Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases.

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.

Flavohemoglobin expression and function in Saccharomyces cerevisiae. No relationship with respiration and complex response to oxidative stress.

The yeast Saccharomyces cerevisiae contains a flavohemoglobin, encoded by the gene YHB1, whose function is unclear. Previous reports presented evidence that its maximal expression requires disruption of mitochondrial respiration and that it plays a role in the response to oxidative stress. We have studied the expression of YHB1 in respiratory deficient cells and in cells exposed to various compounds causing oxidative stress. Several different strains and approaches (spectroscopic detection of the oxygenated form of Yhb1p, beta-galactosidase activity of a YHB1-lacZ fusion, and Northern blot analysis) were used to demonstrate that YHB1 expression and Yhb1p production are not increased by respiration deficiency. YHB1 expression was unchanged in cells challenged with antimycin A or menadione, while it decreased in cells exposed to H2O2, diamide, dithiothreitol, and Cu2+. Transcription of YHB1 is not under the control of the transcriptional factor Yap1p. These results do not support a participation of YHB1 in the genetic response to oxidative stress. We also analyzed the growth phenotypes associated with altered Yhb1p production using YHB1-deleted strains and strains that greatly overproduced Yhb1p. Yhb1p appears to protect cells against the damage caused by Cu2+ and dithiothreitol, while sensitizing them to H2O2. Yhb1p overproduction in a glucose-6-phosphate dehydrogenase-deficient mutant decreased its growth rate. These data indicate that there is a complex relationship(s) between Yhb1p function(s) and cell defense reactions against various stresses.

Characterization of an upstream activation sequence and two Rox1p-responsive sites controlling the induction of the yeast HEM13 gene by oxygen and heme deficiency.

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase, an oxygen-requiring enzyme catalyzing the sixth step of heme biosynthesis. Its transcription has been shown to be induced 40-50-fold in response to oxygen or heme deficiency, in part through relief of repression exerted by Rox1p and in part by activation mediated by an upstream activation sequence (UAS). This report describes an analysis of HEM13 UAS and of the Rox1p-responsive sites by electrophoretic mobility shift assays, DNase I footprinting, and mutational mapping. HEM13 UAS is composed of two subelements: a 16-base pair sequence binding a constitutive factor acting as a transcriptional activator, and a 5'-flanking 20-base pair GC-rich region. Both subelements were required additively for transcription, but each element alone was sufficient for almost normal control by oxygen/heme deficiency. Mutations in both elements decreased the induction ratio 3-4-fold. HEM13 UAS conferred a 2-4-fold oxygen/heme control on a heterologous reporter gene. Two Rox1p-responsive sites, R1 and R3, were identified, which accounted for the 6-7-fold repression by Rox1p. A factor bound to a sequence close to site R3. This DNA-binding activity was only detected in protein extracts of aerobic heme-sufficient ROX1 TUP1 cells, suggesting a possible role in site R3 function.

Probing the active-site residues in Saccharomyces cerevisiae ferrochelatase by directed mutagenesis. In vivo and in vitro analyses.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the insertion of ferrous iron into protoporphyrin, the terminal step in protoheme biosynthesis. The functional/structural roles of 10 invariant amino acid residues were investigated by site-directed mutagenesis in the yeast Saccharomyces cerevisiae ferrochelatase. The mutant enzymes were expressed in a yeast strain lacking the ferrochelatase gene HEM15 and in Escherichia coli. The kinetic parameters of the mutant enzymes were determined for the enzymes associated with the yeast membranes and the enzymes in the bacterial soluble fraction. They were compared with the in vivo functioning of the mutant enzymes. The main conclusions are the following. Glu-314 is critical for catalysis, and we suggest that it is the base responsible for abstracting the N-pyrrole proton(s). His-235 is essential for metal binding. Asp-246 and Tyr-248 are also involved in metal binding in a synergistic manner. The Km for protoporphyrin was also increased in the H235L, D246A, and Y248L mutants, suggesting that the binding sites of the two substrates are not independent of each other. The R87A, Y95L, Q111E, Q273E, W282L, and F308A mutants had 1.2-2-fold increased Vm and 4-10-fold increased Km values for protoporphyrin, but the amount of heme made in vivo was 10-100% of the normal value. These mutations probably affected the geometry of the active center, resulting in improper positioning of protoporphyrin.

Effect of heme and vacuole deficiency on FRE1 gene expression and ferrireductase activity in Saccharomyces cerevisiae.

We have examined the effects of heme or vacuole deficiency on the kinetics of induction of cell surface ferrireductase activity and expression of the FRE1 gene encoding a component of ferrireductase, in response to iron or copper deprivation in S. cerevisiae. Heme deficiency caused a small decrease in the basal expression of FRE1, but did not impair its induction by Fe or Cu limitation. Thus, the absence of ferrireductase activity and its non-inducibility in heme-less cells is not due to the absence of FRE1 expression. Vacuole deficiency led to constitutively high ferrireductase activity slightly induced by Cu limitation, and to high levels of FRE1 expression further inducible by Fe or Cu deprivation. Thus, the vacuole might be a component of the iron signalling pathway.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.

Positive and negative elements involved in the differential regulation by heme and oxygen of the HEM13 gene (coproporphyrinogen oxidase) in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis. Its transcription is increased 40-50-fold in response to oxygen- or heme-deficiency. We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUP1 genes. The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1 delta background). Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner. In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13. Cyp1p had no effect on HEM13 expression in anaerobic cells. Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells. A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation. Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth. The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency. Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency.

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 characteristic 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 beta-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.

Isolation of the gene HEM4 encoding uroporphyrinogen III synthase in Saccharomyces cerevisiae.

We have isolated a genomic DNA fragment that complements the yeast temperature-sensitive cyt mutation, causing respiratory deficiency and accumulation of porphyrins (Sugimura et al., 1966). Partial DNA sequencing of the complementing region and search for similarity in the DNA and protein databases revealed that (1) the gene had been previously isolated by complementation of the mutation ts2326 (Langgut et al., 1986; accession number X04694), and (2) it encodes a protein with 18-23% identity to uroporphyrinogen III synthases from different sources. This enzyme catalyses the fourth step in the heme biosynthetic pathway and we named its gene HEM4. A hem4 delta disruption mutation was constructed which had phenotypes identical to the cyt mutation. Biochemical analysis confirmed the absence of uroporphyrinogen III synthase activity in both hem4 delta and cyt mutant strains.

Isolation of a cDNA encoding chloroplast ferrochelatase from Arabidopsis thaliana by functional complementation of a yeast mutant.

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. It is located in the mitochondria in all eukaryotes and is also found in plastids in plants. Although it has been purified from animals and microorganisms, and genes for it isolated and characterized, very little is known about plant ferrochelatases. We have isolated a cDNA for ferrochelatase from the higher plant Arabidopsis thaliana by functional complementation of a mutant of Saccharomyces cerevisiae defective in this enzyme. The cDNA encodes a protein of 52 kDa, which has 25-35% sequence similarity to ferrochelatases from other organisms. There is an N-terminal extension of about 65 residues, which is almost certainly the chloroplast transit peptide, since the precursor protein, transcribed and translated in vitro, is efficiently imported and processed to the mature size by isolated pea chloroplasts. In contrast, the precursor was not processed by mitochondrial processing peptidase activity, nor could import into isolated yeast mitochondria be demonstrated conclusively, although, presumably, in the rescued yeast mutant, at least some of the Arabidopsis ferrochelatase must be present in the mitochondria. A single transcript the same size as the cDNA was detected in both Arabidopsis leaves and roots, although the amount of message was greater in the photosynthetic tissue. Southern analysis suggests that there is a single gene for chloroplast ferrochelatase in Arabidopsis.

Structure-function studies of yeast ferrochelatase. Identification and functional analysis of amino acid substitutions that increase Vmax and the KM for both substrates.

The molecular basis of the ferrochelatase defects was investigated in two "protoporphyric" and partially heme-deficient yeast mutants. Ferrochelatase, a mitochondrial inner membrane-bound enzyme, catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. The mutant cells made normal amounts of normal-sized ferrochelatase, as detected by immunoblotting. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant strains genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. Substitution of the conserved Ser-169 by Phe caused a 10-fold increase in Vmax and a 45- and 35-fold increase in the KM for protoporphyrin and metal, respectively. Replacement of Ser-174 by Pro produced the same effects, but to a lesser degree. There was a good correlation between the ferrochelatase defects measured in vitro and the heme synthesis deficiencies estimated in vivo. The decreased in vivo heme synthesis is probably due to the lower affinity of the mutant enzymes for iron. We propose that the region identified by the two close mutations contributes to the binding domains of metal and protoporphyrin.

Regulation of Cu,Zn- and Mn-superoxide dismutase transcription in Saccharomyces cerevisiae.

The regulation of Cu,Zn- and Mn-superoxide dismutases (SOD) was investigated by Northern blotting and gene fusions of SOD1 and SOD2 promoters with the beta-galactosidase reporter gene. Cu,ZnSOD expression was increased 3-fold under glucose derepressing conditions, and decreased 4- to 6-fold by oxygen or heme deficiency. MnSOD expression was increased 5-fold by glucose derepression, and decreased 8- to 10-fold by anaerobiosis and 4- to 5-fold by heme deficiency. Induction by paraquat was modest, about 50% for SOD1 and 100% for SOD2; it was apparently independent of the respiratory chain function.

Identification of amino acid changes affecting yeast uroporphyrinogen decarboxylase activity by sequence analysis of hem12 mutant alleles.

The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast 'uroporphyric' mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven 'leaky' mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.

Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae. HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity.

The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis. The gene was isolated by functional complementation of a hem12 mutant. Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da. The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp. We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme. They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions. Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein. These results argue that a single active center exists in uroporphyrinogen decarboxylase.

CYP1 (HAP1) is a determinant effector of alternative expression of heme-dependent transcribed genes in yeast [corrected].

The CYP1 (HAP1) gene of Saccharomyces cerevisiae is known to activate a number of target genes in response to the presence of heme. Several features of the protein, deduced from the sequence of the gene, suggest that CYP1 is a general sensor of the redox state of the cell. To investigate further the function of CYP1, we analysed its effects on the transcription of two genes, HEM13 and 14DM, which are preferentially expressed in anaerobiosis. HEM13 encodes coproporphyrinogen oxidase which catalyses the sixth enzymatic step in the heme biosynthetic pathway and 14DM encodes lanosterol-14-demethylase which is involved in sterol biosynthesis and is a member of the cytochrome P450 family. Isogenic CYP1+ and cyp1 degree deleted strains, either heme-sufficient or heme-deficient (HEM1 disrupted), were grown in aerobic or anaerobic conditions, and transcripts of HEM13 and 14DM were analysed on Northern blots. The results show that in anaerobic and in heme-deficient cells, CYP1 activates the transcription of HEM13 and inhibits that of 14DM. Opposite effects of CYP1 are observed in aerobic, heme-sufficient cells. We concluded that: (i) CYP1 is an efficient activator especially in heme-depleted cells; (ii) CYP1 exerts both positive and negative regulatory effects; (iii) the nature of the regulatory function of CYP1 depends on the target gene; and (iv) for a given gene, the presence or absence of heme or oxygen reverses the sense of CYP1-dependent regulation.

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.

Isolation, sequence, and regulation by oxygen of the yeast HEM13 gene coding for coproporphyrinogen oxidase.

The HEM13 gene of Saccharomyces cerevisiae codes for coproporphyrinogen oxidase (EC 1.3.3.3) catalyzing the sixth enzymic step in the heme biosynthetic pathway. Its expression has been previously shown to be regulated negatively by heme and oxygen. We have isolated the HEM13 gene by functional complementation of a hem13 gene by functional complementation of a hem13 mutant and determined its nucleotide sequence. The open reading frame encodes a protein of 328 amino acids. Its calculated molecular weight (37,673), amino acid composition and amino-terminal sequence predicted from the DNA sequence are in agreement with those determined for the native enzyme (Camadro, J. M., Chambon, H., Jolles, J., and Labbe, P. (1986) Eur. J. Biochem. 156, 579-587). The 5' ends of the HEM13 transcripts were identified by nuclease S1 mapping; induction of HEM13 resulted in an equivalent increase of the level of all the transcripts. 5' deletion analysis revealed that DNA sequence located upstream of 409 nucleotides from the translational initiation codon was needed for depression under oxygen limitation. The loss of induction of coproporphyrinogen oxidase activity by anaerobiosis caused a considerable decrease of heme formation in anaerobic cells.

The effects in vivo of mutationally modified uroporphyrinogen decarboxylase in different hem12 mutants of baker's yeast (Saccharomyces cerevisiae).

Nine new hem12 haploid mutants of baker's yeast (Saccharomyces cerevisiae), totally or partially deficient in uroporphyrinogen decarboxylase activity, were subjected to both genetic and biochemical analysis. The mutations sites studied are situated far apart within the HEM12 gene located on chromosome IV. Uroporphyrinogen decarboxylase activity in the cell-free extracts of the mutants was decreased by 50-100%. This correlated well with the decrease of haem formation and the increased accumulation and excretion of porphyrins observed in vivo. The pattern of porphyrins (uroporphyrin and its decarboxylation products) accumulated in the cells of mutants partially deficient in uroporphyrinogen decarboxylase activity did not differ significantly, although differences in vitro were found in the relative activity of the mutant enzyme at the four decarboxylation steps. The excreted porphyrins comprised mainly dehydroisocoproporphyrin or pentacarboxyporphyrin. In heterozygous hem12-1/HEM12 diploid cells, a 50% decrease in decarboxylase activity led to an increased accumulation of porphyrins as compared with the wild-type HEM12/HEM12 diploid, which points to the semi-dominant character of the hem12-1 mutation. The biochemical phenotypes of both the haploid and the heterozygous diploid resembles closely the situation encountered in porphyria cutanea tarda, the most common human form of porphyria.

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.