Molecular Microbiology (2004) 51 (1), 149–158 doi:10.1046/j.1365-2958.2003.03802.x© 2003 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKMMIMolecular Microbiology1365-2958Blackwell Publishing Ltd, 200351 1149158 Original Article Aberrant cardiolipin metabolism in taz1 mutantZ. Gu et al. Accepted 28 August, 2003. *For correspondence. [email protected]; Tel. ( + 1) 313 577 5202; Fax( + 1) 313 577 6891. Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome Zhiming Gu, 1 Fredoen Valianpour, 2 Shuliang Chen, 1 Frederic M. Vaz, 2 Gertjan A. Hakkaart, 2 Ronald J. A. Wanders 2 and Miriam L. Greenberg 1 * 1 Department of Biological Science, Wayne State University, Detroit, MI 48202, USA. 2 Laboratory of Genetic Metabolic Diseases (GMZ), Academic Medical Center (AMC), University of Amsterdam (UvA), 1100 DE Amsterdam, The Netherlands. SummaryIn eukaryotic cells, the acyl species of the phospho-lipid cardiolipin (CL) are more highly unsaturated thanthose of the other membrane phospholipids. Defec-tive acylation of CL with unsaturated fatty acids anddecreased total CL are associated with Barth syn-drome, an X-linked cardio- and skeletal myopathyattributed to a defect in the gene G4.5 (also known astafazzin). We constructed a yeast mutant ( taz1 ) con-taining a null mutation in the homologue of the human G4.5 gene. The yeast taz1 DDDD mutant was temperaturesensitive for growth in ethanol as sole carbon source,but grew normally on glucose or glycerol plus etha-nol. Total CL content was reduced in the taz1 DDDD mutant,and monolyso-CL accumulated. The predominant CLacyl species found in wild-type cells, C18:1 and C16:1,were markedly reduced in the mutant, whereas CLmolecules containing saturated fatty acids werepresent. Interestingly, CL synthesis increased in themutant, whereas expression of the CL structuralgenes CRD1 and PGS1 did not, suggesting that denovo biosynthetic enzyme activities are regulated byCL acylation. These results indicate that the taz1 DDDD mutant is an excellent genetic tool for the study of CLremodelling and may serve as a model system for thestudy of Barth syndrome.Introduction Cardiolipin (CL) is a phospholipid that is distributed almostexclusively in the mitochondrial inner membrane ofeukaryotic cells, and is important for the optimal perfor-mance of this organelle (Koshkin and Greenberg, 2000;2002; Schlame et al ., 2000). CL has a unique dimericstructure consisting of two phosphatidyl moieties linked bya glycerol bridge. Thus, unlike other membrane phospho-lipids, it has four fatty acyl chains and two phosphategroups. In higher eukaryotes, the fatty acyl groups in CLare more highly unsaturated than those of the other mem-brane phospholipids. How CL acquires its unique acylpattern is not known, but at least two mechanisms arepossible, including substrate sorting/preference by theenzymes of CL de novo synthesis and remodelling ofnewly synthesized CL.Remodelling of CL by a deacylation–reacylation cyclewas first proposed by Schlame and Rustow (1990). In thismodel, newly synthesized CL is deacylated, possibly byphospholipase A, to form monolyso-CL (MLCL), which isthen condensed with acyl-CoA to form mature CL. Evi-dence supporting this model comes from the detection of[ 14 C]-MLCL and [ 14 C]-dilyso-CL (DLCL) in rat liver cellslabelled with [ 14 C]-glycerol 3-phosphate. Labelling ofDLCL was increased or decreased by stimulation or inhi-bition of mitochondrial phospholipase A2, whereas endog-enous CL was resistant to hydrolytic degradation. MLCL,but not DLCL, could be reacylated with [ 14 C]-linoleoyl res-idues derived from 1-palmitoyl-2-[ 14 C]-linoleoyl-phosphati-dylcholine in mitochondrial extracts from rat liver cells. Inaddition, exogenous CL was also reacylated with [ 14 C]-linoleoyl residues. These data are consistent with a deacy-lation–reacylation cycle (Schlame and Rustow, 1990).Further evidence for this mechanism is apparent in stud-ies with an inhibitor of de novo phospholipid synthesis,cyclopentenylcytosine (CPEC). Treatment of H9c2 cardiacmyoblasts with CPEC did not affect incorporation of[ 14 C]-linoleic acid into CL, suggesting that incorporationof linoleic acid into CL is not likely to be mediated by denovo CL synthesis (Hatch and McClarty, 1996). Finally,isolated intact rat heart mitochondria contain coenzymeA-dependent acyltransferases that reacylate MLCL to CL(Ma et al ., 1999). Taken together, these studies suggestthat the unique fatty acid composition of CL is most prob-ably achieved by remodelling.Recent evidence for the importance of CL remodellingis provided by biochemical analyses of cells from patientswith Barth syndrome, a severe X-chromosome-linked dis-order characterized by cardiac and skeletal myopathy,150 Z. Gu et al. © 2003 Blackwell Publishing Ltd, Molecular Microbiology , 51 , 149–158 neutropenia, abnormal mitochondria and increased levelsof organic acids in the urine (Barth et al ., 1983; 1996;Bolhuis et al ., 1991; D’Adamo et al ., 1997). The diseaseis often fatal in childhood because of cardiac failure orsepsis (Barth et al ., 1983; Bolhuis et al ., 1991). The genethat is defective in Barth syndrome, G4.5 (also known astafazzin) has been cloned (Bione et al ., 1996), andsequence analysis of the gene suggested that it encodesa putative phospholipid acyltransferase (Neuwald, 1997).A decrease in CL levels and reduced incorporation oflinoleic acid (18:2) into both CL and the precursor lipidphosphatidylglycerol were observed in fibroblasts fromBarth syndrome patients, which led to the hypothesis thatthe G4.5 gene product is required for CL remodelling(Vreken et al ., 2000). Further analysis of the lipid compo-sition in Barth syndrome cells showed a lack of tetralino-leoyl-CL (L 4 -CL) (Schlame et al ., 2002; Valianpour et al .,2002a,b), the most predominant CL species in mitochon-dria from normal skeletal and cardiac muscles.How the CL remodelling defect associated with Barthsyndrome leads to the related pathologies is not known.To begin to address this question, we constructed a yeastmutant ( taz1 ) defective in the homologue of the Barthsyndrome gene G4.5 . The yeast Saccharomyces cerevi-siae is currently the most tractable eukaryote in which tocharacterize CL remodelling. The yeast CL biosyntheticpathway is typical of that of higher eukaryotes (Schlameand Greenberg, 1997; Schlame and Hostetler, 1997). Thegenes