Abstract
To compare roles of specific enzymes in supply of NADPH for cellular biosynthesis, collections of yeast mutants were constructed by gene disruptions and matings. These mutants include haploid strains containing all possible combinations of deletions in yeast genes encoding three differentially compartmentalized isozymes of NADP+-specific isocitrate dehydrogenase and in the gene encoding glucose-6-phosphate dehydrogenase (Zwf1p). Growth phenotype analyses of the mutants indicate that either cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p) or the hexose monophosphate shunt is essential for growth with fatty acids as carbon sources and for sporulation of diploid strains, a condition associated with high levels of fatty acid synthesis. No new biosynthetic roles were identified for mitochondrial (Idp1p) or peroxisomal (Idp3p) NADP+-specific isocitrate dehydrogenase isozymes. These and other results suggest that several major presumed sources of biosynthetic reducing equivalents are non-essential in yeast cells grown under many cultivation conditions. To develop an in vivo system for analysis of metabolic function, mammalian mitochondrial and cytosolic isozymes of NADP+-specific isocitrate dehydrogenase were expressed in yeast using promoters from the cognate yeast genes. The mammalian mitochondrial isozyme was found to be imported efficiently into yeast mitochondria when fused to the Idp1p targeting sequence and to substitute functionally for Idp1p for production of alpha-ketoglutarate. The mammalian cytosolic isozyme was found to partition between cytosolic and organellar compartments and to replace functionally Idp2p for production of alpha-ketoglutarate or for growth on fatty acids in a mutant lacking Zwf1p. The mammalian cytosolic isozyme also functionally substitutes for Idp3p allowing growth on petroselinic acid as a carbon source, suggesting partial localization to peroxisomes and provision of NADPH for beta-oxidation of that fatty acid.
Highlights
Reducing equivalents in the form of NADPH are required by many enzymes in central biosynthetic pathways, whereas the enzymatic sources of biosynthetic reducing equivalents are believed to be limited in number
An earlier study of a zwf1 homozygous diploid mutant indicated that this enzyme is not essential for the significant increase in fatty acid biosynthesis that occurs during sporulation [4]
Strains lacking Zwf1p were identified by glucose-6-phosphate dehydrogenase assays (Fig. 1B), and the ZWF1 gene disruption was confirmed by Southern blot analysis (Fig. 1C)
Summary
Yeast Strains and Growth Conditions—Yeast strains used in this study were the parental haploid strain S173-6B (MATa leu 112 his ura trp1-289, 13) and previously constructed mutants of this strain containing deletions and URA3 insertions in the IDP1 and/or IDP2 loci [7, 8]. The yeast IDP3 gene was disrupted as described by Henke et al [9] using transformation with a construct generated by PCR containing a selectable kanMX4 gene flanked by 5Ј- and 3Ј-noncoding regions of IDP3. To construct plasmids for expression of mammalian mitochondrial NADPϩ-specific isocitrate dehydrogenase in yeast, a 100-bp EcoRI restriction fragment was removed from the coding region of the yeast IDP1 gene [7] and replaced with a 1.6-kbp EcoRI cDNA fragment containing the coding region and 3Ј-noncoding sequences for the pig mitochondrial enzyme [20]. For expression of mammalian cytosolic NADPϩ-specific isocitrate dehydrogenase in yeast, a 190-bp BclI/EcoRI restriction fragment was removed from the coding region of the yeast IDP2 gene [8] and replaced with a 1.7-kbp EcoRI cDNA fragment containing the coding region and 3Ј-noncoding sequences for the rat cytosolic enzyme [23]. Immunoreactivity was detected using radiolabeled protein A [7] or the enhanced chemiluminescent method (ECL, Amersham Pharmacia Biotech) and autoradiography
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