No abstract

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.

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.

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.

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.