Metal binding to Saccharomyces cerevisiae ferrochelatase.

Ferrochelatase is the terminal enzyme in the heme biosynthetic pathway. It catalyzes the insertion of ferrous iron into protoporphyrin IX to produce protoheme IX. The crystal structures of ferrochelatase from Saccharomyces cerevisiae in free form, in complex with Co(II), a substrate metal ion, and in complex with two inhibitors, Cd(II) and Hg(I), are presented in this work.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%.

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.

Negative control of yeast coproporphyrinogen oxidase synthesis by heme and oxygen.

Coproporphyrinogen oxidase (EC 1.3.3.

Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase.

We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1-5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.

Modified uroporphyrinogen decarboxylase activity in a yeast mutant which mimics porphyria cutanea tarda.

The isolation of a new mutant Sm1 strain of yeast, Saccharomyces cerevisiae, is described: this strain was partially defective in haem formation and accumulated large amounts of Zn-porphyrins. Genetic analysis showed that the porphyrin accumulation was under the control of a single nuclear recessive mutation. Biochemical analysis showed that the main porphyrins accumulated in the cells were uroporphyrin and heptacarboxyporphyrin, mostly of the isomer-III type.

Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis.

Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed.