Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

MIRIAM L. GREENBERG

Abstract

Cardiolipin (CL) is an anionic phospholipid synthesized in the mitochondrial inner membrane. Perturbation of CL metabolism leads to Barth syndrome (BTHS), a life threatening genetic disorder. I utilized genetic, biochemical and cell biological approaches in yeast to elucidate the cellular functions of CL. Understanding the functions of CL is expected to shed light on the pathology and possible treatments for BTHS.

BTHS is caused by mutations in TAZ1, which encodes a CL remodeling enzyme called tafazzin. BTHS patients exhibit a wide range of clinical presentations, indicating that physiological modifiers influence the BTHS phenotype. A targeted synthetic lethality screen was performed to identify physiological modifiers of CL deficiency. Using this approach, synthetic genetic interactions of CL mutants were identified with genes encoding mitochondrial outer membrane proteins, specifically in the TOM, MDM and SAM complexes, which are involved in mitochondrial protein import, suggesting that CL plays a role in this process. Consistent with this, we showed that CL is present in the outer mitochondrial membrane and is involved in assembly of outer membrane protein complexes in yeast as well as in BTHS lymphoblasts. In addition to this, we showed that CL mutants interacted with genes encoding mitochondrial protein import complexes of the inner membrane, including the TIM and PAM complexes. To identify the role of CL in maintaining mitochondrial morphology, a targeted synthetic lethality screen was performed to determine if CL mutants genetically interacted with mutants defective in this function. The crd1Δ mutant genetically interacted with genes required for mitochondrial fusion and fission, suggesting a common cellular function. In addition to genes involved in mitochondrial fusion, crd1Δ genetically interacted with the UPS and GET complex mutants suggesting shared cellular functions with these as well. Unlike the UPS complex, a role for the GET complex in CL metabolism is unknown. My studies indicated that levels of CL were decreased in the get2Δ mutant, thus identifying a novel role of Get2p in the regulation of CL levels in yeast. In summary, the genetic interaction studies identified functions that could be physiological modifiers of CL deficiency in yeast, and could possibly point to modifiers of the BTHS phenotype.

Based on the genetic interactions of CL mutants with mitochondrial fusion mutants, we tested if CL plays a role in mitochondrial fusion. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, I hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. Previous studies have shown that CL and mitochondrial PE have overlapping functions, and the loss of both is synthetically lethal. In the current study, we showed that the loss of both CL and mitochondrial PE exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.

Recent studies have shown that cells lacking CL exhibit decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. Consistent with this finding, we showed that crd1Δ cells exhibit a growth defect on acetate medium, consistent with a defect in the TCA and glyoxylate cycles. A defect in the TCA cycle, and decreased mitochondrial functions, leads to activation of the retrograde (RTG) pathway. While the crd1Δ mutant exhibits these mitochondrial defects, it fails to activate the RTG pathway, as the expression of CIT2 in crd1Δ is not upregulated at elevated temperature. Consistent with the RTG defect, crd1Δ cells exhibit glutamate auxotrophy at elevated temperature. We also find that overexpression of RTG2, a positive regulator of the RTG pathway, and deletion of BHM2, a negative regulator of the RTG pathway, rescues the ts phenotype of crd1Δ. The RTG pathway is required for expression of genes that replenish TCA cycle metabolites. In addition to the RTG pathway, the β-oxidation pathway can also compensate for the defect in the TCA cycle by replenishing intermediates such as acetyl-CoA. Interestingly, crd1Δ exhibits growth defects on oleic acid medium, suggesting that cells lacking CL have a defect in the β-oxidation pathway. Taken together, my studies suggest that CL mutants have defects in the TCA and glyoxylate cycles and in the β-oxidation pathway, which cannot be alleviated due to defective activation of the RTG pathway. Identifying the function of CL in RTG signaling and metabolic pathways will facilitate understanding of specific metabolic deficiencies in BTHS patients.

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