The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.     

The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae

The biosynthesis of yeast 5-aminolevulinate (ALA) synthase, a mitochondrial protein encoded by the nuclear HEM1 gene, has been studied in vitro in a cell-free translation system and in vivo in whole cells. In vitro translation of mRNA hybrid-selected by the cloned HEM1 gene, or of total RNA followed by immunoprecipitation with anti-(ALA synthase) antibody yielded a single polypeptide of higher molecular mass than the purified ALA synthase. This larger form, also seen in pulse-labeled cells, can be post-translationally processed by isolated mitochondria. These results show that the cytoplasmically made ALA synthase is synthesized with a cleavable extension which was estimated to be about 3.5 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The complete nucleotide sequence of the HEM1 gene and its flanking regions was determined. The 5' ends of the HEM1 mRNAs map from -76 to -63 nucleotides upstream of the translation initiation codon. The open reading frame of 1644 base pairs encodes a protein of 548 amino acids with a calculated Mr of 59,275. The predicted amino-terminal sequence of the protein is strongly basic (five basic and no acidic amino acids within the first 35 residues), rich in serine and threonine and must represent the transient presequence that targets this protein to the mitochondria. Comparison of deduced amino acid sequences indicates a clear homology between the mature yeast and chick embryo liver ALA synthases.     

Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.     

Ornithine decarboxylase activity and cell cycle regulation in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the specific activity of the enzyme ornithine decarboxylase (ODC) was correlated with overall growth status. The activity of ODC was highest in actively growing cells, whereas the specific activity was lower in slow-growing cultures limited for nitrogen or inhibited by low concentrations of cycloheximide. Specific activities of ODC were also low in cultures arrested in the stationary phase (in the G1 portion of the cell cycle) by starvation for required nutrients. Although correlated with overall growth, ODC activity was not required for growth or cell cycle regulation. Cells continued to grow in the presence of the polyamine spermidine or spermine, which markedly reduced ODC specific activities. Thus, high levels of ODC activity were not necessary for growth, nor were decreased ODC specific activities sufficient to cause cells to arrest in G1. Conversely, one agent (o-phenanthroline) which causes growing cells to arrest in G1 did so with no effect on ODC specific activity. Therefore, ODC specific activity changes are not necessary for cell cycle regulation but simply reflect the normal growth status of cells.     

The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.     

Uroporphyrinogen decarboxylase: complete human gene sequence and molecular study of three families with hepatoerythropoietic porphyria.

A deficiency in uroporphyrinogen decarboxylase (UROD) enzyme activity, the fifth enzyme of the heme biosynthetic pathway, is found in patients with sporadic porphyria cutanea tarda (s-PCT), familial porphyria cutanea tarda (f-PCT), and hepatoerythropoietic porphyria (HEP). Subnormal UROD activity is due to mutations of the UROD gene in both f-PCT and HEP, but no mutations have been found in s-PCT. Genetic analysis has determined that f-PCT is transmitted as an autosomal dominant trait. In contrast, HEP, a severe form of cutaneous porphyria, is transmitted as an autosomal recessive trait. HEP is characterized by a profound deficiency of UROD activity, and the disease is usually manifest in childhood. In this study, a strategy was designed to identify alleles responsible for the HEP phenotype in three unrelated families. Mutations of UROD were identified by direct sequencing of four amplified fragments that contained the entire coding sequence of the UROD gene. Two new missense mutations were observed at the homoallelic state: P62L (proline-to-leucine substitution at codon 62) in a Portuguese family and Y311C (tyrosine-to-cysteine substitution at codon 311) in an Italian family. A third mutation, G281E, was observed in a Spanish family. This mutation has been previously described in three families from Spain and one from Tunisia. In the Spanish family described in this report, a paternal uncle of the proband developed clinically overt PCT as an adult and proved to be heterozygous for the G281E mutation. Mutant cDNAs corresponding to the P62L and Y311C changes detected in these families were created by site-directed mutagenesis. Recombinant proteins proved to have subnormal enzyme activity, and the Y311C mutant was thermolabile.     

Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5′ untranslated region. “Uroporphyric” mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype chracteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12 The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the β-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.     

Farnesyl diphosphate synthetase. Molecular cloning, sequence, and expression of an essential gene from Saccharomyces cerevisiae

Farnesyl diphosphate (FPP) synthetase is a key enzyme in isoprenoid biosynthesis which supplies C15 precursors for several classes of essential metabolites including sterols, dolichols, and ubiquinones. The structural gene for FPP synthetase was isolated on a 4.5-kilobase EcoRI genomic restriction fragment from the yeast Saccharomyces cerevisiae. The clone encodes a 40,483-dalton polypeptide of 342 amino acids with a high degree of similarity to the protein encoded by a putative rat liver clone of FPP synthetase (Clarke, C. F., Tanaka, R. D., Svenson, K., Wamsley, M., Fogelman, A. M., and Edwards, P. A. (1987) Mol. Cell Biol. 7, 3138-3146) and to an active site protein fragment from avian liver FPP synthetase (Brems, D. N., Bruenger, E., and Rilling, H. C. (1981) Biochemistry 20, 3711-3718). When cloned into the yeast shuttle vector YRp17, the 4.5-kilobase EcoRI fragment directed a 2-3-fold over-expression of FPP synthetase activity in transformed yeast cells. The levels of expression were independent of culture growth phase and orientation of the insert, indicative of a functional promoter in the clone. Disruption of the FPP synthetase gene from a diploid yeast strain, followed by dissection and analysis of tetrads, demonstrates that the gene is an essential, single copy number gene in yeast. The gene for FPP synthetase resides on chromosome XI as judged from Southern blots of separated yeast chromosomes.     

Characterization of two new genes essential for vegetative growth in Saccharomyces cerevisiae: nucleotide sequence determination and chromosome mapping

Based on nucleotide sequence determination, we have identified two new yeast genes FUN80 and FUN81 located on chromosome XIII. They are both essential for cellular growth but their function is still unknown. FUN80 is closely linked to the ARGRI (or ARG80) gene while FUN81 is located next to the ARGRII (or ARG81) gene. Interestingly, the proteins encoded by these two genes have a long stretch of acidic amino acids within their C-terminal portions.     

HYS2, an essential gene required for DNA replication in Saccharomyces cerevisiae.

To investigate cell cycle regulation at the S or G2 phase in Saccharomyces cerevisiae, we have isolated mutants displaying supersensitivity to hydroxyurea (HU), a chemical that inhibits DNA replication. Such mutants, which we have named hydroxyurea sensitive (hys), defined four linkage groups and we characterized the hys2 mutation in this study. The hys2-1 mutant displays temperature sensitive growth and a constellation of phenotypes indicating defective DNA metabolism. At the restrictive temperature, hys2-1 cells arrest as large budded cells with a single nucleus at the neck of the bud and a short spindle. The hys2-1 mutant exhibits increased rates of chromosome loss and recombination. Additionally, hys2-1 appears to accumulate incompletely replicated DNA that can be detected by a pulse field electrophoresis assay. Finally, deletion of RAD9 in a hys2-1 strain decreases the percentage of arrested cells, suggesting that an intact RAD9-checkpoint is required for the cell cycle arrest in hys2-1 cells. HYS2 encodes a 55 kDa protein that is essential for viability at all temperatures. Taken together, these data suggest that Hys2 plays a role in DNA replication.     

AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.     

The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15

The HEM15 gene in Saccharomyces cerevisiae encodes ferrochelatase (EC 4.99.1.1, protoheme ferrolyase), a mitochondrial inner membrane-bound enzyme which catalyzes the insertion of ferrous ion into protoporphyrin IX, the last step in protoheme biosynthesis. The gene was isolated by functional complementation of a hem15 mutant. Sequence analysis of a 2.9-kilobase genomic DNA fragment revealed an open reading frame of 1179 nucleotides, plus a gene coding for a tRNA(Val)(GUU) and delta elements downstream from the 3'-end of HEM15. The open reading frame encodes a precursor form of the protein containing a 31-amino acid presequence. The mature enzyme contains 362 amino acid residues; its calculated molecular weight (40,900) and predicted amino-terminal sequence agree with those determined from the purified protein. It is relatively abundant in lysine (9%) and contains no apparent transmembrane segment. Disruption of the HEM15 gene led to non-viable cells in certain genetic background. Northern (RNA) analysis showed a slight (1.5-2-fold) repression of HEM15 expression by glucose.     

Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae.

Pyruvate decarboxylase is the key enzyme in alcoholic fermentation in yeast. Two structural genes, PDC1 and PDC5 have been characterized. Deletion of either of these genes has little or no effect on the specific pyruvate decarboxylase activity, but enzyme activity is undetectable in mutants lacking both PDC1 and PDC5 (S. Hohmann and H. Cederberg, Eur. J. Biochem. 188:615-621, 1990). Here I describe PDC6, a gene structurally closely related to PDC1 and PDC5. The product of PDC6 does not seem to be required for wild-type pyruvate decarboxylase activity in glucose medium; delta pdc6 mutants have no reduced specific enzyme activity, and the PDC6 deletion did not change the phenotype or the specific enzyme activity of mutants lacking either or both of the other two structural genes. However, in cells grown in ethanol medium the PDC6 deletion caused a reduction of pyruvate decarboxylase activity. Northern (RNA) blot analysis showed that PDC6 is weakly expressed, and expression seemed to be higher during growth in ethanol medium. This behavior remained obscure since pyruvate decarboxylase catalyzes an irreversible reaction. Characterization of all combinations of PDC structural gene deletion mutants, which produce different amounts of pyruvate decarboxylase activity, showed that the enzyme is also needed for normal growth in galactose and ethanol medium and in particular for proper growth initiation of spores germinating on ethanol medium.     

Coordinate regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae.

Regulation of the activity of the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) was measured in vitro by using membrane preparations from wild-type and mutant strains of Saccharomyces cerevisiae. PSD specific activity was not affected by carbon source, and on all carbon sources, the highest specific activity was observed in cells entering the stationary phase of growth. However, PSD activity was found to be regulated in response to soluble precursors of phospholipid biosynthesis. PSD specific activity was reduced to about 63% of the level observed in unsupplemented wild-type cells when the cells were grown in the presence of 75 microM inositol. The presence of 1 mM choline alone had no repressing effect, but the presence of 1 mM choline and 75 microM inositol together led to further repression to a level of about 28% of the derepressed activity. Regulatory mutations known to affect regulation or expression of genes encoding phospholipid-synthesizing enzymes also affected PSD specific activity. opi1 mutants, which are constitutive for a number of phospholipid-biosynthetic enzymes, were found to have high, constitutive levels of PSD. Likewise, in ino2 or ino4 regulatory mutants, PSD activity was found to be at the fully repressed level regardless of growth condition. Regulation of PSD activity was also affected in several structural-gene mutants under conditions of impaired phosphatidylcholine biosynthesis. Together, these data strongly suggest that PSD expression is controlled by the mechanism of general control of phospholipid biosynthesis that regulates many enzymes of phospholipid biosynthesis.     

Ornithine decarboxylase from Saccharomyces cerevisiae. Purification, properties, and regulation of activity

Ornithine decarboxylase has been purified 1,500-fold to homogeneity from a spe2 mutant of Saccharomyces cerevisiae which lacks S-adenosylmethionine decarboxylase and is derepressed for ornithine decarboxylase. The ornithine decarboxylase is a single polypeptide (Mr = 68,000) and requires a thiol and pyridoxal phosphate for activity. Addition of 10(-4) M spermidine and 10(-4) M spermine to the growth medium reduces the activity of the enzyme by 90% in 4 h. However, immunoprecipitation studies showed that the extracts of polyamine-treated cells contain as much enzyme protein as normal cell extracts. This loss of ornithine decarboxylase activity is probably due to a post-translational modification of enzyme protein because we found no evidence for any inhibitor of activity in the polyamine-treated cells.