Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype
2021; Elsevier BV; Volume: 298; Issue: 1 Linguagem: Inglês
10.1016/j.jbc.2021.101462
ISSN1083-351X
AutoresPaulína Káňovičová, Petra Čermáková, Dominika Kubalová, Lenka Bábelová, Petra Veselá, Martin Valachovič, Jakub Zahumenský, Anton Horváth, J Malínský, Mária Balážová,
Tópico(s)Genetic Neurodegenerative Diseases
ResumoBarth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies. Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies. Mitochondria represent a highly specialized and dynamic functional organelle comprised of two structurally distinct lipid bilayers, the inner and outer mitochondrial membranes. Two negatively charged phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL) are highly specific for mitochondria. CL is an atypical phospholipid with four acyl chains, which exhibits numerous functions in mitochondrial bioenergetics and biogenesis (1Basu Ball W. Neff J.K. Gohil V.M. The role of nonbilayer phospholipids in mitochondrial structure and function.FEBS Lett. 2018; 592: 1273-1290Crossref PubMed Scopus (73) Google Scholar). PG is an obligatory CL precursor and usually a low-abundance phospholipid. Outside mitochondria, PG fulfills essential roles in specific membranes of some eukaryotic organisms (2Dugail I. Kayser B.D. Lhomme M. Specific roles of phosphatidylglycerols in hosts and microbes.Biochimie. 2017; 141: 47-53Crossref PubMed Scopus (14) Google Scholar, 3Furse S. Is phosphatidylglycerol essential for terrestrial life?.J. Chem. Biol. 2017; 10: 1-9Crossref PubMed Scopus (18) Google Scholar). For example, it is the preferred phospholipid in thylakoid membranes of photosynthetic organisms (4Sato N. Roles of the acidic lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol in photosynthesis: Their specificity and evolution.J. Plant Res. 2004; 117: 495-505Crossref PubMed Scopus (93) Google Scholar) or an important component of the pulmonary surfactant in mammals (5Günther A. Ruppert C. Schmidt R. Markart P. Grimminger F. Walmrath D. Seeger W. Surfactant alteration and replacement in acute respiratory distress syndrome.Respir. Res. 2001; 2: 353-364Crossref PubMed Scopus (191) Google Scholar, 6Voelker D.R. Numata M. Phospholipid regulation of innate immunity and respiratory viral infection.J. Biol. Chem. 2019; 294: 4282-4289Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In addition, there is a growing body of evidence that PG can, at least partially, substitute some functions of CL when it is missing (7Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), or that it may in fact be PG itself that performs some of the functions originally attributed to CL (8Vaena de Avalos S. Su X. Zhang M. Okamoto Y. Dowhan W. Hannun Y.A. The phosphatidylglycerol/cardiolipin biosynthetic pathway is required for the activation of inositol phosphosphingolipid phospholipase C, Isc1p, during growth of Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280: 7170-7177Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 9Baile M.G. Sathappa M. Lu Y.-W. Pryce E. Whited K. McCaffery J.M. Han X. Alder N.N. Claypool S.M. Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.J. Biol. Chem. 2014; 289: 1768-1778Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In general, a decrease in mitochondrial CL content at the expense of PG or other anionic lipids is the most frequently described pathological change in CL profile associated with a large number of diseases (10Chicco A.J. Sparagna G.C. Role of cardiolipin alterations in mitochondrial dysfunction and disease.Am. J. Physiol. Cell Physiol. 2007; 292: C33-44Crossref PubMed Scopus (478) Google Scholar).Final steps of CL biosynthesis include remodeling of the immature CL molecule. The newly synthesized CL is first converted to monolyso-CL (MLCL) and subsequently reacylated to mature CL by a transacylase or acyltransferase (11Claypool S.M. Koehler C.M. The complexity of cardiolipin in health and disease.Trends Biochem. Sci. 2012; 37: 32-41Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). This way, an optimal, tissue-specific fatty acid composition of CL is achieved. Mutations in the MLCL transacylase tafazzin, encoded by the TAZ gene, were identified as the primary cause of the X chromosome-linked recessive disease called Barth syndrome (BTHS). Clinically, BTHS is characterized by abnormal mitochondria, dilated cardiomyopathy, neutropenia, skeletal myopathy, growth delay, exercise intolerance, and increased level of organic acid in the urine (7Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 8Vaena de Avalos S. Su X. Zhang M. Okamoto Y. Dowhan W. Hannun Y.A. The phosphatidylglycerol/cardiolipin biosynthetic pathway is required for the activation of inositol phosphosphingolipid phospholipase C, Isc1p, during growth of Saccharomyces cerevisiae.J. Biol. Chem. 2005; 280: 7170-7177Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As a result of TAZ mutations, BTHS patients exhibit decreased levels of CL and accumulate MLCL in mitochondria. Besides that, both PG and CL of these patients contain reduced amounts of linoleic acid (C18:2), a characteristic acyl chain found in mature mammalian CL (12Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (300) Google Scholar).Several relevant studies have demonstrated the importance of the yeast Saccharomyces cerevisiae as a model for studying lipid biosynthesis disorders, including BTHS. Steps of CL synthesis are highly evolutionarily conserved; therefore, the yeast taz1Δ mutant has been used as a simple model to study defects resulting from altered remodeling and deficiency of CL (13Gu Z. Valianpour F. Chen S. Vaz F.M. Hakkaart G.A. Wanders R.J.A. Greenberg M.L. Aberrant cardiolipin metabolism in the yeast taz1 mutant: A model for Barth syndrome.Mol. Microbiol. 2004; 51: 149-158Crossref PubMed Scopus (172) Google Scholar). Indeed, heterologous expression of the human TAZ gene was sufficient to rescue the phenotype of taz1Δ yeast cells (14Ma L. Vaz F.M. Gu Z. Wanders R.J.A. Greenberg M.L. The human TAZ gene complements mitochondrial dysfunction in the yeast taz1Delta mutant. Implications for Barth syndrome.J. Biol. Chem. 2004; 279: 44394-44399Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), which made the yeast BTHS model attractive for studies focused on molecular mechanisms underlying the BTHS pathology. There is however a remarkable difference between human BTHS and yeast taz1Δ cells, a significantly lower PG content in the latter (12Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (300) Google Scholar). The amount of PG in wild-type yeast is at the limit of detection, even under conditions of increased CL biosynthesis during active respiration (15Simocková M. Holic R. Tahotná D. Patton-Vogt J. Griac P. Yeast Pgc1p (YPL206c) controls the amount of phosphatidylglycerol via a phospholipase C-type degradation mechanism.J. Biol. Chem. 2008; 283: 17107-17115Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar), and it is not much increased in the absence of Taz1 (9Baile M.G. Sathappa M. Lu Y.-W. Pryce E. Whited K. McCaffery J.M. Han X. Alder N.N. Claypool S.M. Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.J. Biol. Chem. 2014; 289: 1768-1778Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Gu Z. Valianpour F. Chen S. Vaz F.M. Hakkaart G.A. Wanders R.J.A. Greenberg M.L. Aberrant cardiolipin metabolism in the yeast taz1 mutant: A model for Barth syndrome.Mol. Microbiol. 2004; 51: 149-158Crossref PubMed Scopus (172) Google Scholar). This aspect could be of crucial importance, as recent data indicated that PG levels per se affect mitochondrial morphology and function (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar, 17Matsumura A. Higuchi J. Watanabe Y. Kato M. Aoki K. Akabane S. Endo T. Oka T. Inactivation of cardiolipin synthase triggers changes in mitochondrial morphology.FEBS Lett. 2018; 592: 209-218Crossref PubMed Scopus (16) Google Scholar). Low levels of PG thus reduce the reliability of the yeast taz1Δ mutant as a plausible BTHS model, as it cannot be ruled out that the relatively higher PG participates in generating a complex BTHS phenotype in mammals.PG level in yeast is controlled by PG-phosphate (PGP) synthase Pgs1 and PG-specific phospholipase Pgc1 through an effective mechanism capable of fast, wide-range, and bidirectional PG regulation (18Kubalová D. Káňovičová P. Veselá P. Awadová T. Džugasová V. Daum G. Malínský J. Balážová M. The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol.FEMS Yeast Res. 2019; 19foz045Crossref Scopus (2) Google Scholar). Deletion of PGC1 leads, under conditions of sustained Pgs1 activity, to nonspecific accumulation of PG without distinct side effects on the amounts of other phospholipids, including CL, but with apparent adverse effects on mitochondrial fusion and respiration (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar). Therefore, in this study, we tested whether the deletion of PGC1 in taz1Δ cells could generate a yeast BTHS model that would better simulate the PG/CL ratio detected in mammalian cells.As an alternative approach to regulate PG levels in the yeast BTHS model, we tested the effect of valproic acid (VPA) treatment on the analyzed yeast strains. VPA is a broad-spectrum antiepileptic drug that has been widely used for more than 60 years and is approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorders and neuralgia. Although the mechanism of its therapeutic effect is not yet clear, it is known that among other effects, VPA inhibits de novo synthesis of inositol from glucose-6-phosphate by indirectly blocking myo-inositol phosphate synthase (19Yu W. Daniel J. Mehta D. Maddipati K.R. Greenberg M.L. MCK1 is a novel regulator of myo-inositol phosphate synthase (MIPS) that is required for inhibition of inositol synthesis by the mood stabilizer valproate.PLoS One. 2017; 12e0182534Crossref Scopus (15) Google Scholar). In yeast, inositol inhibits PGP synthase, Pgs1, catalyzing the rate-limiting step of PG de novo synthesis. Accordingly, increased biosynthesis of PG and CL in response to VPA treatment has been reported in yeast during fermentation (20Ju S. Greenberg M.L. Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis.Mol. Microbiol. 2003; 49: 1595-1603Crossref PubMed Scopus (40) Google Scholar). Here we report that VPA changes the content of anionic phospholipids in yeast grown on nonfermentable carbon source only moderately, but is capable of restoring the coupling between the electron transport and ATP synthesis, affected in taz1Δ and pgc1Δtaz1Δ mutants.ResultsPhospholipid characterization of pgc1Δtaz1Δ double mutantIn humans, the cellular PG content is in general higher compared with yeast (12Vreken P. Valianpour F. Nijtmans L.G. Grivell L.A. Plecko B. Wanders R.J. Barth P.G. Defective remodeling of cardiolipin and phosphatidylglycerol in Barth syndrome.Biochem. Biophys. Res. Commun. 2000; 279: 378-382Crossref PubMed Scopus (300) Google Scholar, 21Lu Y.-W. Galbraith L. Herndon J.D. Lu Y.-L. Pras-Raves M. Vervaart M. Van Kampen A. Luyf A. Koehler C.M. McCaffery J.M. Gottlieb E. Vaz F.M. Claypool S.M. Defining functional classes of Barth syndrome mutation in humans.Hum. Mol. Genet. 2016; 25: 1754-1770Crossref PubMed Scopus (39) Google Scholar). Following the TAZ gene disruption, PG levels in human cells further increase (21Lu Y.-W. Galbraith L. Herndon J.D. Lu Y.-L. Pras-Raves M. Vervaart M. Van Kampen A. Luyf A. Koehler C.M. McCaffery J.M. Gottlieb E. Vaz F.M. Claypool S.M. Defining functional classes of Barth syndrome mutation in humans.Hum. Mol. Genet. 2016; 25: 1754-1770Crossref PubMed Scopus (39) Google Scholar). Under conditions of sustained production, in principle there are two ways how to increase the generally low PG content in yeast—either its utilization as a CL precursor or its direct degradation can be compromised. Accumulation of PG in a yeast strain lacking the CL synthase Crd1 rescued some defects caused by the loss of CL (7Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Deletion of PGC1 gene coding for PG-specific phospholipase C (15Simocková M. Holic R. Tahotná D. Patton-Vogt J. Griac P. Yeast Pgc1p (YPL206c) controls the amount of phosphatidylglycerol via a phospholipase C-type degradation mechanism.J. Biol. Chem. 2008; 283: 17107-17115Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) resulted also in PG accumulation, but at normal CL levels in mitochondria (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar). Therefore, to prepare a yeast BTHS model with an elevated PG content, we deleted PGC1 gene in taz1Δ strain. All the experiments were performed in media without inositol to stimulate PGP synthase, Pgs1 (22Zhong Q. Greenberg M.L. Regulation of phosphatidylglycerophosphate synthase by inositol in Saccharomyces cerevisiae is not at the level of PGS1 mRNA abundance.J. Biol. Chem. 2003; 278: 33978-33984Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23He Q. Greenberg M.L. Post-translational regulation of phosphatidylglycerolphosphate synthase in response to inositol.Mol. Microbiol. 2004; 53: 1243-1249Crossref PubMed Scopus (29) Google Scholar), and with nonfermentable carbon sources, to stimulate mitochondrial activity.As expected, mitochondria of the pgc1Δtaz1Δ mutant exhibited a combined lipid composition phenotype: (i) similar to the taz1Δ strain, the defect in CL remodeling led to the MLCL accumulation in the double deletion strain and (ii) the absence of Pgc1 resulted in increased levels of PG in this strain, probably mainly at the expense of PC fraction (Fig. 1A). Besides the PG accumulation and PC depletion, no statistically significant difference between the taz1Δ and pgc1Δtaz1Δ strains was detected.Previously, we reported differences between the fatty acid composition of PG accumulated in pgc1Δ and crd1Δ strains (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar). Similarly to these strains, the acyl chain composition of mitochondrial PG in taz1Δ significantly differed from the wild type (Fig. 1B). Specifically, in PG isolated from the mitochondria of taz1Δ cells, we found increased palmitoleic acid (C16:1; 145 ± 30% of the wild type value) and oleic acid (C18:1; 125 ± 14% of the wild-type value). This increase was fully at the expense of stearic acid (C18:0; 33 ± 10% of the wild-type value), which was similar to the changes detected in pgc1Δ and pgc1Δtaz1Δ strains. Consistent with previously published data (9Baile M.G. Sathappa M. Lu Y.-W. Pryce E. Whited K. McCaffery J.M. Han X. Alder N.N. Claypool S.M. Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.J. Biol. Chem. 2014; 289: 1768-1778Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Gu Z. Valianpour F. Chen S. Vaz F.M. Hakkaart G.A. Wanders R.J.A. Greenberg M.L. Aberrant cardiolipin metabolism in the yeast taz1 mutant: A model for Barth syndrome.Mol. Microbiol. 2004; 51: 149-158Crossref PubMed Scopus (172) Google Scholar), the CL-bound fatty acids content in the analyzed strains depended solely on Taz1 activity, as pgc1Δ profile was indistinguishable from wild type, and similarly, pgc1Δtaz1Δ profile was comparable with taz1Δ strain. In the latter two strains, we observed a pronounced decrease in unsaturated palmitoleic (C16:1) and oleic acid (C18:1) and an increase in saturated palmitic (C16:0) and stearic acid (C18:0) bound to CL molecules. Similarly, the fatty acid composition of MLCL was not affected by the further deletion of PGC1 gene (Fig. 1B).Sterols and sterol esters in pgc1Δtaz1Δ double mutantBesides defects in CL remodeling, decreased cholesterol synthesis has been observed in lymphoblasts of BTHS patients after serum starvation (24Spencer C.T. Bryant R.M. Day J. Gonzalez I.L. Colan S.D. Thompson W.R. Berthy J. Redfearn S.P. Byrne B.J. Cardiac and clinical phenotype in Barth syndrome.Pediatrics. 2006; 118: e337-e346Crossref PubMed Scopus (171) Google Scholar, 25Hauff K.D. Hatch G.M. Reduction in cholesterol synthesis in response to serum starvation in lymphoblasts of a patient with Barth syndrome.Biochem. Cell Biol. 2010; 88: 595-602Crossref PubMed Scopus (12) Google Scholar). Therefore, we tested levels of ergosterol and its derivatives in the yeast mutants mimicking BTHS.We compared the neutral lipid content in wild type, pgc1Δ, taz1Δ, and pgc1Δtaz1Δ strains. Significantly decreased ergosterol levels in both strains lacking TAZ1 gene were detected. Approx. 20% drop of ergosterol content was detected in these strains independent of the presence of Pgc1 (Fig. 2A). The analysis also revealed an increased fraction of sterol esters (SE) in the neutral lipids of all deletion mutants analyzed. In both single deletion mutants, SE fraction was significantly overrepresented if compared with the wild type. Importantly, this fraction further increased in pgc1Δtaz1Δ cells (Fig. 2B), suggesting different mechanisms of SE elevation in each of the single mutants.Figure 2TAZ1 and/or PGC1 deletions affect ergosterol metabolism. Wild type, pgc1Δ, taz1Δ, and pgc1Δtaz1Δ strains (see Table 1 for details) were cultivated in SMDGE I- medium for 24 h. Yeast homogenates prepared by zymolyase treatment were used for neutral lipid extraction. Extracted lipids were separated by TLC and scanned at 193 nm. Ergosterol content (A) and a relative fraction of SE in total neutral lipids (TAG+SE; B) were determined. Data represent mean values from eight independent experiments ±SEM. Statistically significant differences between mutant strains and wild type, or pgc1Δtaz1Δ and taz1Δ strain, or pgc1Δtaz1Δ and pgc1Δ strain are marked. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. SE, sterol esters; TAG, triacylglycerol; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of elevated PG on mitochondrial morphology and function in pgc1Δtaz1Δ cellsPhospholipid composition of mitochondrial membranes affects the organelle morphology and function. In cells and tissues of BTHS patients, abnormal mitochondrial ultrastructure and increased mass of mitochondria were observed (26Xu Y. Sutachan J.J. Plesken H. Kelley R.I. Schlame M. Characterization of lymphoblast mitochondria from patients with Barth syndrome.Lab. Invest. 2005; 85: 823-830Crossref PubMed Scopus (118) Google Scholar, 27Clarke S.L.N. Bowron A. Gonzalez I.L. Groves S.J. Newbury-Ecob R. Clayton N. Martin R.P. Tsai-Goodman B. Garratt V. Ashworth M. Bowen V.M. McCurdy K.R. Damin M.K. Spencer C.T. Toth M.J. et al.Barth syndrome.Orphanet J. Rare Dis. 2013; 8: 23Crossref PubMed Scopus (204) Google Scholar, 28Gonzalvez F. D'Aurelio M. Boutant M. Moustapha A. Puech J.-P. Landes T. Arnauné-Pelloquin L. Vial G. Taleux N. Slomianny C. Wanders R.J. Houtkooper R.H. Bellenguer P. Møller I.M. Gottlieb E. et al.Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation.Biochim. Biophys. Acta. 2013; 1832: 1194-1206Crossref PubMed Scopus (116) Google Scholar, 29Schlame M. Cardiolipin remodeling and the function of tafazzin.Biochim. Biophys. Acta. 2013; 1831: 582-588Crossref PubMed Scopus (107) Google Scholar). Similarly in yeast taz1Δ mutant, aberrant cristae morphology and swollen mitochondria have been observed (9Baile M.G. Sathappa M. Lu Y.-W. Pryce E. Whited K. McCaffery J.M. Han X. Alder N.N. Claypool S.M. Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.J. Biol. Chem. 2014; 289: 1768-1778Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 30Claypool S.M. Boontheung P. McCaffery J.M. Loo J.A. Koehler C.M. The cardiolipin transacylase, tafazzin, associates with two distinct respiratory components providing insight into Barth syndrome.Mol. Biol. Cell. 2008; 19: 5143-5155Crossref PubMed Scopus (86) Google Scholar). To further characterize the pgc1Δtaz1Δ strain, we compared mitochondria of this strain with those of the wild type, pgc1Δ, and taz1Δ strains.To do this, we cultivated wild type, pgc1Δ, taz1Δ, and pgc1Δtaz1Δ yeast in SMDGE medium without inositol for 24 h and visualized mitochondria in situ using Mitotracker-Red fluorescent dye (Fig. 3A). As expected, aberrant mitochondria were occasionally observed in taz1Δ cells. The aberrant mitochondria were of a characteristic ring shape. The frequency of this aberration was almost tripled in the double deletion strain (Fig. 3, A and B). As apparent at the ultrastructural level, the aberrant mitochondria contained excessive amounts of juxtaposed extended membranes/cristae. At the same time, the morphology of "normal," rod-like mitochondria of the same strain did not significantly differ from the wild type (Fig. 4), with the following exception: deletion of PGC1 gene in wild-type strain increased mitochondrial fragmentation as we also reported previously (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar). In accordance with this observation, a similar tendency could be recognized when mitochondrial morphology of taz1Δ and pgc1Δtaz1Δ strains were compared.Figure 3Deletion of PGC1 increases occurrence of morphologically aberrant mitochondria in taz1Δ cells. Mitochondrial morphology was visualized by Mitotracker Red staining in wild type, pgc1Δ, taz1Δ, and pgc1Δtaz1Δ cells grown in SMDGE I- medium for 24 h. Maximum intensity projections of six consecutive confocal sections are presented (A). Abnormal, ring-shaped mitochondria were detected in mutants lacking TAZ1 gene (arrowheads). The occurrence of ring mitochondria was quantified in each strain (B). Sets of 200 cells were analyzed in three biological replicates of the experiment. Statistically significant differences between mutant strains and the wild type or pgc1Δtaz1Δ and taz1Δ strain are marked. Bar: 5 μm. ∗∗∗p < 0.001. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Mitochondria in pgc1Δtaz1Δ mutant represent two populations of differential fine structure. Ultrathin sections of pgc1Δtaz1Δ cells imaged by transmission electron microscopy are presented. Normal, rod-like mitochondria (A and B) and aberrant, ring-shaped mitochondria (C–G) are shown in cellular context (A–C) and in detail (D–G). Relevant cellular compartments are marked in all figures. Bars: 1 μm (A–C), 500 nm (D–G). C, cytoplasm; LD, lipid droplet; M, mitochondrion; N, cell nucleus; V, vacuole.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The morphology of mitochondria is tightly connected with mitochondrial functionality. For example, the aforementioned mitochondrial fragmentation in pgc1Δ cells was accompanied by decreased coupling between electron transport and ATP synthesis (16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. Biophys. Acta. 2016; 1857: 34-45Crossref PubMed Scopus (19) Google Scholar). Similarly, the formation of aberrant, flat mitochondrial sheets correlated with extensive respiratory defects of the crd1Δ strain lacking cardiolipin synthase. These included suboptimal respiratory chain function due to destabilization of respiratory chain supercomplexes (7Jiang F. Ryan M.T. Schlame M. Zhao M. Gu Z. Klingenberg M. Pfanner N. Greenberg M.L. Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J. Biol. Chem. 2000; 275: 22387-22394Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 9Baile M.G. Sathappa M. Lu Y.-W. Pryce E. Whited K. McCaffery J.M. Han X. Alder N.N. Claypool S.M. Unremodeled and remodeled cardiolipin are functionally indistinguishable in yeast.J. Biol. Chem. 2014; 289: 1768-1778Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 16Pokorná L. Čermáková P. Horváth A. Baile M.G. Claypool S.M. Griač P. Malínský J. Balážová M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.Biochim. B
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