Cardiolipin (CL) is the signature lipid of the mitochondrial membrane and plays a key role in mitochondrial physiology and cell viability. The importance of CL is underscored by the finding that the severe genetic disorder Barth syndrome results from defective CL composition and acylation. Disruption of PGS1, which encodes the enzyme that catalyses the committed step of CL synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol and CL. The pgs1Δ mutant exhibits severe growth defects at 37°C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1Δ that grew at 37°C. The present review summarizes our analysis of suppression of pgs1Δ growth defects by a mutant that has a loss-of-function mutation in KRE5, a gene involved in cell wall biogenesis.
- anionic phospholipid
- cardiolipin (CL)
- cell wall biogenesis
- mitogen-activated protein kinase (MAPK)
Cardiolipin (CL) is an acidic glycerophospholipid with a unique dimeric structure consisting of four fatty acyl chains. It is ubiquitous in eukaryotes and primarily found in the mitochondrial inner membrane (reviewed in ). In vitro studies indicate that CL interacts with a large number of mitochondrial proteins and is required for optimal enzyme activities of the mitochondrial respiratory chain, NADH dehydrogenase (complex I), ubiquinol:cytochrome c reductase (complex III), cytochrome c oxidase (complex IV), the ATP synthase (complex V) and the carrier proteins for phosphate and adenine nucleotides (reviewed in ).
The importance of CL is underscored by the finding that mutations in the human tafazzin gene G4.5, that cause Barth syndrome, lead to defective CL remodelling . Barth syndrome is a life-threatening genetic disorder characterized by cardiomyopathy, neutropenia, skeletal myopathy and respiratory chain defects. The human tafazzin gene G4.5 encodes a transacylase that transfers linoleic acid from other phospholipids to CL . Mutations in tafazzin lead to a dramatic decrease in CL levels and reduced incorporation of linoleic acid (18:2) into CL and its precursor PG (phosphatidylglycerol) . Tetralinoleoyl-CL, the most predominant CL species in mitochondria from normal skeletal and heart muscle, is almost completely absent from Barth syndrome cells . This was the first direct association of aberrant CL metabolism with a human illness and confirmed the importance of CL in cellular function.
The past several years have witnessed an exciting increase in in vivo studies to elucidate the role of CL in mitochondrial function and cell viability. Identification of yeast genes encoding CL biosynthetic enzymes greatly facilitated this research. CRD1 encodes CL synthase [5–7] and PGS1, which was originally thought to encode phosphatidylserine synthase  codes for the first enzyme of the CL pathway, phosphatidylglycerolphosphate synthase . Identification of these genes allowed the construction of deletion mutants deficient in CL biosynthesis. Disruption of PGS1 results in the complete loss of both PG and CL [8,9]. The crd1Δ mutant has no detectable CL but accumulates PG [5–7,10–12]. Deletion of the yeast homologue of the human tafazzin gene, TAZ1, leads to decreased CL, aberrant CL acyl species and accumulation of monolysocardiolipin . In vivo comparisons of cell functions in wild-type and CL-deficient mutants confirmed the importance of CL in mitochondria. Mitochondria from the crd1Δ mutant, which lacks CL, exhibit a reduced membrane potential resulting in a decrease in the import of proteins into the mitochondria. Decreased activities of respiratory enzymes including ATP/ADP translocase, ATPase and cytochrome c oxidase are also observed . Biochemical analysis of mitochondrial bioenergetics revealed that CL-deficient mitochondria are coupled only under optimal conditions, and coupling is decreased during osmotic stress, increased respiration rate or elevated temperature [14–16]. In addition, CL stabilizes respiratory chain supercomplexes and individual complexes and is required to prevent the formation of the resting state of cytochrome c oxidase in the membrane .
In addition to mitochondrial bioenergetics, CL was found to play an essential role in maintaining cell viability at elevated temperatures. Mutants deficient in CL synthesis exhibit temperature-sensitive growth. The taz1Δ mutant is temperature-sensitive for growth on ethanol but grows well on other carbon sources at elevated temperatures . The crd1Δ mutant loses viability on both fermentable and non-fermentable carbon sources at elevated temperatures and does not form colonies from single cells [10,12,17]. The pgs1Δ mutant exhibits the most severe growth defects and cannot grow at all at 37°C, even on glucose [9,18]. While defective energetic coupling at elevated temperatures in crd1Δ [14,15] and taz1Δ  may explain temperature sensitivity of these mutants in non-fermentable medium, the reason for loss of viability on glucose is not known.
Defective cell wall in mutants lacking mitochondrial anionic phospholipids
In order to understand the essential functions of CL at elevated temperature, we took the genetic approach of isolating spontaneous suppressor mutants of pgs1Δ that grow at elevated temperatures. One of the suppressors was identified as a loss-of-function mutation of KRE5, a gene involved in cell wall biogenesis. This points to a connection between temperature-sensitive growth of CL-deficient mutants and cell wall defects , and is consistent with a large-scale screen to identify genes involved in cell wall biogenesis by Lussier et al. , who reported that disruption of the promoter of PGS1 leads to hypersensitivity to cell wall-perturbing agents such as zymolyase, Calcofluor White, papulacandin and caffeine.
The yeast cell wall is an essential organelle, which determines the shape and maintains cell integrity during growth and morphogenesis. It contains almost equal amounts of mannoproteins and glucans, including the fibrous β-1,3-glucan and the branched β-1,6-glucan. The third component, chitin represents <1% of the cell wall (reviewed in ). Chitin is enriched in bud scars with only a minor portion uniformly dispersed in the lateral wall. In response to weakening of the cell wall, chitin deposition is increased (reviewed in ). Together with β-1,3-glucan, it confers mechanical strength to the cell wall (reviewed in ).
Biochemical analysis of the cell wall in pgs1Δ and the kre5 suppressor strain provided clues to the suppression mechanism. β-1,3-Glucan is dramatically reduced in pgs1Δ . As a result, pgs1Δ mutant cells become enlarged and rounded. In the kre5 suppressor strain, β-1,3-glucan was dramatically increased, growth in the presence of Calcofluor White was improved, cell size and growth at elevated temperature were all restored . In addition, osmotic stabilization with sorbitol suppressed temperature sensitivity of all CL-deficient mutants at elevated temperatures , suggesting that deficiency in mitochondrial anionic phospholipids results in cell wall defects that lead to a temperature-sensitive growth phenotype. These findings are intriguing, as they suggest certain mitochondrial functions that require PG and CL are necessary for cell wall construction.
A connection between mitochondrial functions and cell wall biogenesis has been implicated in several previous studies as well. In addition to PGS1, Lussier et al. reported that mutations in four other genes with mitochondria-associated functions, IMP2′, IFM1, SMP2 and COX11, have cell wall defects . Three of these genes (IFM1, SMP2 and COX11) are required for mitochondrial DNA stability [22–24]. ECM10, a gene with unknown function identified in the same screen with cell wall defects , was found to localize in mitochondrial nucleoids and its overexpression results in extensive mitochondrial DNA aggregation . A genomewide screening for deletion mutants that exhibit increased resistance to K1 killer toxin, indicative of alterations in the cell surface, identified 17 deletion mutants affecting genes for respiration and ATP metabolism . All of the mutants are respiratory-deficient and four are involved in mitochondrial genome maintenance. Screening for new yeast mutants affecting mannosylphosphorylation of cell wall mannoproteins identified two genes with mitochondrial functions, YFH1 and PHB2 . YFH1 is the yeast homologue of the human frataxin gene implicated in the neurodegenerative disorder Friedreich's ataxia. Deletion of YFH1 results in excessive mitochondrial iron accumulation . PHB2 encodes a subunit of the prohibitin complex (Phb1p–Phb2p), the evolutionarily conserved protein that is involved in the regulation of mitochondrial morphology, proliferation and segregation and influences the replicative life span [29–31].
The identification of cell wall defects in mutants with mitochondrial dysfunction suggests that mitochondria may play a general role in the regulation of cell wall biogenesis. The decreased β-1,3-glucan level in the pgs1Δ mutant suggests an interesting connection between mitochondrial anionic phospholipids and glucan synthesis. Further investigation of the β-1,3-glucan biosynthetic pathway in CL-deficient mutants may reveal the important role of mitochondrial anionic lipids in the regulation of glucan synthesis. β-1,3-Glucan is synthesized by GS (β-1,3-glucan synthase) localized on the plasma membrane . GS is composed of a catalytic subunit encoded by the two homologous genes FKS1 and FKS2  and a regulatory subunit, the small GTPase, Rho1p . The Rho-type GTPase is generally regulated by switching between a GDP-bound inactive state and a GTP-bound active state. Various factors are involved in the regulation of β-1,3-glucan synthesis by Rho1p in yeast cells. The putative cell surface sensor protein Wsc1p stimulates nucleotide exchange of Rho1p through the GDP/GTP exchange factor, Rom2p . Lrg1p, a GTPase-activating protein, promotes formation of GDP-bound Rho1p thus negatively regulating β-1,3-glucan synthesis . Thus overexpression of WSC1 or ROM2 , or loss of function of LRG1  restores the impaired β-1,3-glucan synthesis observed in GS mutants. In addition, post-translational modification of Rho1p by the geranylgeranyl group is also required for binding of Rho1p to GS and activation of GS activity . Other factors affect β-1,3-glucan synthesis by regulation of the catalytic subunit of GS. Movement of Fks1p driven by actin is required for the construction of a uniform and solid cell wall . Transcription of FKS2 is up-regulated in response to cell wall stress . Increased β-1,3-glucan level in the presence of the kre5 suppressor mutation is probably due to an increased expression of FKS2 in response to defective β-1,6-glucan synthesis.
How does loss of mitochondrial anionic lipids affect cell wall construction?
Several proteins involved in β-1,3-glucan synthesis were found to have dual localization in the plasma membrane and mitochondria. The catalytic and regulatory subunits of GS are localized in the plasma membrane at the site of cell wall synthesis . Gas1p, a putative β-1,3-glucan remodelling enzyme, is attached to the plasma membrane via a glycosyl-phosphatidylinositol anchor . Interestingly, Fks1p, Rho1p  and Gas1p [41,42] are also found in mitochondria. It is not known if dual plasma membrane/mitochondrial localization of these enzymes has any physiological relevance. It is tempting, however, to assume that mitochondria are required for the maturation or modification of those enzymes, in which case mitochondrial dysfunction would result in decreased enzyme activity and cell wall defects. Alternatively, mitochondrial dysfunction may trigger signals that prevent proper mobilization of these enzymes to the site of cell wall biosynthesis.
Alternatively, cell wall defects observed in CL-deficient mutants could be an indirect result of the perturbation of signalling transduction towards cell wall biogenesis. Yeast cell wall biogenesis is a highly regulated process that involves elaborate control mechanisms. All major signalling pathways such as the PKC (protein kinase C) MAPK (mitogen-activated protein kinase) pathway, the cAMP-dependent PKA pathway and the HOG1 MAPK pathway are directly or indirectly involved in cell wall construction (reviewed in ).
The Pkc1p kinase controls a highly conserved MAPK signal-transduction pathway that regulates cell wall biogenesis (reviewed in ). This pathway consists of a cascade of phosphorylation reactions initiated with the activation of Pkc1p. Pkc1p then activates a MAPK module involving the MEK (MAPK/ERK kinase) kinase Bck1p, the redundant MEK, MAPK kinases Mkk1p and Mkk2p and the MAPK Slt2p. Signalling through the MAPK cascade results in dual phosphorylation and activation of Slt2p. Currently, two transcription factors have been identified as direct targets of Slt2p: Rlm1p, which mediates the activation of genes involved in cell wall synthesis  and the SBF [SCB (Swi4p–Swi6p cell cycle box)-binding factor] complex for cell-cycle regulation, which is also implicated in the regulation of cell wall formation [45,46]. Disruption of PKC1 results in autolysis due to lack of cell wall integrity . Mutants with disruption in any of the other protein kinases exhibit sorbitol remediable temperature-sensitive growth [48–50], a phenotype also observed in CL-deficient mutants. The PKA signalling transduction pathway and the Hog1 MAPK pathway are also implicated in the modulation of cell wall construction (reviewed in ), even though the mechanism of how these pathways affect cell wall biogenesis remains unknown. It will be interesting to determine whether these signalling transduction pathways function properly in CL-deficient mutants.
A loss-of-function allele of KRE5, a gene involved in cell wall biogenesis, was isolated and identified as an extragenic suppressor of pgs1Δ that allows it to grow at elevated temperatures. Characterization of pgs1Δ and the suppressor strain strongly suggests that temperature sensitivity of CL-deficient mutants was primarily due to defective cell wall integrity. This is the first demonstration of defective cell wall biosynthesis in mutants lacking mitochondrial anionic phospholipids PG and CL. Our findings thus provide new insights into the essential functions of these lipids and point to a regulatory role for mitochondria in cell wall biogenesis.
Seventh Yeast Lipid Conference: Independent Meeting held at Swansea Clinical School, Swansea, Wales, U.K., 12–14 May 2005. Organized and Edited by D. Kelly, S. Kelly and D. Lamb (Swansea, U.K.).
Abbreviations: CL, cardiolipin; GS, β-1,3-glucan synthase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PG, phosphatidylglycerol
- © 2005 The Biochemical Society