, separated by 2D BN/SDS–PAGE, and immunoblotted for TAZ.
Figure 5.
Figure 5.
Mammalian TAZ is protease resistant and associates with IMS-facing leaflets. (A) Mitochondria were titrated with increasing amounts of digitonin to differentially solubilize mitochondrial compartments. After centrifugation, equal volumes of the pellet (P) and supernatant (S) fractions were resolved by SDS–PAGE and immunoblotted. (B) Quantified band intensities for two markers per compartment were combined and plotted as the percent detected in supernatant (mean ± SEM; n = 7). (C) Mitochondria, osmotically ruptured mitoplasts, or detergent-solubilized mitochondria from the indicated source were incubated without or with protease, resolved by SDS–PAGE, and immunoblotted. (D) Mitochondria were solubilized with 0.1% (v/v) SDS and where indicated, heated at 95°C for 5 min prior to adding pronase E, proteinase K or trypsin. Equal volumes of each sample were resolved by SDS–PAGE and immunoblotted. (E) Mitochondria were solubilized with digitonin and fractionated into a supernatant (S1) and pellet (P1) by centrifugation. Material not extracted by digitonin (P1) was further solubilized with TX-100 and separated into a supernatant (S2) and pellet (P2) by centrifugation. Fractions were resolved by SDS–PAGE and immunoblotted as indicated. (F) In organello import of radiolabeled [35S]-TAZ into 293 Flp-In mitochondria at the indicated temperature in the presence or absence of a membrane potential. Following import, mitochondria were treated with trypsin, resolved by SDS–PAGE, and bands detected by phosphoimaging. Where indicated, mitochondria were osmotically ruptured in the presence or absence of trypsin. P, precursor (5% of precursor protein + 100 μg mitochondria).
Figure 6.
Figure 6.
Both termini of TAZ are in the IMS. (A) Cartoon of epitope tagged TAZ constructs. (B) Whole cell extracts (30 μg) were immunoblotted for the indicated proteins. (C) Phospholipids from the indicated cells were labeled with 32Pi and separated by TLC. (D) Quantification of the MLCL:CL ratio (mean ± SEM; n = 3). Significant differences compared with WT 293 Flp-In cells were determined by one-way ANOVA. n.s. = differences not significant. (E and F) The protease accessibility of epitope tags added to the NH2 (E) or COOH (F) termini of TAZ was determined as in Figure 5C. (G) tazTALEN.19 cells overexpressing WT or TAZ-APEX2 were labeled in vivo with H2O2 and DAB followed by OsO4 and EM imaging. Scale bars are provided in each panel.
Figure 7.
Figure 7.
The R57L allele is highly unstable. (A) Sequence alignment of TAZ from the indicated species encompassing human Arg57 and His69 (white face on black background) and the HXXXXD motif (underlined). (B) Whole cell extracts (20 μg) from 5 to 6 clones transfected with the indicated TAZ alleles were immunoblotted. (C) Relative mRNA level of TAZ analyzed by the comparative CT (ΔΔCT) method, represented as mean fold change (2ΔΔCT) ± SD n ≥ 4 relative to 293 Flp-In TAZ expression. (D) In organello import of radiolabeled precursor R57L TAZ into freshly isolated 293 Flp-In mitochondria as described in Figure 5F. (E) tazTALEN cells overexpressing WT, R57L and H69Q alleles were incubated for 4 h with 20 μm of the proteasome inhibitor, MG132. 40 μg of whole cell lysates were resolved by SDS–PAGE and immunoblotted for the indicated proteins. (F) Whole cell extracts (20 μg) were collected after incubation with cycloheximide for the indicated times, and the amount of TAZ remaining assessed by immunoblot. The half-lives of the indicated TAZ alleles were calculated using the mean of five individual repetitions. See also Supplementary Material, Figure S5.
Figure 8.
Figure 8.
The H69Q mutant is catalytically dead. (A) Mitochondria (20 μg) from 293 Flp-In, tazTALEN and tazTALEN cells overexpressing WT, R57L and H69Q alleles were immunoblotted as indicated. (B) In organello import of radiolabeled precursor H69Q TAZ into freshly isolated 293 Flp-In mitochondria as described in Figure 5F. (C) Assembly of H69Q as determined by 2D BN/SDS–PAGE. (D) Lyso-PC to PC transacylase activities were determined in mitochondria isolated from 293 Flp-In, tazTALEN and tazTALEN cells overexpressing the indicated TAZ alleles (mean ± SEM, n = 3). Significant differences are indicated. n.s., not significant. The relative amounts of CL (E) and MLCL (F), and the subsequent MLCL:CL ratio (G), in tazTALEN cells overexpressing WT, R57L and H69Q were determined as previously described. See also Supplementary Material, Figure S5.
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