Characterizing tafazzin and Barth syndrome mutant tafazzins

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To begin dissecting the processes of cardiolipin (CL) remodeling, this proposal focuses on the characterization of the putative CL acyltransferase, tafazzin (Tazlp), the mutant gene product associated with Barth syndrome (BTHS). The long-term goal of this research is to ascertain how CL is remodeled, the molecular players involved in this process, what steps are regulated, and the consequences of both normal and abnormal CL remodeling with respect to mitochondria! function. This will provide a basis to understand the role of Taz1 p in BTHS. Moreover, this work will shed light on additional cardiomyopathies, including diabetic cardiomyopathy, which has recently been shown to involve deficits in CL synthesis and increased CL cat a bo I ism. The goal of the first specific aim is to identify lipids and proteins that either directly interact or associate with Taz1 p in a complex. Preliminary results indicate that Taz1 p binds noncovalently to phospholipids and/or fatty acyl chains and assembles into several large macromolecular complexes. To identify potential lipid ligands, a battery of biochemical and genetic approaches will be employed including use of recombinant Tazlp, defined phospholipids, ELISA, Fat Blots, BN-PAGE, and a dual affinity tagged Taz1 p construct. The dual affinity tagged Taz1 p construct will additionally be employed to reveal interacting proteins by standard biochemical procedures. Results from this aim are expected to provide insight into the CL remodeling pathway in which Tazlp participates, including potentially identifying the substrate and target specificity of Taz1 p, other components involved in this process, and potentially novel functions of Taz1 p. For the second specific aim, a panel of BTHS mutants, occurring at identical or conserved residues between the human and yeast orthologs, will be investigated for their ability to localize to and within the mitochondrion correctly, associate with proteins and/or lipids as identified in aim 1, and function. Through the systematic comparison with wild type Taz1 p, the molecular basis for a Taz1 p mutant and BTHS will be provided. For those BTHS mutations that occur at residues unique to human Taz1 p, a cell culture model system will be developed that will allow the molecular dissection of this subset of BTHS mutants; the goal of specific aim 3. Results from this study are important for public health because cardiac disease affects the general population and this study will provide insight into basic mechanisms leading to cardiac disease.