GENETIC APPROACHES TO ANALYZING THE INSERTION & TOPOLOGY

我们将使用基因融合的方法来研究拓扑结构 完整膜蛋白的排列。碱性的熔融 磷酸酶(AP)到这类蛋白质的周质结构域 AP酶活性高，而AP与细胞质的融合 结构域的酶活性较低。通过分离大量的 这种融合到完整的膜蛋白，MALF和 先导肽，我们希望推导出它们的拓扑结构 膜蛋白。β-半乳糖苷酶(BetaGz)与人乳清蛋白的融合 膜蛋白的周质结构域显示低β-Gz 活性，而将BetaGz融合到细胞质结构域 同样的蛋白质具有很高的活性--情况正好相反 美联社。膜蛋白与BetaGz融合蛋白的分析 还应提供有关拓扑的信息。 一种将BetaGz融合到具有低BetaGz活性的MALF， 据推测，由于其膜插入，将被用来获得 并对改变MALF插入能力的突变体进行了表征 进入细胞膜。该菌株Lac+衍生物的筛选 应该同时产生MARF基因的突变和未连接的 将对突变体进行表征，并应提供有关1)的信息 MALF中的信号负责其膜插入和2) 插入过程中涉及的任何细胞组件。

We will use a gene fusion approach to study the topological arrangement of integral membrane proteins. Fusions of alkaline phosphatase (AP) to periplasmic domains of such proteins give high AP enzymatic activity, while fusions of AP to cytoplasmic domains give low enzymatic activity. By isolating a large number of such fusions to the integral membrane proteins, MalF and leader peptidase, we hope to derive the topology of these membrane proteins. Fusions of betagalactosidase (betaGz) to periplasmic domains of membrane proteins exhibit low beta-Gz activity, while fusions of betaGz to cytoplasmic domains of the same protein give high activity - the converse of the situation with AP. Analysis of betaGz fusions to membrane proteins should also give information on topology. One betaGz fusion to MalF which has low betaGz activity, presumably due to its membrane insertion, will be used to obtain and characterize mutants which alter the ability of MalF to insert into the membrane. Selection of Lac+ derivatives of this strain should yield both mutations in the malF gene and unlinked mutants will be characterized and should yield information on 1) the signals in MalF responsible for its membrane insertion and 2) any cellular components that are involved in the insertion process.