Kinetic and physiological effects of alterations in homologous isocitrate-binding sites of yeast NAD(+)-specific isocitrate dehydrogenase.

Yeast NAD(+)-specific isocitrate dehydrogenase is an allosterically regulated octameric enzyme composed of four each of two homologous but nonidentical subunits designated IDH1 and IDH2. Models based on the crystallographic structure of Escherichia coli isocitrate dehydrogenase suggest that both yeast subunits contain isocitrate-binding sites. Identities in nine residue positions are predicted for the IDH2 site whereas four of the nine positions differ between the IDH1 and bacterial enzyme sites. Thus, we speculate that the IDH2 site is catalytic and that the IDH1 site may bind but not catalytically alter isocitrate. This was examined by kinetic analyses of enzymes with independent and concerted replacement of residues in each yeast IDH subunit site with the residues that differ in the other subunit site. Mutant enzymes were expressed in a yeast strain containing disrupted IDH1 and IDH2 loci and affinity-purified for kinetic analyses. The primary effects of various residue replacements in IDH2 were reductions of 30->300-fold in V(max) values, consistent with the catalytic function of this subunit. In contrast, replacement of all four residues in IDH1 produced a 17-fold reduction in V(max) under the same assay conditions, suggesting that the IDH1 site is not the primary catalytic site. However, single or multiple residue replacements in IDH1 uniformly increased half-saturation concentrations for isocitrate, implying that isocitrate can be bound at this site. Both subunits appear to contribute to cooperativity with respect to isocitrate, but AMP activation is lost only with residue replacements in IDH1. Overall, results are consistent with isocitrate binding by IDH2 for catalysis and with isocitrate binding by IDH1 being a prerequisite for allosteric activation by AMP. The effects of residue substitutions on enzyme function in vivo were assessed by analysis of various growth phenotypes. Results indicate a positive correlation between the level of IDH catalytic activity and the ability of cells to grow with acetate or glycerol as carbon sources. In addition, lower levels of activity are associated with increased production of respiratory-deficient (petite) segregants.

Increased synthesis and decreased stability of mitochondrial translation products in yeast as a result of loss of mitochondrial (NAD(+))-dependent isocitrate dehydrogenase.

We have previously demonstrated that the yeast Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh) binds specifically and with high affinity to the 5'-untranslated leader sequences of mitochondrial mRNAs in vitro and have proposed a role for the enzyme in the regulation of mitochondrial translation [Elzinga, S.D.J. et al. (2000) Curr. Genet., in press]. Although our studies initially failed to reveal any consistent correlation between idh disruption and mitochondrial translational activity, it is now apparent that compensatory extragenic suppressor mutations readily accumulate in idh disruption strains thereby masking mutant behaviour. Now, pulse-chase protein labelling of isolated mitochondria from an Idh disruption mutant lacking suppressor mutations reveals a strong (2-3-fold) increase in the synthesis of mitochondrial translation products. Strikingly, the newly synthesised proteins are more short-lived than in mitochondria from wild-type cells, their degradation occurring with a 2-3-fold reduced half-life. Enhanced degradation of translation products is also a feature of yeast mutants in which tethering/docking of mitochondrial mRNAs is disturbed. We therefore suggest that binding of Idh to mitochondrial mRNAs may suppress inappropriate translation of mitochondrial mRNAs.

Yeast mutants of glucose metabolism with defects in the coordinate regulation of carbon assimilation.

The enzymes of the glyoxylate cycle and gluconeogenesis are tightly regulated by transcriptional, posttranscriptional, and posttranslational mechanisms in Saccharomyces cerevisiae. We have previously identified four genes, ACN8, ACN9, ACN17, and ACN18, whose mutant phenotype includes two- to fourfold elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism. The affected enzymes are elevated on nonfermentable carbon sources but are still fully repressed by glucose. Catabolite inactivation of the cytosolic malate dehydrogenase is not affected in the mutants. Instead, the phenotype appeared to be manifested primarily at the level of transcription. The ACN8, ACN17, and ACN18 genes were isolated by functional complementation of the respective mutant's inability to utilize acetate as a carbon and energy source, and these genes were shown to encode subunits of metabolic enzymes. ACN8 was identical to FBP1, which encodes the gluconeogenic enzyme, fructose 1,6-bisphosphatase, while ACN17 and ACN18 were identical to the SDH2 and SDH4 genes, respectively, that encode subunits of the respiratory chain and tricarboxylic acid cycle enzyme, succinate dehydrogenase. Mutants defective in other glyoxylate cycle and gluconeogenic enzymes also display the elevated enzyme phenotype, indicating that the enzyme superinduction is a general property of gluconeogenic dysfunction. Glucose 6-phosphate levels were diminished in the mutants, suggesting that endogenous glucose synthesis can regulate the expression of gluconeogenic enzymes.

Genetic and biochemical interactions involving tricarboxylic acid cycle (TCA) function using a collection of mutants defective in all TCA cycle genes.

The eight enzymes of the tricarboxylic acid (TCA) cycle are encoded by at least 15 different nuclear genes in Saccharomyces cerevisiae. We have constructed a set of yeast strains defective in these genes as part of a comprehensive analysis of the interactions among the TCA cycle proteins. The 15 major TCA cycle genes can be sorted into five phenotypic categories on the basis of their growth on nonfermentable carbon sources. We have previously reported a novel phenotype associated with mutants defective in the IDH2 gene encoding the Idh2p subunit of the NAD+-dependent isocitrate dehydrogenase (NAD-IDH). Null and nonsense idh2 mutants grow poorly on glycerol, but growth can be enhanced by extragenic mutations, termed glycerol suppressors, in the CIT1 gene encoding the TCA cycle citrate synthase and in other genes of oxidative metabolism. The TCA cycle mutant collection was utilized to search for other genes that can suppress idh2 mutants and to identify TCA cycle genes that display a similar suppressible growth phenotype on glycerol. Mutations in 7 TCA cycle genes were capable of functioning as suppressors for growth of idh2 mutants on glycerol. The only other TCA cycle gene to display the glycerol-suppressor-accumulation phenotype was IDH1, which encodes the companion Idh1p subunit of NAD-IDH. These results provide genetic evidence that NAD-IDH plays a unique role in TCA cycle function.

Acn9 is a novel protein of gluconeogenesis that is located in the mitochondrial intermembrane space.

Previous studies have indicated that the Acn9 protein is involved in gluconeogenesis. Yeast mutants defective in the ACN9 gene display phenotypes identical with mutants defective in metabolic enzymes required for carbon assimilation. These phenotypes include the inability to utilize acetate as a carbon and energy source, elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis and acetyl-CoA mobilization, and a deficiency in de novo synthesis of glucose from ethanol. The ACN9 gene was isolated by functional complementation of the acetate growth defect of an acn9 mutant. The open reading frame corresponds to YDR511w, and encodes a protein of unknown function. Homologs have been identified in human, mouse, and nematode databases. Two mutant alleles were sequenced. The mutations altered amino acid residues that are conserved among members of the new gene family. ACN9 gene expression was slightly repressed by glucose, and the level of the transcript was approximately 100-fold lower than that of glyoxylate or tricarboxylic acid cycle enzymes. A functional epitope-tagged form of Acn9 was expressed to study expression and the subcellular localization of the protein. The tagged protein was localized to the mitochondrial intermembrane space.

An unassembled subunit of NAD(+)-dependent isocitrate dehydrogenase is insoluble and covalently modified.

The NAD(+)-dependent isocitrate dehydrogenase of Saccharomyces cerevisiae is an octamer composed of four Idh1p subunits and four Idh2p subunits. Isocitrate dehydrogenase functions in the tricarboxylic acid cycle and has also been reported to bind to the 5' nontranslated region of mitochondrially encoded mRNAs. Mutants defective in either or both of these subunits are unable to grow on the nonfermentable carbon source, acetate, but will utilize glycerol or ethanol. Mutant strains lacking Idh2p maintain normal if not elevated levels of mitochondrial Idh1p. In addition to the mature unassembled Idh1p subunit, a complex of bands in the 85- to 170-kDa range (Idh1p-Cpx) is observed using NAD-IDH antiserum. Both Idh1p and Idh1p-Cpx are insoluble within the mitochondrion and are associated with the mitochondrial inner membrane. A histidine-tagged form of Idh1p was expressed in yeast strains. Chemical amounts of the Idh1p-Cpx could be purified from strains lacking Idh2p but not from strains containing normal levels of Idh2p. The data indicate that Idh1p-Cpx is an aggregated and cross-linked form of Idh1p that may be oxidized within the mitochondrion as a consequence of its aborted assembly.

Genes of succinyl-CoA ligase from Saccharomyces cerevisiae.

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.

Mutations in the IDH2 gene encoding the catalytic subunit of the yeast NAD+-dependent isocitrate dehydrogenase can be suppressed by mutations in the CIT1 gene encoding citrate synthase and other genes of oxidative metabolism.

During a screen for respiration competent yeast mutants that were unable to grow with acetate as a carbon source, two idh2 cit1 double mutants were identified. These strains were defective in the catalytic subunit of the NAD(+)-dependent isocitrate dehydrogenase and citrate synthase of the tricarboxylic acid (TCA) cycle. The strains harboring the idh2 alleles from these strains had two unusual phenotypes. First, their growth on many nonfermentable carbon sources was much poorer than strains containing other idh2 mutations. Second, the poor growth phenotype could be suppressed by the presence of mutations in CIT1 and other genes encoding oxidative functions. Spontaneous suppressor mutants that restore fast growth on glycerol medium to strains harboring two idh2 alleles were isolated, and a large percentage of the suppressor mutations have been identified within the CIT1 gene and at several other loci. Elevated levels of several TCA cycle proteins were observed in these idh2 mutants that were not observed in the presence of suppressing cit1 mutations. Citrate and isocitrate concentrations were also elevated in the idh2 mutants, but probably not to toxic levels. Five idh2 alleles were sequenced to understand the defects of the two classes of mutations. Sequence analysis indicated that the poor growth phenotype was caused by the loss of Idh2p protein. Similarly, eight cit1 alleles were sequenced to understand their characteristics as glycerol suppressors of idh2. These and other studies indicate that any mutation within CIT1 was capable of suppressing the idh2 mutations. Several models to explain these interactions are discussed.

Mutants of Saccharomyces cerevisiae with defects in acetate metabolism: isolation and characterization of Acn- mutants.

The two carbon compounds, ethanol and acetate, can be oxidatively metabolized as well as assimilated into carbohydrate in the yeast Saccharomyces cerevisiae. The distribution of acetate metabolic enzymes among several cellular compartments, mitochondria, peroxisomes, and cytoplasm makes it an intriguing system to study complex metabolic interactions. To investigate the complex process of carbon catabolism and assimilation, mutants unable to grow on acetate were isolated. One hundred five Acn- ("ACetate Nonutilizing") mutants were sorted into 21 complementation groups with an additional 20 single mutants. Five of the groups have defects in TCA cycle enzymes: MDH1, CIT1, ACO1, IDH1, and IDH2. A defect in RTG2, involved in the retrograde communication between the mitochondrion and the nucleus, was also identified. Four genes encode enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1, MDH2, and PCK1. Five other genes appear to be defective in regulating metabolic activity since elevated levels of enzymes in several metabolic pathways, including the glyoxylate cycle, gluconeogenesis, and acetyl-CoA metabolism, were detected in these mutants: ACN8, ACN9, ACN17, ACN18, and ACN42. In summary, this analysis has identified at least 22 and as many as 41 different genes involved in acetate metabolism.

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes.

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae.

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.

Association of glyoxylate and beta-oxidation enzymes with peroxisomes of Saccharomyces cerevisiae.

Although peroxisomes are difficult to identify in Saccharomyces cerevisiae under ordinary growth conditions, they proliferate when cells are cultured on oleic acid. We used this finding to study the protein composition of these organelles in detail. Peroxisomes from oleic acid-grown cells were purified on a discontinuous sucrose gradient; they migrated to the 46 to 50% (wt/wt) sucrose interface. The peroxisomal fraction was identified morphologically and by the presence of all of the enzymes of the peroxisomal beta-oxidation pathway. These organelles also contained a significant but minor fraction of two enzymes of the glyoxylate pathway, malate synthase and malate dehydrogenase-2. The localization of malate synthase in peroxisomes was confirmed by immunoelectron microscopy. It is postulated that glyoxylate pathway enzymes are readily and preferentially released from peroxisomes upon cell lysis, accounting for their incomplete recovery from isolated organelles. Small uninduced peroxisomes from glycerol-grown cultures were detected on sucrose gradients by marker enzymes. Under these conditions, catalase, acyl-coenzyme A oxidase, and malate synthase cofractionated at equilibrium close to the mitochondrial peak, indicating smaller, less dense organelles than those from cells grown on oleic acid. Peroxisomal membranes from oleate cultures were purified by buoyant density centrifugation. Three abundant proteins of 24, 31, and 32 kilodaltons were observed.

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.

Lipid synthesis in inositol-starved Saccharomyces cerevisiae.

Lipid synthesis was analyzed in an inositol-requiring mutant of Saccharomyces cerevisiae (MC13). Both rates and cellular amounts of [U-14C]acetate incorporation into phospholipids, triacylglycerols, free sterols and steryl esters were elevated in an inositol-starved culture compared to the supplemented control at a time when the deprived culture was losing viability (inositol-less death). The rates at a later time were greatly reduced. During the period when de novo lipid synthesis was high in the starved culture, phospholipid turnover and presumed conversion to triacylglycerols was also accelerated; no differences were apparent in the turnover of the sterol fractions between the two cultures. No change in the fractional percent of ergosterol or of the sterol precursors could be attributed to inositol starvation. The synthesis and maintenance of membrane lipids (phospholipids and free sterols) and their coupling in cellular metabolism are discussed in light of these results.

Enrichment for auxotrophic mutants in Saccharomyces cerevisiae using the cell wall inhibitor, echinocandin B.

Echinocandin B has been shown to inhibit fungal cell wall synthesis. This report describes the use of echinocandin B to enrich for nutritional auxotrophs in a mutagenized strain of Saccharomyces cerevisiae. Up to 20-fold enrichment levels for total auxotrophs were achieved after a single round of treatment with echinocandin; this level of enrichment is the highest of all procedures for which a specific mutant strain is not required.

Inhibition of sterol transmethylation by S-adenosylhomocysteine analogs.

Structural analogs of S-adenosylhomocysteine were tested in vitro for inhibition of the yeast S-adenosylmethionine:delta 24-sterol-C-methyltransferase enzyme. A wide inhibitory range by these compounds was observed, suggesting which structural features of the parent compound are important for binding to the enzyme. No analog tested had inhibitory activity specific only for this enzyme. The most active compound was sinefungin, a metabolite of Streptomyces griseolus, which was also able to inhibit growth of yeast cultures. Sterol extracts of cells grown in the presence of sinefungin revealed a dramatic increase in the levels of zymosterol, the sterol substrate in the transmethylation under study, and a concomitant decrease in the levels of ergosterol. Evidence is presented that sinefungin is transported inside the cell by the same permease as S-adenosylmethionine. We conclude that sinefungin is blocking the in vivo methylation of sterols in yeast. The implications of this finding are discussed.