Absence of Cardiolipin Results in Temperature Sensitivity, Respiratory Defects, and Mitochondrial DNA Instability Independent of pet56

Abstract

Cardiolipin (CL) is a dimeric phospholipid localized primarily in the mitochondrial membrane. Previous studies have shown that yeast cells containing a disruption of CRD1, the structural gene encoding CL synthase, exhibit temperature-sensitive colony formation and multiple mitochondrial defects. A recent report (Zhang, M., Su, X., Mileykovskaya, E., Amoscato, A. A., and Dowhan, W. (2003) J. Biol. Chem. 278, 35204 -35210) suggested that defects associated with CL deficiency may result from the reduced expression of PET56 in crd1⌬ mutant backgrounds and should be reevaluated. In the current study, we present evidence that CL deficiency leads to mitochondrial DNA instability, loss of viability, and defects in oxidative phosphorylation at elevated temperatures. The observed mutant phenotypes are characteristic of crd1⌬ mutant cells of both PET56 and pet56 backgrounds and are complemented by an episomal copy of CRD1 but not by expression of the PET56 gene. Phosphatidylglycerol is elevated in crd1⌬ mutant cells when grown in the presence of fermentable and non-fermentable carbon sources, although the extent of the increase is higher in nonfermentable medium. An increase in the ratio of phosphatidylethanolamine to phosphatidylcholine was also apparent in the mutant. These findings demonstrate that CRD1, independent of PET56, is required for optimal mitochondrial function and for an essential cellular function at elevated temperatures.Cardiolipin (CL), 1 a unique phospholipid with dimeric structure, is ubiquitous in eukaryotes and is primarily found in the mitochondrial inner membrane (1). It plays a key role in mitochondrial bioenergetics by optimizing the activities of enzymes in the oxidative phosphorylation pathway (1), stabilizing the supercomplexes of Saccharomyces cerevisiae respiratory chain complexes III and IV (2), preventing rate-dependent uncoupling, and providing osmotic stability in yeast mitochondria (3). CL is also involved in mitochondrial biogenesis, possibly via assisting protein import into mitochondria (4) and maintaining optimal mitochondrial internal structure (5). Defective remodeling of CL is associated with Barth syndrome, a severe genetic disorder characterized by cardiomyopathy, neutropenia, skeletal myopathy, and respiratory chain defects (6). The phenotype of Barth syndrome is dependent upon multiple factors that are not well understood (7,8). Elucidation of the functions of CL will help to clarify the abnormalities associated with this disorder.We have observed that the yeast crd1⌬ mutant, which lacks CL synthase and has no detectable CL, loses cell viability during growth at elevated temperatures (4, 9). Crd1⌬ mutant cells cannot form colonies at 37°C from single cells seeded on YPD (1% yeast extract, 2% peptone, and 2% dextrose) plates (9). In addition, crd1⌬ mutant cells grown in fermentable or non-fermentable carbon sources also segregate large numbers of petites (respiratory incompetent cells) after prolonged culture at elevated temperatures (4). CL is, thus, essential for maintaining...

Loss of Function ofKRE5Suppresses Temperature Sensitivity of Mutants Lacking Mitochondrial Anionic Lipids

“…Mutants lacking only CL exhibit a strain-dependent decrease in mtDNA stability at elevated temperature (Jiang et al, 2000;Zhong et al, 2004). We have noticed that crd1⌬ mutants from different strain backgrounds differ greatly with respect to the temperature at which growth is defective and mtDNA becomes unstable (Zhong et al, 2004). In addition, crd1⌬ was less thermotolerant on synthetic medium than on rich medium (Zhong et al, 2004).…”

“…The taz1⌬ mutant is temperature sensitive for growth on ethanol but grows well on other carbon sources at elevated temperature . The crd1⌬ mutant loses viability on both fermentable and nonfermentable carbon sources at elevated temperature, and it does not form colonies from single cells seeded on YPD plates (Jiang et al, 1999(Jiang et al, , 2000Zhong et al, 2004). The pgs1⌬ mutant exhibits the most severe growth defects and cannot grow at all at 37°C, even on glucose (Chang et al, 1998a;Dzugasova et al, 1998).…”

“…Disruption of PGS1, the structural gene encoding PGP synthase, results in the complete loss of both PG and CL (Janitor et al, 1996;Chang et al, 1998a). The crd1⌬ mutant, which lacks CL synthase, has no detectable CL but accumulates PG (Jiang et al, 1997;Chang et al, 1998b;Tuller et al, 1998;Jiang et al, 2000;Pfeiffer et al, 2003;Zhong et al, 2004). The human taffazin gene (TAZ1), which is associated with Barth syndrome, encodes a transacylase that may be involved in the remodeling of CL (Xu et al, 2003).…”

“…Mutant cells of crd1⌬ grown in the presence of fermentable or nonfermentable carbon sources segregate large numbers of petites (respiratory incompetent cells) after prolonged culture at elevated temperature (Jiang et al, 2000;Zhong et al, 2004). The pgs1⌬ mutant was initially determined to be "petite lethal" because the mutant cells did not survive ethidium bromide mutagenesis, which induces petite formation (Janitor and Subik, 1993;Dzugasova et al, 1998).…”

“…Zhong et al, 2004). The pgs1⌬ mutant was initially determined to be "petite lethal" because the mutant cells did not survive ethidium bromide mutagenesis, which induces petite formation (Janitor and Subik, 1993;Dzugasova et al, 1998).…”