Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins

Abstract

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215-232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

Figure 1.

Taz1p is a mitochondrial resident. (A) Fractions were prepared from the wt strain through a series of differential centrifugations. 50 μg of each fraction was separated by SDS-PAGE and analyzed by immunoblot using antisera specific for the indicated subcellular organelle. n = 2. (B) Taz1p localizes to the IM and OM in immunogold-labeled ultrathin cryosections of the parental wt strain. n, nucleus; ne, nuclear envelope; pm, plasma membrane; m, mitochondria. Arrows, OM; arrowheads, IM. Bars, 0.1 μm.

Figure 2.

Taz1p nonperipherally associates with the IM, OM, and contact sites. (A) Wt mitochondria were analyzed by alkali extraction using 0.1 M carbonate at the indicated pH values. Equal volumes of the pellet (P) and TCA-precipitated supernatant (S) fractions were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted for the indicated mitochondrial markers. n = 3. (B) Sonicated mitochondrial membrane vesicles were prepared from wt mitochondria for fractionation on linear sucrose gradients (0.85–1.8 M). Fractions were collected from heavy (fraction 1; bottom) to light density (fraction 16; top), and 5 μg of each fraction was immunoblotted as indicated. Chemiluminescent images were also collected and two exposures per blot were quantified. For each individual mitochondrial marker, the amount in each fraction is expressed as the percentage of the sum of the signals for that marker in all of the fractions. For both the IM and OM, the mean ± SD of three different markers of each compartment are presented (IM represents AAC, Cox2p, and cytochrome c ₁; OM represents OM45p, porin, and Tom70p). The immunoblots and derived data are from a single representative experiment. n = 3.

Figure 3.

Three epitope-tagged Taz1p constructs are functional. (A) Schematics of the three constructs, with potential TM domains indicated. (B) After steady-state labeling with ³²P_i, phospholipids were extracted from the indicated strains, separated by TLC, and revealed by phosphoimaging. The migration of phospholipids is indicated (PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid). (C) The relative abundance of MLCL was determined for each strain. The amount of MLCL in each strain is expressed as a percentage of the total phospholipids in each strain. Mean ± SEM. n = 6. Δtaz1 yeast accumulate significant amounts of MLCL relative to wt yeast (P ≤ 0.001), as determined by t-test. Control Δtaz1 yeast transformants ([Vector Alone]) accumulate significant amounts of MLCL relative to Δtaz1 yeast transformed with wt Taz1p ([WT Taz1p]) or any of the epitope-tagged constructs (P < 0.001) as determined by one way analysis of variance (ANOVA), with Holm–Sidak pairwise comparisons. (D) Purified mitochondria from the indicated yeast strains were analyzed by alkali extraction as before, except that Taz1p was identified by immunoblotting with monoclonal antibodies specific for the appropriate epitope tag. (E) Quantitation was performed as previously described. The percentage of Taz1p present in the derived supernatants after carbonate extraction was determined as follows: S/(S + P) × 100, where S is the volume of Taz1p detected in the supernatant at a given pH and P is the volume associated with the pellet at the same pH. Mean ± SD. n = 3.

Figure 4.

Taz1p is an interfacial membrane protein facing the IMS. Intact mitochondria, mitochondria subjected to osmotic shock (mitoplasts), or mitochondria solubilized with 0.1% Triton X-100 were incubated alone or in the presence of the indicated concentration of proteinase K. Equivalent amounts of each sample (designated by the indicated quantity of starting material) were resolved by SDS-PAGE and immunoblotted as indicated. The source of the mitochondria for each image is wt (A), Δtaz1 (B), MycTaz (C), TazMycTaz (D), and TazHA (E). The three gray arrows highlight Taz1p fragments generated during the TCA precipitation step performed to inactivate proteinase K, which involves the removal of increasing amounts of the C terminus. The white arrow, which designates an ∼27-kD fragment generated upon addition of low amounts of proteinase K to mitoplasts, lacks at least the N-terminal 154 amino acids, and contains the C terminus, which is stabilized in 0.1% Triton X-100. The black arrow reveals an ∼24-kD fragment only generated upon addition of high concentrations of proteinase K to wt mitoplasts. To visualize the Taz1p fragments, overexposed immunoblots are presented, except for D, where the indicated exposure lengths for the anti-Myc immunoblots were spliced together using Photoshop. Asterisks in A and B highlight background bands identified in the Δtaz1 mitochondria immunoblots (B). (F) Drawing summarizing the localization and membrane association of Taz1p. n = 3.

Figure 5.

Yeast Taz1p harboring authentic BTHS mutations that occur in the putative interfacial membrane anchor of Taz1p localize to mitochondria, but are nonfunctional. (A) ClustalW alignment of the putative interfacial membrane anchor (boxed) of Taz1p from human, mouse, and S. cerevisiae. The BTHS mutations are indicated at the top, with mutations occurring at conserved and identical residues provided in green and red, respectively. (B) The relative expression of the four different BTHS mutants (three clones/mutant) was determined from whole-cell extracts by immunoblotting for Taz1p (bottom) with KDH serving as a loading control (top). (C) The same as B, except that three clones derived from a humanized yeast Taz1p were analyzed next to the three Taz1p mutants harboring single BTHS mutations occurring at conserved residues. (D and E) Steady-state ³²P labeling and analyses were performed as described in Fig. 3 (B and C). Δtaz1 yeast accumulate significant amounts of MLCL relative to wt yeast (P ≤ 0.001), as determined by t-test. All of the BTHS mutants, with the notable exception of the humanized Taz1p, demonstrate a statistically significant accumulation of MLCL relative to Δtaz1 transformed with wt Taz1p ([WT Taz1p]; P < 0.001) as determined by one-way ANOVA, with Holm–Sidak pairwise comparisons. Mean ± SEM. n = 4, except for humanized Taz1p, where n = 3. (F) Fractions were prepared from the indicated yeast strains through a series of differential centrifugations. 50 μg of each fraction was separated by SDS-PAGE and analyzed by immunoblot using antisera specific for the indicated subcellular organelle. n = 2.

Figure 6.

Altered membrane association of the BTHS Taz1p mutants results in two fates: matrix mistargeting or aberrant complex assembly. Mitochondria isolated from the indicated strains were analyzed by sonication (A) or alkali extraction (B), as previously described. (C) Quantitation and data analyses were performed as described in Fig. 3 E. The asterisks indicate a statistically significant increase in the release into the supernatant of each of the BTHS Taz1p mutants, relative to endogenous Taz1p (P < 0.001) as determined by one-way ANOVA, with Holm–Sidak pairwise comparisons. Mean ± SD. n = 3. (D) 100 μg mitochondria from the indicated strain was solubilized in 1.5% (wt/vol) digitonin and subjected to blue native–PAGE (6–16% acrylamide), and then Taz1p was detected by immunoblotting. The black, green, and blue arrows highlight 160-, 220-, and 280-kD Taz1p-containing complexes identified in wt and [WT Taz1p] mitochondrial extracts. The red arrow highlights a 460-kD complex distinctly observed in mitochondrial extracts derived from the G230R Taz1p mutant. n = 4. (E) Mitochondria derived from the indicated strains were treated exactly as described in Fig. 4. n = 3. For simplicity, only one set of control immunoblots is presented in B and E. The controls for every source of mitochondria are provided in Fig. S4. Fig. S4 is available at http://www.jcb.org/cgi/content/full/jcb.200605043/DC1.

Figure 7.

Model of Taz1p function and the defect associated with the two distinct classes of BTHS Taz1p variants. (A) Taz1p (blue) associates with IMS-facing membranes via a TM-like loop. Here, it transfers fatty acyl groups (light green) to MLCL (gray) and/or lyso-PC (light blue), forming CL and PC, respectively. (B) The V223D, V224R, and I226P Taz1p mutations inactivate a putative stop–transfer signal (red squiggle) resulting in Taz1p mistargeting to the mitochondrial matrix. In this compartment, Taz1p is unable to function (red X), potentially caused by the absence of its physiological lipid target within the matrix. (C) The stop–transfer activity is preserved in the G230R BTHS mutant; however, possibly because of charge interactions between the positively charged Arg and negatively charged phospholipid headgroups, the mutant Taz1p exhibits an altered association with mitochondrial membranes. This results in aberrant complex assembly (unidentified components depicted in yellow, purple, and dark green) and loss of function (red X).