|dc.description.abstract||Barth syndrome (BTHS) is an X-linked disease characterized by cardio- and skeletal myopathy, hypotonia, growth delay, neutropenia, and 3-methylglutaconic aciduria. Patients have mutations in the TAZ gene on chromosome Xq28 (G4.5) most of which are presumed to result in a loss-of-function of the protein product, tafazzin (TAZ). As TAZ is involved in the remodeling of the phospholipid, cardiolipin (CL), the resultant lipid profile in the absence of its function shows decreased CL levels, accumulated monolyso-CL, and the remaining CL contains an altered acyl chain composition. Unfortunately, the lack of an antibody against endogenous TAZ has prevented a detailed cell biologic and biochemical characterization of the endogenous protein. This in turn has impeded a molecular understanding of the protein’s role in BTHS pathogenesis. Here, we have developed three mouse monoclonal antibodies capable of detecting endogenous TAZ in mammals. A complicating aspect of mammalian TAZ research is the presence of many predicted alternatively spliced variants, of which only the hTAZ –exon5 was able to fully restore aberrant CL profile in the Saccharomyces cerevisiae Δtaz1 mutant. Importantly, each TAZ antibody has the ability to detect every predicted isoform as determined by epitope mapping. Our results also show that only one isoform of TAZ is normally expressed in human fibroblasts, HEK293 Flp-In cells, amd mouse heart and liver, despite the reported detection of mRNA corresponding to multiple splice variants.
Using mouse tissues, 293 Flp-In cells, and immortalized fibroblasts derived from healthy and BTHS patients, we demonstrate that mammalian TAZ is a highly protease-resistant and localized to the mitochondria where it associates non-integrally with membranes and assembles in a range of complexes. Like its yeast counterpart, TAZ in humans and rodents associates with the IMS-facing leaflets of the inner and outer mitochondrial membrane, Finally, using a novel mammalian BTHS cell culture model established via TALEN-mediated genome editing, we demonstrate that the loss-of-function mechanisms for two pathogenic alleles, R57L and H69Q, when overexpressed in tazTALEN cells, are the same as originally modeled and defined in yeast. Thus, our results reveal that tazTALEN cells can serve as a convenient platform to systematically dissect the loss-of-function mechanisms that underlie other BTHS variants, especially those that cannot be modeled in yeast due to lack of conservation. Combined with the generation of antibodies against endogenous TAZ, this work provides a major leap forward in our ability to characterize this enigmatic protein, to assign and discern the functions of TAZ and its numerous variants, and to study its role in CL metabolism.||