Abstract
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.
Highlights
In eukaryotic cells cardiolipin (CL)1 and its synthesis are predominantly localized to the mitochondrial inner membrane [1,2,3]
The committed and rate-limiting step in CL biosynthesis in Saccharomyces cerevisiae is catalyzed by phosphatidylglycerophosphate (PG-P) synthase yielding PG-P which is rapidly dephosphorylated [16]
The characteristic CDPalcohol/phosphatidyltransferase signature [41] is present between positions 106 and 136 of the CL synthase. This motif is present in the PG-P synthases shown in Fig. 2 and is found in phosphatidylserine synthases from Bacillus subtilis and S. cerevisiae, in phosphatidylinositol synthase from S. cerevisiae, and in numerous other bacterial PG-P synthases [40]; this motif is not found in either the phosphatidylserine synthase [42] or CL synthase [43] of E. coli
Summary
Materials—All chemicals were reagent grade or better. Restriction endonucleases and DNA-modifying enzymes were from Promega Corp., New England Biolabs, Stratagene, and Boehringer Mannheim. DNA Manipulations—Methods for plasmid and genomic DNA preparation, restriction enzyme digestion, DNA ligation, E. coli and yeast transformation, DNA sequencing, and sequence analysis were carried out as described previously [22]. For both analytical and preparative purposes, PCR was performed after optimizing conditions [31]. Labeling and Analysis of Phospholipids—Cells grown in CSM plus 2% glucose with auxotrophic selection when appropriate were labeled with [32P]orthophosphate for six generations, the phospholipids extracted in the presence of carrier lipid (30 g of equal ratio of phospholipids including phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, PG, and CL) as described previously [22]. AmpR pBluescript, TRP1, ampR lacIЈOPZЈ, ampR, ColE1 origin PGAL1, URA3, ampR, 2-m circle, ColE1 origin CLS1 (genomic), derivative of pYES2 PGAL1-CLS1 (PCR product), derivative of pYES2 Very late AcMNPV polyhedrin promoter (PPH), ampR, ColE1 origin, recombination sequences ORF603 and ORF1629 PPH-CLS1, derivative of pVL1392 a Partial list of genetic markers
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