The Taz1p transacylase is imported and sorted into the outer mitochondrial membrane via a membrane anchor domain

Abstract

Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome.

Fig 1

Taz1p import is not dependent on the Δψ and is first imported through the TOM complex. (A) Radiolabeled Taz1p was incubated with wild-type mitochondria, and aliquots were removed at the indicated time points and incubated with 20 μg/ml of trypsin. Valinomycin at 1 μM and FCCP at 50 μM were added to the reaction buffer to dissipate the Δψ. Samples were separated by SDS-PAGE, and imported Taz1p was detected by autoradiography. Imported Taz1p was quantitated using Bio-Rad Quantity One software, with the last time point in the energized mitochondria set to 100%. (B) Taz1p was imported into isolated yeast mitochondria that were pretreated with 20 μg/μl of trypsin. p, precursor; m, mature form of Su9-DHFR. (C) Import of Taz1p into isolated Δtom20 and Δtom70 mitochondria.

Fig 2

Taz1p import is independent of the TIM22 and TIM23 translocons. (A to C) Taz1p was imported into tim23-6, tim22-4, tim10-1, and tim10-1tim9^S mitochondria as described for Fig. 1A. (D) Taz1p was imported into WT mitochondria followed by cross-linking (XL) with 1 mM DSP and then coimmunoprecipitated (IP) with antibodies directed against Tim10p, Tom40p, cytochrome b₂, CCP1, Tim22p, and Tim23p. Cross-links were broken with BME prior to loading the samples to the SDS-PAGE gel to confirm that the conjugates were Taz1p.

Fig 3

Taz1p is first imported into the outer mitochondrial membrane. (A) WT mitochondria (15 mg/ml) were solubilized with the indicated concentration of digitonin, and the soluble proteins were separated from the mitochondrial pellet by centrifugation. Outer (porin and OM45) and inner (Tim45p and AAC) membrane markers were averaged, and cyt b₂ marked the intermembrane space. Equal amounts were analyzed by immunoblotting and quantitated with Bio-Rad Quantity 1 software. The percentage solubilized was calculated as [(S)/(S + P) × 100] for each detergent concentration (mean ± SD, n = 3). Immunoblots are provided in Fig. S2A to D in the supplemental material. (B) Same as panel A, with tim10-1 mitochondria. (C) Same as panel A, with tim23-6 mitochondria. (D) Taz1p was imported into WT mitochondria for 15 min, and then fractionation was investigated for the imported Taz1p as in panel A. Markers included imported porin and Tim54p.

Fig 4

Taz1p^V224R is imported into the matrix via the Tim23 pathway. (A) Taz1p and Taz1p^V224R were synthesized in vitro and imported into WT mitochondria, which were subjected to osmotic shock, followed by centrifugation to separate the supernatant (S) from the mitoplast pellet (P). Mitochondria were also treated with 20 μg/ml of proteinase K or 20 μg/ml of proteinase K plus 1% Triton X-100. As a control, M was included. (B) Radiolabeled Taz1p^V224R was imported into both WT and tim23-6 mitochondria in the presence and absence of a membrane potential, followed by protease treatment to remove nonimported precursor. (C) Taz1p^V224R was imported as in panel B in WT, tim10-1, and tim10-1tim9^S mitochondria. (D) Taz1p and Taz1p^V224R were imported into WT mitochondria, and the samples were solubilized in 1% digitonin and separated on a 6 to 16% blue native gel.

Fig 5

The membrane anchor of Taz1p is required for mitochondrial localization. (A) Taz1pΔMA^215–232, which lacks the membrane anchor, was expressed in a Δtaz1 strain and the truncated protein was detected by immunoblot analysis in a total protein lysate. The immunoblot analysis of WT TAZ1 and aconitase (Aco1) serve as controls for blotting and loading, respectively. (B) The lysate (T) of strains from panel A was fractionated into the M and PMS fractions, followed by immunoblotting. Cytosolic Hsp70 served as an additional control for fractionation. (C) Taz1pΔMA^215–232 was imported into isolated WT, tim10-1, and tim10-1tim9^S mitochondria and subjected to trypsin treatment to remove nonimported precursor. Samples in WT mitochondria were also subject to carbonate extraction (Na₂CO₃) and separated in supernatant (S) and pellet (P) fractions by centrifugation. Fragments that were initially resistant to protease were labeled “protected.” (D) Taz1pΔMA^215–232 was imported as for panel C into WT, Δtom20, and Δtom70 mitochondria.

Fig 6

The membrane anchor region and its flanking sequences are sufficient for mitochondrial import. (A) Constructs were made in which the membrane anchor (MA) of Taz1p (amino acids 215 to 232) and flanking sequences (amino acids 215 to 265, 204 to 265, and 204 to 232) were inserted between two DHFR moieties and imported into isolated WT mitochondria. Nonimported precursor was removed by protease treatment. (B) The V224R mutation was introduced, creating DHFR-MA^{204–265/V224R}-DHFR, and imported as for panel A. (C) Imported DHFR-MA^204–265-DHFR and DHFR-MA^{204–265/V224R}-DHFR were localized within mitochondria by osmotic shock in the presence and absence of proteinase K and Triton X-100. Controls include Tim54p for the intermembrane space and Su9-DHFR for the matrix.