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

Abstract

Disruption of PGS1, which encodes the enzyme that catalyzes the committed step of cardiolipin (CL) synthesis, results in loss of the mitochondrial anionic phospholipids phosphatidylglycerol (PG) and CL. The pgs1⌬ mutant exhibits severe growth defects at 37°C. To understand the essential functions of mitochondrial anionic lipids at elevated temperatures, we isolated suppressors of pgs1⌬ that grew at 37°C. One of the suppressors has a loss of function mutation in KRE5, which is involved in cell wall biogenesis. The cell wall of pgs1⌬ contained markedly reduced ␤-1,3-glucan, which was restored in the suppressor. Stabilization of the cell wall with osmotic support alleviated the cell wall defects of pgs1⌬ and suppressed the temperature sensitivity of all CL-deficient mutants. Evidence is presented suggesting that the previously reported inability of pgs1⌬ to grow in the presence of ethidium bromide was due to defective cell wall integrity, not from "petite lethality." These findings demonstrated that mitochondrial anionic lipids are required for cellular functions that are essential in cell wall biogenesis, the maintenance of cell integrity, and survival at elevated temperature. INTRODUCTIONCardiolipin (CL), a unique anionic phospholipid with dimeric structure, is ubiquitous in eukaryotes and primarily found in the mitochondrial inner membrane . CL plays a key role in mitochondrial bioenergetics (Jiang et al., 2000; Greenberg, 2000, 2002;Schlame et al., 2000;Pfeiffer et al., 2003) and is also involved in mitochondrial biogenesis (Kawasaki et al., 1999;Jiang et al., 2000). Defective remodeling of CL is associated with Barth syndrome, a severe genetic disorder characterized by cardiomyopathy, neutropenia, skeletal myopathy, and respiratory chain defects (Vreken et al., 2000). The phenotype of Barth syndrome is dependent upon multiple factors that are not well understood (Barth et al., 1983(Barth et al., , 1996. Elucidation of the functions of CL will help to clarify the abnormalities associated with this disorder.The biosynthesis of CL is conserved in eukaryotic organisms. It occurs via three enzymatic reactions , including formation of phosphatidylglycerolphosphate (PGP) from CDP-DAG and glycerol-3-P, dephosphorylation of PGP to phosphatidylglycerol (PG), and condensation of CDP-DAG and PG to form CL. 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). Deletion of the yeast homolog of this gene, TAZ1, leads to decreased CL, aberrant CL acyl species, and accumulation of monolysocardiolipin . Mutants deficient in CL biosynthesis exhibit growth defects at elevat...

Translational Regulation of Nuclear Gene COX4 Expression by Mitochondrial Content of Phosphatidylglycerol and Cardiolipin in Saccharomyces cerevisiae

“…In addition, pgs1⌬ cells carrying this plasmid remained unable to grow on a nonfermentable carbon source (YPEG medium), indicating that the lack of PG and CL caused permanent damage to cells, which prevented the return of respiratory competence even after supplementation with PG. Consistent with this observation is the report that some yeast cells deficient in PG and CL lose mtDNA (74). We confirmed the lack of mitochondrially encoded mRNA transcripts by RT-PCR analysis of YZD1 (pgs1⌬) cells in contrast to wild-type cells, consistent with YZD1 cells lacking most if not all their mtDNA (data not shown).…”

“…Yeast mutants (pgs1⌬) deficient in both PG and CL display more severe defects in mitochondrial function, such as slow growth on a fermentable carbon source and total inability to respire on nonfermentable carbon sources. One of the reasons for the lack of growth of pgs1⌬ cells on nonfermentable carbon sources appears to be due to an initial reduction in the level of respiratory components (48) and eventual loss of mitochondrial DNA (mtDNA) (74).…”

Synthetic Lethal Interaction of the Mitochondrial Phosphatidylethanolamine and Cardiolipin Biosynthetic Pathways in Saccharomyces cerevisiae

“…PE is also required for the formation of glycosylphosphatidylinositol-linked proteins (4,36), which play an essential role in cell viability by maintaining structural integrity of the cell wall. A recent report from our laboratory indicated an important role of CL and its precursor, phosphatidylglycerol, in maintaining cell wall integrity (37). Based on these results, we speculated that loss of viability of crd1⌬psd1⌬ cells could be the result of compromised cell wall stability.…”

Histoplasma capsulatumα-(1,3)-glucan blocks innate immune recognition by the β-glucan receptor

“…For analysis by f luorescence microscopy, 300-nm sections were immunolabeled with antibodies MOPC-104E and a monoclonal ␤-(1,3)-glucan-specific antibody (Biosupplies Australia, Parkville, Australia), probed with f luorophore-conjugated anti-IgM and anti-IgG secondary antibodies, respectively, then visualized with an Olympus BX-60 microscope (Center Valley, PA) with a ϫ100 objective. The ␤-(1,3)-glucan monoclonal antibody has previously been shown to be specific for ␤-(1,3)-linked glucans (34) and has been used in biochemical and immunof luorescence studies of fungal cell walls (26,35). Cell wall glucan perimeters were produced by manually tracing the outermost boundary of f luorescence after importing f luorophore-specific micrographs into ImageJ (National Institutes of Health, Bethesda, MD).…”