Identification of novel mitochondrial localization signals in human Tafazzin, the cause of the inherited cardiomyopathic disorder Barth syndrome.

Mutations in the gene tafazzin (TAZ) result in Barth syndrome (BTHS). Patients present with hypotonia, cyclic neutropenia, 3-methyglutaconic aciduria, and cardiomyopathy, which is the major cause of mortality. The recessive, X-linked TAZ gene encodes a mitochondrial membrane-associated phospholipid modifying enzyme, which adds unsaturated fatty acid species to monolysocardiolipin to generate mature cardiolipin in the mitochondrial membrane that is essential for mitochondrial morphology and function. To identify intrinsic mitochondrial localization sequences in the human TAZ protein, we made sequential TAZ peptide-eGFP fusion protein expression constructs and analyzed the localization of eGFP fluorescence by confocal microscopy. We assessed these fusion proteins for mitochondrial localization through cotransfection of H9c2 cells with plasmids encoding organellar markers linked to TdTomato. We have identified two peptides of TAZ that are independently responsible for mitochondrial localization. Using CRISPR-generated TAZ knock out cell lines, we found that these peptides are able to direct proteins to mitochondria in the absence of endogenous TAZ. These peptides are not located within the predicted enzymatic clefts of TAZ, implying that some BTHS disease causing mutations may affect mitochondrial localization without affecting transacylase activity. These novel peptides improve our understanding of TAZ intracellular trafficking, provide insight into the molecular basis of BTHS and provide molecular reagents for developing targeted mitochondrial therapies.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids.

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.