Defining functional classes of Barth syndrome mutation in humans

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.

Impaired Cardiolipin Biosynthesis Prevents Hepatic Steatosis and Diet-Induced Obesity

“…The mouse monoclonal antibody raised against TAZ (Clone 2G3F7) was affinity purified by Steven Claypool’s laboratory at the Johns Hopkins University School of Medicine (15). …”

Mechanism for Remodeling of the Acyl Chain Composition of Cardiolipin Catalyzed by Saccharomyces cerevisiae Tafazzin

“…His-77 is located in the HX 4 D motif, a conserved structural motif in acyltransferase, and the H77Q mutant is a typical example of the class 4 mutants (23). We prepared the H77Q mutant and isolated and determined its transacylation activity in the reaction PC (18: (42) recently demonstrated that the human equivalent of the H77Q mutant (H69Q in human) remarkably lacks the transacylation activity, indicating conservation of the functional importance of this histidine.…”

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

“…The ‘reduced OsO 4 ’ procedure, which involves addition of 1% (wt/vol) potassium ferrocyanide immediately prior to staining and produces stronger contrast on membranes relative to other cellular components, has also been used for staining DAB reaction product from APEX2 (refs. 23,24). Finally, Joesch et al reported that reducing tissue samples using sodium hydrosulfite before OsO 4 staining greatly enhances the EM-level contrast of APEX2 staining 41 .…”

“…For example, we confirmed that a fusion of APEX to the mitochondrial calcium uniporter was functional for transporting calcium into the mitochondrial matrix 12 . Claypool and co-workers examined lipid metabolite ratios to confirm the activity of an APEX2 fusion to tafazzin, a transacylase enzyme 24 . Sandin and co-workers showed that an APEX2 fusion to the protein caveolin rescued the formation of caveole 27 .…”

“…This Protocol uses Durcupan ACM resin, but most alternative resins are compatible with APEX2 samples, including CY212 (ref. 31), Epon 19,24 , Spurr’s, or Procure 812 (ref. 42).…”