PROTEIN TRANSLOCATION IN YEAST

我们的主要目标是了解某些特定的 不同类别的蛋白质被选择性地靶向并跨 酿酒酵母的内质网膜。 催化这一过程的细胞机械中的酵母成分将 经过生化鉴定、表征和提纯。在随后的 遗传分析克隆的基因将被扰乱并研究其影响 在体内和体外。具体地说：i)一种酵母体外蛋白 易位试验将用于提纯可溶性，以及 膜结合转位因子。进一步的体外检测将是 旨在阐明它们的功能。编码这些因子的基因将 使用对纯化材料产生的分子探针进行克隆。Ii) 此外，我们将提纯和克隆这些酵母的类似物 哺乳动物蛋白质中已经很好定义的成分 易位系统，即SRP和SRP受体，预计将被 也存在于酵母中。三)i)或ii)中确定的基因将是 并且产生的空等位基因的表型将是 特色化的。一旦确定了分泌受损的表型，它就会 将被用来分离条件等位基因。四)相互作用的组成部分 将通过分离ts或ts的基因外抑制物来鉴定 CS或体外工程突变。五)“反向遗传” 上面概述的方法将得到生物化学的补充 用经典遗传方法分离的突变体的特征 筛选酵母转位机制中的变化。 归根结底，生化和遗传相结合的方法的力量应该 导致对蛋白质易位的完整的分子描述 机械及其基本和调节成分，并使我们能够 了解蛋白质跨膜迁移的机制 膜。拟议的研究显然是最基本的性质和 毫无疑问，这将是一个具有深远意义的 从细胞和分子的角度认识生理和病理 水平。

Our primary objective is understanding the mechanism by which certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum membrane in the yeast Saccharomyces cerevisiae. Yeast components of the cellular machinery that catalyzes this process will be biochemically identified, characterized and purified. In subsequent genetic analyses the cloned genes will be perturbed and the effects studied in vivo and in vitro. Specifically: i) A yeast in vitro protein translocation assay will be used to purify soluble, as well as membrane-bound translocation factors. Further in vitro assays will be designed to elucidate their function. The genes encoding such factors will be cloned using molecular probes generated to the purified material. ii) Additionally, we will purify and clone the yeast analogs of those components that are already well defined in the mammalian protein translocation system, namely SRP and SRP receptor, which are expected to be present also in yeast. iii) Genes identified in i) or ii) will be disrupted and the phenotype of the resulting null alleles will be characterized. Once a secretion-impaired phenotype has been identified, it will be used to isolate conditional alleles. iv) Interacting components will be identified by isolation of extragenic suppressors of either ts or cs or of in vitro engineered mutations. v) The 'reverse genetic' approaches outlined above will be complemented by a biochemical characterization of mutants isolated by classical genetic means by screening for alterations in the yeast translocation machinery. Ultimately, the power of a combined biochemical and genetic approach should lead to a complete molecular description of the protein translocation machinery and its essential and modulatory constituents, and allow us to gain an understanding of the mechanism of protein movement across membranes. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.