Open Access Dissertation
Date of Award
Miriam L. Greenberg
Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome.
In order to fully understand the cellular response to the loss of CL, genome-wide expression profiling was carried out in the yeast CL mutant crd1Δ. The results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. The crd1Δ mutant exhibits decreased activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1, which encode proteins involved in the export of mitochondrial generated Fe-S co-factors to the cytosol and a transcription factor that regulates several antioxidant genes, respectively, did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.
Consistent with the role of CL in mitochondrial Fe-S biogenesis, perturbation of CL synthesis leads to decreased Yfh1 protein, a putative iron donor for mitochondrial Fe-S cluster assembly, and reduced activities of Fe-S enzymes aconitase and sulfite reductase, which are required for the synthesis of glutamate and sulfur-containing amino acids (methionine and cysteine). The data presented in this study show that the synthesis of glutamate and cysteine are decreased in crd1Δ. Interestingly, both amino acids are required for the synthesis of an essential antioxidant, glutathione (GSH), a tripeptide of glutamate, cysteine, and glycine. The growth defect of crd1Δ at elevated temperature and in the presence of oxidants is rescued by GSH supplementation, which is consistent with decreased synthesis of GSH. Collectively, these findings indicate that GSH deficiency in crd1Δ is due to the depletion of precursors for GSH synthesis, which is caused by defective Fe-S biogenesis.
To obtain an understanding of CL functions that might explain the cellular defects in BTHS, a screen was carried out to identify genes that, when overexpressed, suppress the growth deficiency of crd1Δ in galactose-containing media at elevated temperature. The suppressor screen utilizing a high copy genomic DNA library led to the identification of fifty putative suppressors of crd1Δ. Five plasmids were intially sequenced, and interestingly, each plasmid contained the same region of chromosome III, spanning four genes. One of the genes was LEU2, which is involved in leucine biosynthesis, a process that is perturbed in the crd1Δ mutant. This supports the finding that perturbation of leucine synthesis likely contributes to the growth defect of crd1Δ.
Patil, Vinay A., "The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism" (2013). Wayne State University Dissertations. 611.