PROTEIN PROTEIN INTERACTIONS IN POLYISOPRENE SYNTHESIS

这项建议将确定蛋白质-蛋白质相互作用在 酿酒酵母类异戊二烯生物合成途径的研究 双杂交遗传筛选。含有异戊二烯的化合物，如 甾醇、泛醌、二十二醇和戊烯基化蛋白质是必需的。 细胞功能，由常见的前体分子合成。 这一途径中不同酶之间的相互作用可能会引导这些 适当的聚异戊二烯产品的前体。同时， 这项工作将试图确定两个编码作用于 这条路。第一个是异二聚六氢戊二烯的一个亚基- 辅酶Q合成的二磷酸合成酶。已知的亚基 异源二聚体将作为酵母双杂交的靶序列 不仅可以识别与复合体有关的蛋白质的筛选 还包括假定的第二亚单位 活动。然后，活性六烯基-PP合成酶异二聚体 表达、纯化和鉴定。待鉴定的第二个基因 编码二醇生物合成的顺式戊烯基转移酶。两个酵母 开放阅读框与首次报道的顺式- 将对黄分枝杆菌的戊烯基转移酶序列进行活性测试。 这些蛋白质将作为酵母双杂交筛选的靶标， 并对活性蛋白进行纯化和鉴定。

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.