Asymmetrical distribution of cardiolipin in yeast inner mitochondrial membrane triggered by carbon catabolite repression

131

Cardiolipin (CL) is an acidic phospholipid present almost exclusively in membranes harboring respiratory chain complexes. We have previously shown that, in Saccharomyces cerevisiae, CL provides stability to respiratory chain supercomplexes and CL synthase enzyme activity is reduced in several respiratory complex assembly mutants. In the current study, we investigated the interdependence of the mitochondrial respiratory chain and CL biosynthesis. Pulse-labeling experiments showed that in vivo CL biosynthesis was reduced in respiratory complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) and oxidative phosphorylation complex V (ATP synthase) assembly mutants. CL synthesis was decreased in the presence of CCCP, an inhibitor of oxidative phosphorylation that reduces the pH gradient but not by valinomycin or oligomycin, both of which reduce the membrane potential and inhibit ATP synthase, respectively. The inhibitors had no effect on phosphatidylglycerol biosynthesis or CRD1 gene expression. These results are consistent with the hypothesis that in vivo CL biosynthesis is regulated at the level of CL synthase activity by the ⌬pH component of the proton-motive force generated by the functional electron transport chain. This is the first report of regulation of phospholipid biosynthesis by alteration of subcellular compartment pH. Cardiolipin (CL)1 is an acidic glycerophospholipid with a unique dimeric structure consisting of four fatty acyl chains (1). It is almost exclusively present in membranes designed to generate an electrochemical potential gradient for ATP synthesis, including the mitochondrial inner membrane and bacterial plasma membrane. CL plays a vital role in mitochondrial structure and function by providing osmotic stability to mitochondrial membranes (2) and by specifically interacting with many inner membrane proteins (reviewed in Refs. 3 and 4). Normal levels of CL are required for optimal mitochondrial bioenergetic functions (5, 6). Decreased CL levels are observed in cells undergoing apoptosis (7-9) and aging (10, 11) and in fibroblasts of Barth syndrome patients (12). Thus, a detailed understanding of the regulation of CL levels will provide important insights into the manifestation of these conditions. CL is associated with major proteins of the mitochondrial respiratory chain including NADH dehydrogenase (complex I) (13), ubiquinol:cytochrome c reductase (complex III) (13-17), and cytochrome c oxidase (complex IV) (18, 19), the ATP synthase (complex V) (20), and the carrier proteins for phosphate (21) and adenine nucleotides (22). CL modulates the catalytic activities of proteins, as seen in the case of the ADP-ATP carrier (5, 23, 24) and complex IV (25), and/or provides stability as reported for complex III (17) and complex IV (19). CL binds specifically and irreversibly to cytochrome c (26), providing a membrane attachment site for cytochrome c and limiting the soluble pool of the protein. X-ray crystallography studies have further confirmed the specific interaction...

Section: Cardiolipin (Cl)supporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Cardiolipin Biosynthesis and Mitochondrial Respiratory Chain Function Are Interdependent

Gohil

Hayes

Matsuyama

et al. 2004

131

“…It has been used to specifically visualize mitochondria in living cells using fluorescence microscopy and for quantification of CL in mitochondria of whole cells (1). Wild type cells when treated with this dye clearly show fluorescent structures (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In eukaryotic cells cardiolipin (CL) 1 and its synthesis are predominantly localized to the mitochondrial inner membrane (1)(2)(3). Due to its cellular distribution, CL is thought to be involved in critical cellular functions such as cytochrome c oxidase function (4), binding of matrix Ca 2ϩ (5), maintenance of mitochondrial membrane permeability (6,7), and mitochondrial protein import (8 -12).…”

mentioning

confidence: 99%

Isolation and Characterization of the Gene (CLS1 ) Encoding Cardiolipin Synthase in Saccharomyces cerevisiae

Chang¹,

Heacock²,

Mileykovskaya³

et al. 1998

197

193

In eukaryotic cells, cardiolipin (CL) synthase catalyzes the final step in the synthesis of CL from phosphatidylglycerol and CDP-diacylglycerol. CL and its synthesis are localized predominantly to the mitochondrial inner membrane, and CL is generally thought to be an essential component of many mitochondrial processes. By using homology searches for genes potentially encoding phospholipid biosynthetic enzymes, we have cloned the gene (CLS1) encoding CL synthase in Saccharomyces cerevisiae. Overexpression of the CLS1 gene under its endogenous promoter or the inducible GAL1 promoter in yeast and expression of CLS1 in baculovirus-infected insect cells resulted in elevated CL synthase activity. Disruption of the CLS1 gene in a haploid yeast strain resulted in the loss of CL synthase activity, no detectable CL, a 5-fold elevation in phosphatidylglycerol levels, and lack of staining of mitochondria by a dye with high affinity for CL. The cls1::TRP1 null mutant grew on both fermentable and non-fermentable carbon sources but more poorly on the latter. The level and activity of cytochrome c oxidase was normal, and a dye whose accumulation is dependent on membrane proton electrochemical potential effectively stained the mitochondria. These results definitively identify the gene encoding the CL synthase of yeast.

“…Therefore, in contrast to the other phospholipids, synthesis of the mitochondrial lipid CL increases during the stationary phase during growth in glucose, a time in which mitochondrial development increases to respond to the switch from fermentative to respiratory growth. In fact, the relative CL composition doubles as cells enter the stationary phase (35,36). CL may play an important role in cell survival during the stationary growth phase, as both pgs1 and crd1 null mutants lose viability during prolonged liquid culture at elevated temperature (19).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Phosphatidylglycerophosphate Synthase by Inositol in Saccharomyces cerevisiae Is Not at the Level of PGS1 mRNA Abundance

Zhong

Greenberg

2003

Phosphatidylglycerophosphate synthase catalyzes the committed step in the synthesis of the mitochondrial phospholipid cardiolipin. We showed previously that phosphatidylglycerophosphate synthase activity in Saccharomyces cerevisiae is increased in conditions favoring mitochondrial development and during growth in the absence of inositol. Interestingly, the regulatory effects of inositol were not altered in ino2, ino4, or opi1 mutants suggesting that regulation in response to inositol is not at the level of gene transcription. We report here that steady state mRNA levels of the PGS1 gene, which encodes phosphatidylglycerophosphate synthase, were not altered by inositol or choline. Growth in the presence of the inositol-depleting drug valproate led to an increase in phosphatidylglycerophosphate synthase activity unaccompanied by increased PGS1 mRNA. PGS1 mRNA abundance was not decreased in ino2 or ino4 mutants and was unaffected in an opi1 mutant. Therefore, regulation of phosphatidylglycerophosphate synthase by inositol is not mediated at the level of mRNA abundance and does not require the INO2-INO4-OPI1 regulatory circuit. PGS1 was increased in glycerol/ethanol compared with glucose media and was maximally expressed as cells entered the stationary phase. Deletion of the mitochondrial genome did not affect PGS1 expression. Thus, whereas inositol controls phosphatidylglycerophosphate synthase activity, regulation of PGS1 expression occurs primarily in response to mitochondrial development cues.