RETRIEVAL OF TRANSMEMBRANE PROTEIN TO THE ER

真核细胞的分泌途径由一系列不同的 蛋白质以矢量方式在其间移动的细胞器。尽管 蛋白质和脂质的大量进出， 细胞器维持一组蛋白质，这些蛋白质定义了其独特的结构 和功能特性。维持这些常驻蛋白质似乎 至少部分地通过使用以 蛋白质沿着一条逆行运输路线回到它的正确位置沿着。 我们以前已经确定了一个双赖氨酸基序在COOH末端 I型和III型膜蛋白，赋予在ER中的驻留。 同样地，C-末端四肽KDEL维持可溶性蛋白质在细胞中的表达。 急诊室的内腔。我们最近发现，双赖氨酸基序，如 KDEL基序是指导蛋白质转运的检索信号 回到急诊室。 已经获得了初步数据，即在第一和第二氨基酸处的15个氨基酸序列。 II型ER膜蛋白的氨基末端，不变的p33形式 链（Ii），对于将该蛋白质靶向ER是必需的。的 该建议的主要目标是通过以下方式表征该靶向基序： 广泛的定点诱变，并确定它是否保持其他 II型膜蛋白在ER移植时。接下来我们将 研究这个基序是否以一种 类似于双赖氨酸基序。 标记物接受的翻译后修饰的类型和速率 具有附加的双赖氨酸和KDEL基序的蛋白质表明， 蛋白质可以很好地从整个系统的多个位置取回。 以蒸馏假说预测的方式堆叠高尔基体（Rothman， 1981年）。为了研究这个问题，我们将比较和对比 用KDEL、双赖氨酸或Iip 33靶向基序标记的标记蛋白 相互之间，以及各种细胞器的已知居民， 亚细胞分级分离和免疫荧光和电子显微镜。 通过这种方式，我们将绘制由每个这些引导的检索路径 基序，并评估各种后ER隔室在 这些过程。 我们的长期目标是实现分子描述的机制 识别具有这些ER靶向基序的膜蛋白 并被送到急诊室为此，我们将开始制定一项 基因筛选，以确定酵母突变体是缺乏这种 过程这种突变体的互补性应该使我们能够分离出 在这个排序过程中涉及的组件的数量，从而大大 提高我们对分泌途径的细胞器 建立和维护。

The secretory pathway of eukaryotic cells consists of a series of distinct organelles between which proteins are moved in a vectorial manner. Despite the extensive movement of proteins and lipids into and out of them, each organelle maintains a set of proteins which define its unique structural and functional properties. Maintaining these resident proteins appears to be achieved at least in part by use of retrieval signals that target a protein back to its correct location along a retrograde transport route. We have previously identified a double lysine motif at the COOH terminus of type I and III membrane proteins that confers residency in the ER. Likewise a C-terminal tetra-peptide KDEL maintains soluble proteins in the lumen of the ER. We have recently shown that the double lysine motif, like the KDEL motif is a retrieval signal directing the transport of proteins back to the ER. Preliminary data has been obtained that a 15 amino acid sequence at the amino terminus of a type II ER membrane protein, the p33 form of invariant chain (Ii), is necessary for the targeting of this protein to the ER. The major goal of this proposal is to characterize this targeting motif by extensive site directed mutagenesis and to determine if it maintains other type II membrane proteins in the ER when transplanted. Next we will investigate whether this motif directs retrieval to the ER in a manner analogous to the double lysine motif. The types and rates of post-translational modifications received by marker proteins with appended double lysine and KDEL motifs suggest these proteins may well be retrieved from multiple locations throughout the Golgi stack in a manner predicted by the distillation hypothesis (Rothman, 1981). To investigate this we will compare and contrast the location of marker proteins tagged with KDEL, double lysine or Iip33 targeting motifs to each other and to known residents of the various organelles by use of subcellular fractionation and immunofluorescence and electron microscopy. In this way we will chart the retrieval path way directed by each of these motifs, and evaluate the role the various post-ER compartments play in these processes. Our long term goal is to achieve a molecular description of the mechanism by which membrane proteins with these ER targeting motifs are recognized and retrieved to the ER. To this end we will begin work to devise a genetic screen to identify yeast mutants that are deficient in this process. Complimentation of such mutants should allow us to isolate of a number of components involved in this sorting process, and thereby greatly improve our understanding of how the organelles of the secretory pathway are established and maintained.