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Cloning and identification of HEM14, the yeast gene for mitochondrial protoporphyrinogen oxidase
D M Glerum1, A Shtanko, A Tzagoloff
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Abstract:
A respiratory-defective mutant (C54) of Saccharomyces cerevisiae was found to have a phenotype consistent with a mutation in either mitochondrial protoporphyrinogen oxidase or ferrochelatase. The mutant is grossly deficient in hemes, accumulates protoporphyrin and is rescued by exogenous heme. The increased levels of protoporphyrin at the expense of heme is indicative of a block in one of the two last steps of the heme biosynthetic pathway. Complementation of C54 by a known ferrochelatase mutant suggested that the defect was most likely in HEM14 encoding protoporphyrinogen oxidase. A plasmid capable of complementing C54 was obtained by transformation with a yeast genomic plasmid library. A partial sequence of the insert identified the gene as reading frame YER014 of yeast chromosome V (GenBank Accession Number U18778). This reading frame codes for a protein homologous to human protoporphyrinogen oxidase. Disruption of this gene elicits a respiratory defect and accumulation of protoporphyrin. The phenotype of the null mutant together with the homology of YER014p to human protoporphyrinogen oxidase provide compelling evidence that YER014 is HEM14.
Insights
Researchers identified a yeast mutant with a defect in heme biosynthesis. The study pinpoints HEM14, encoding protoporphyrinogen oxidase, as the responsible gene, crucial for cellular respiration and heme production.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Heme biosynthesis is essential for cellular respiration.
- Mutations in the final steps of heme synthesis can cause respiratory defects.
- Protoporphyrinogen oxidase (PPO) and ferrochelatase are key enzymes in the terminal heme pathway.
Purpose of the Study:
- To identify the specific gene responsible for a respiratory-defective phenotype in Saccharomyces cerevisiae mutant C54.
- To characterize the role of the identified gene in heme biosynthesis and cellular respiration.
- To confirm the identity of the gene through complementation and disruption studies.
Main Methods:
- Phenotypic analysis of a respiratory-defective yeast mutant (C54).
- Complementation assays with known ferrochelatase mutants.
- Screening of a yeast genomic plasmid library for complementing clones.
- Gene sequencing and homology analysis.
- Gene disruption to create a null mutant.
Main Results:
- Mutant C54 accumulated protoporphyrin and was deficient in heme, indicating a block in the late heme pathway.
- Complementation suggested the mutation was in HEM14, encoding protoporphyrinogen oxidase.
- A complementing plasmid contained yeast chromosome V reading frame YER014, homologous to human PPO.
- Disruption of YER014 resulted in a respiratory defect and protoporphyrin accumulation, confirming it as HEM14.
Conclusions:
- The yeast gene YER014 encodes protoporphyrinogen oxidase (HEM14).
- HEM14 is essential for heme biosynthesis and cellular respiration in Saccharomyces cerevisiae.
- The identified gene YER014p is homologous to human protoporphyrinogen oxidase, suggesting conserved function.