Biomedical subjects

M Góra

Publications and source records attributed to M Góra.

10 recordsLinked to original sources

PubMed2000

Suppressors of translation initiation defect in hem12 locus of Saccharomyces cerevisiae.

A system for the positive selection of transational initiation suppressors in S. cerevisiae has been developed. A mutant with an ATA initiation codon in the HEM12 gene, encoding uroporphyrinogen decarboxylase, was used to select cis- and trans-acting suppressors. These suppressors partially restore growth on nonfermentable carbon sources, such as glycerol, but still allow the accumulation of porphyrins. All extragenic suppressors are mapped to the SUI1 locus, encoding initiation factor eIF1. The effect of the hem12 mutation is also partially reversed by the known SUI3 suppressor encoding the beta subunit of eIF2. In contrast, the sui2 suppressor encoding the a subunit of eIF2 does not affect the hem12 phenotype. The intragenic suppressors are able to restore the translation of hem12 due to the generation of additional, in frame AUG codons upstream of the hem12-14 mutation. Mutational analysis of the HEM12 leader sequence was also performed to determine the role of small open reading frames (uORFs) present upstream of the HEM12 ORF. Studies on the expression of integrated hem12-1/4-lacZ fusion, devoid of all upstream ATGs, indicate a lack of regulatory effect of uORFs on HEM12 translation.

Alleles↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1996

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.

Alleles↗

PubMed1971