PROTEIN PROTEIN INTERACTIONS IN POLYISOPRENE SYNTHESIS

这项提案将确定蛋白质-蛋白质相互作用， 利用酵母菌合成类异戊二烯途径 双杂交遗传筛选 含类异戊二烯的化合物，例如 甾醇、泛醌、多萜醇和异戊烯化蛋白质起着重要的作用 细胞功能，并从共同的前体分子合成。 在这一途径中各种酶之间的相互作用可能会引导这些 合适的聚类异戊二烯产物的前体。 与此同时， 这项工作将试图确定两个基因编码酶的作用， 这条路。 第一个是异二聚体六异戊二烯基的亚基， 辅酶Q合成的二磷酸合酶。 已知的亚基 异源二聚体将作为酵母双杂交中靶序列 筛选不仅可以鉴定复合物中涉及的蛋白质， 上面假设的，但也是推定的第二亚基所必需的， 活动 然后，活性的六异戊二烯基-PP合酶异二聚体将是 表达、纯化和表征。 第二个待鉴定的基因 编码长萜醇生物合成的顺式异戊二烯基转移酶。 两种酵母 开放阅读框架与首次报道的顺式- 异戊烯基转移酶序列。将检测黄体的活性。 这些蛋白质将被用作酵母双杂交筛选的靶标， 并对活性蛋白进行纯化和表征。

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.