Genetic regulation and enzymatic reaction of DI!2 and DIT1, two sporulation-specific genes of saccharomyces cerevisiae

The basic aim of the project was to elucidate the exact biochemical role of two genes, DIT1 and DIT2, that were discovered ion our laboratory. The two genes are transcribed only during a short time in mid-late sporulation. Their biological function is biosynthesis of the outermost layer of the yeast spore wall which confers resistance aginst digestive enzymes and harsh environmental conditions to the spore. The spore is not only a means of reshuffling genes (sexual reproduction) but also of dissemination of the species. In the first part of this project, we showed that Dit2p (which shows sequence homology with the large superfamily of cytochromes P450) is in fact the third P450 enzyme of yeast. Like the other two, it is localized in the endoplasmic reticulum and depends on cytochrome P450 reductase. We established an in vitro system for the enzymatic reaction catalyzed by Dit2p using a purified microsomal fraction from yeast ectopically expressing DIT2. Dit2p is an atypical P450 enzyme as it catalyzes an oxidase-type reaction. Two molecules of the substrate, N-formyl tyrosine are oxidised to form one molecule of Bis-N-formyl dityrosine. Co-substrates are NADPH and molecular oxygen. Water (but not hydrogen peroxide) is formed as a by-product. Oxygen is not incorporated into the substrate. The product, Bis-N-formyl dityrosine, is further metabolized by de-formylation and epimerization. DL-dityrosine is part of the highly cross-linked macromolecule forming the outer layer of the spore wall. Partial conversion to the D stereochemical form contributes to the nearly total resistance of the spore against proteases. The role of the second gene, DIT1, is catalyzing the step before oxidation, i.e. formylation. Interestingly this formylation is not dependent on a functional tetrahydrofolate biosynthetic pathway. However, we could not establish an in vitro system for this reaction and, therefore, the exact molecular details of the formylation remain obscure. We also investigated trancriptional regulation of the two genes, DIT1 and DIT2, which are transcribed from a common intergenic region on chromosome IV. We identified a new vegetative repressor of the DIT genes, Spt10p, and showed that a deletion of this gene has the same effect as the three recessive alles which were found in the original mutant screen. Interestingly, SPT10 is not only a repressor of the DIT intergenic region in vegetative cells but is also needed for normal sporulation. The deletion of SPT10 displays a broad spectrum of pleiotropic phenotypes, including irregular segregation of chromosomes in meiosis and mitosis. Sequence homology indicates that SPT10 could be a histone deacetylase. We identified a new cis-acting repressor element, DRE, in the DIT intergenic region and showed that a still unknown protein, which is not Spt10p, binds to this palindromic element. These results, althouhg they have no immediate industrial or medical application, are important new insights into gene function and gene regulation in a very basic eukaryotic developmental system.