This proposal will identify protein-protein interactions in the isoprenoid biosynthetic pathway of Saccharomyces cerevisiae using yeast two-hybrid genetic screening. Isoprenoid-containing compounds such as sterols, ubiquinone, dolichol, and prenylated proteins serve essential cellular functions and are synthesized from common precursor molecules. Interactions between various enzymes in this pathway may channel these precursors to the appropriate polyisoprenoid products. Concurrently, this work will attempt to identify two genes encoding enzymes acting in this pathway. The first is a subunit of the heterodimeric hexaprenyl- diphosphate synthase of Coenzyme Q synthesis. The known subunit of the heterodimer will serve as a target sequence in yeast two-hybrid screening which may identify not only proteins involved in the complexes postulated above, but also the putative second subunit necessary for activity. The active hexaprenyl-PP synthase heterodimer would then be expressed, purified and characterized. The second gene to be identified encodes the cis-prenyltransferase of dolichol biosynthesis. Two yeast open reading frames highly similar to the first reported cis- prenyltransferase sequence from M. luteus will be tested for activity. These proteins will be used as targets for yeast two-hybrid screening, and active protein will be purified and characterized.
