Characterization of tafazzin splice variants from humans and fruit flies

Abstract

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin.

FIGURE 1.

Isoforms of tafazzin from humans and Drosophila. The schematic representation shows aligned tafazzin isoforms with the conserved regions in gray and the hydrophobic segments in black. Drosophila tafazzins differ by their N-terminal variable regions. Human tafazzins differ by the presence/absence of exons 5 and 7.

FIGURE 2.

Intracellular localization of tafazzin isoforms. HeLa cells were transfected with plasmids expressing HA-tagged isoforms of tafazzin. For confocal microscopy, cells were stained with HA antibody to visualize tafazzin and with MitoTracker to visualize mitochondria.

FIGURE 3.

Intracellular localization of dTAZ-B. HeLa cells were transfected with a plasmid expressing HA-tagged dTAZ-B and subsequently analyzed by confocal microscopy. The intracellular distribution of dTAZ-B was compared with the distribution of mitochondria (A), the endoplasmic reticulum (B), and the Golgi apparatus (C). Polyclonal antibody raised against Drosophila tafazzin detected two bands in purified Drosophila membranes. The band that corresponds to dTAZ-B remains present in the ΔTAZ mutant (D). WT, wild type.

FIGURE 4.

Transacylase activity of tafazzin isoforms. Different tafazzins were expressed in Sf9 insect cells, mitochondria were isolated, and transacylase activities were measured in different assay systems. Transfections with wild-type baculovirus served as control (None). Substrate pairs included 16:0-[¹⁴C]18:2-PC + monolyso-CL (A), CL (bovine heart) + lyso-[¹⁴C]PC (B), PC (with the specified acyl group) + lyso-[¹⁴C]PC (C), 16:0-18:2-phosphatidylglycerol + lyso-[³H]phosphatidic acid (PA) (D), and 16:0-18:2-PC + lyso-[¹⁴C]PC (E).

FIGURE 5.

Transgenic expression of tafazzin isoforms in ΔTAZ Drosophila. Imprecise excision of the P-element from fly strain KG02529 created flies with deletion of dTAZ-A (ΔTAZ) (12). Then, various isoforms of tafazzin were expressed in the ΔTAZ mutant (ΔTAZ + insert). The graphs show the motor function of various transgenic fly strains measured by climbing assay. In this assay, a fly population is distributed between seven tubes according to its climbing activity. Flies in tube 1 have the lowest climbing activity, and flies in tube 7 have the highest climbing activity.

FIGURE 6.

Phenotype of transgenic fly strains. The P-element was excised from fly strain KG02529, creating either wild-type flies (WT, precise excision of P element) or flies with deletion of dTAZ-A (ΔTAZ, imprecise excision of P element) (12). Then, various isoforms of tafazzin were expressed in the ΔTAZ mutant (ΔTAZ + insert). A, male fertility was quantified and expressed as percent of wild-type fertility. B, respiratory activity of isolated mitochondria was measured in the presence of ADP and succinate and expressed as percent of wild-type activity. C, CL was analyzed by MALDI-TOF mass spectrometry, and the amount of (16:1)₂(18:2)₂-CL was quantified with the internal standard (14:0)₄-CL after correcting for ¹³C isotope effects (20). D, the ratio of 68:4-CL (m/z = 1400) over 68:6-CL (m/z = 1396) was determined from MALDI-TOF mass spectra after correction for the type II ¹³C isotope effect (20).

FIGURE 7.

C₆₈-CL species from various Drosophila strains. Mitochondrial lipid extracts were analyzed by MALDI-TOF mass spectrometry. The figure shows the spectral region that contains the C₁₆-C₁₆-C₁₈-C₁₈-CL species of wild-type flies (WT), tafazzin mutant flies (ΔTAZ), and several transgenic flies with the expression of tafazzin isoforms in the ΔTAZ background (ΔTAZ+). Red arrowheads mark peaks at m/z = 1396 ((16:1)₂(18:2)₂-CL, i.e. remodeled CL), and green arrowheads mark peaks at m/z = 1400 (mainly 16:0-16:1-18:1-18:2-CL, i.e. non-remodeled CL).

FIGURE 8.

Complex formation and membrane association of hTAZ-Δ5 and hTAZ-FL. A–C, HA-tagged constructs of hTAZ-Δ5 and hTAZ-FL were expressed in 293T cells. Mitochondria were isolated and solubilized with digitonin, and solubilized proteins were separated by BN-PAGE followed by Coomassie staining (A) or Western blot analysis with the HA antibody (B and C). Protein bands corresponding to respiratory complexes III₂, IV, and V, are indicated. D, isolated mitochondria were treated with alkali (0.1 m NaHCO₃), and the release of tafazzin was measured by Western blot and quantified by densitometric analysis.

FIGURE 9.

Proteinase K treatment of 293T cell mitochondria. HA-tagged constructs of hTAZ-Δ5 and hTAZ-FL were expressed in 293T cells, and mitochondria were isolated and treated with proteinase K (0, 20, 200 ng/ml) in the absence or presence of digitonin (1 g/g protein) or Triton X-100 (0.1%). Samples were analyzed by Western blot for tafazzins (HA), mitochondrial porin (VDAC), and subunit NDUS3 of complex I (CoI).