Screening Of Human Genes Which Specifically Inhibits Sporulation In Yeast Cells

In response to nutrient limitation,diploid cells of the budding yeast Saccharomyces cerevisiae undergo meiosis,and differentiate into a dormant and stress resistant form of haploid cell termed a spore.Sporulation is a developmental process that do not happen in mammals.However,the process includes several biological events which are conserved in eukaryotic cells,such as nutrient sensing,meiosis,and re-organization of membrane trafficking.Therefore,We developed a method to screen human genes which would specifically inhibit yeast sporulation.In this screening,human genes were individually expressed in yeast cells and their effects on sporulation were monitored.We examined totally 3,128 genes and found 12 genes which strongly inhibiting sporulation but did not inhibit vegetative growth.Among them,4-hydroxyphenylpyruvate dioxygenase(HPD)exhibited unique property: its expression resulted in a failure of the formation of the outermost layer of the spore wall termed the dityrosine layer.The dityrosine layer is mainly composed of the bisformyl dityrosine.Because bisformyl dityrosine is a fluorescence molecule,spores emit blue fluorescence under UV light.However,expression of HPD causes a loss of the fluorescence in spores.Intensity of dityrosine fluorescence was increased in the media cultured HPD expressing spores,suggesting that dityrosine containing molecules leak from developing spores.HPD produces homogentisate(HGA)from a tyrosine metabolite,4-hydroxyphenylpyruvate.Our results suggest that incorporation of HGA into the spore wall,probably via its oxidation,causes a defect in dityrosine layer assembly.These findings provide insight into the mechanism of the dityrosine layer.Expression the other candidate genes resulted in failure in entering in meiosis.These candidates are involved in various cellular processes;some are functionally uncharacterized.Further analysis using yeast cells would help to reveal functions of the gene products.Overall,our screen is useful not only for understanding the sporulation process but also for finding novel functions of human proteins.