Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

MIRIAM L. GREENBERG

Abstract

The phospholipid cardiolipin (CL) is crucial for many cellular functions and signaling pathways, both inside and outside of mitochondria. My thesis focuses on the role of CL in energy metabolism. Many reactions of electron transport and oxidative phosphorylation, the transport of metabolites needed for these processes, and the stabilization of electron transport chain supercomplexes, require CL. Recent studies indicate that CL is required for the synthesis of iron-sulfur (Fe-S) co-factors, which are essential for numerous metabolic pathways. Activation of carnitine-acetylcarnitine translocase, which transports acetyl-CoA into the mitochondria, is CL dependent. The presence of substantial amounts of CL in the peroxisomal membrane suggests that CL may be important for peroxisomal functions. Understanding the role of CL in energy metabolism may identify physiological modifiers that exacerbate the loss of CL and underlie the variation in symptoms observed in Barth syndrome, a genetic disorder of CL metabolism.

In order to identify biochemical pathways exacerbated by the loss of CL, I carried out a Synthetic Genetic Array (SGA) screen of the yeast CL mutant crd1Δ. The results indicated that crd1Δ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Previous studies have shown that synthesis of acetyl-CoA depends primarily on pyruvate conversion in the mitochondria and the cytosol. The crd1Δ mutant exhibited decreased acetyl-CoA levels and decreased growth on acetate as a sole carbon source. Gene expression and protein levels of PDH were increased, but PDH specific activity remained unaltered. These findings suggest that defective ability to convert acetate to acetyl-CoA and possibly decreased enzymatic activity of PDH may account for perturbed acetyl-CoA synthesis in CL-deficient cells.

Consistent with a requirement for CL in acetyl-CoA synthesis, perturbation of CL synthesis leads to decreased activity of carnitine-acetylcarnitine translocase, a transporter found in the mitochondrial membrane specific for import of acetylcarnitine into the mitochondria. Growth of crd1Δ at elevated temperature and on acetate medium is restored by supplementation of carnitine, acetylcarnitine, or oleate. Interestingly, synthetic lethality was observed between crd1Δ and mutants in the glyoxylate cycle, suggesting that this cycle is essential to replenish TCA cycle intermediates in CL-deficient cells. The studies described in this thesis are the first to demonstrate that CL is required for synthesis and transport of acetyl-CoA.

To obtain an understanding of tafazzin function, an SGA screen was carried out to identify mutants that synthetically interact with taz1Δ. Interesting interactions were observed with phospholipase B, ornithine carbamoyltransferase, and genes required for mitochondrial iron homeostasis and vacuolar protein sorting. These findings suggest that tafazzin may be involved in cellular processes other than CL remodeling.

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