PROTEIN TRANSLOCATION IN YEAST

我们的主要目标是了解某些 蛋白质的种类被选择性地靶向并跨膜转运。 内质网膜在酵母酿酒酵母。 催化这一过程的细胞机器的酵母成分将 进行生物化学鉴定、表征和纯化。在随后 基因分析克隆的基因将受到干扰，并研究其影响。 在体内和体外。 i）酵母体外蛋白质 易位测定将用于纯化可溶性的，以及 膜结合易位因子。 进一步的体外试验将在 旨在阐明其功能。 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）第二阶段 此外，我们将纯化和克隆这些酵母类似物， 在哺乳动物蛋白质中已经明确定义的成分 易位系统，即SRP和SRP受体，预计将被 也存在于酵母中。 iii）i）或ii）中鉴定的基因将是 破坏，并且所得无效等位基因的表型将被破坏。 表征了 一旦鉴定出分泌受损的表型， 将用于分离条件等位基因。 ㈣相互作用的组成部分 将通过分离ts或 CS或体外工程化突变。 （五）“反向遗传” 上述方法将由生物化学补充， 通过经典遗传方法分离的突变体的表征， 筛选酵母易位机制的改变。 最终，生物化学和遗传学相结合的方法的力量应该 导致蛋白质易位的完整分子描述 机械及其基本和调节成分，并允许我们 了解蛋白质运动的机制， 膜。 拟议的研究显然是最基本的性质， 毫无疑问，这将对一个 了解细胞和分子水平的生理学和病理学 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.