MOLECULAR BASIS OF YEAST KILLER PROTOXIN GENE EXPRESSION

我们正试图阐明潜在的基本分子机制。 1型杀手的生产和对一种真菌病毒毒素的特异性免疫 酿酒酵母中的dsRNA系统。我们的方法是一种坐标方法，利用 包括生化和基因技术。我们预计这一结果来自于 这些研究将为各种相互关联的 酵母中与蛋白质分泌和内切相关的几个方面 在高等真核生物中的蛋白质加工，并将进一步发展我们的 了解这种真菌病毒系统的分子生物学， 尤其是在毒素作用和免疫机制方面 功能。 K1系统由胞质包装的分段dsrna基因组组成。 在病毒样颗粒(VLP)中。两个主要成分是4.7 千碱基对(Kb)L-双链RNA，编码VLP的主要衣壳蛋白； 1.9kb的M-dsRNA。在以前的研究中，我们确定了34.8千道尔顿(Kd) 原毒素作为M-dsRNA的主要翻译产物。直通基因 利用前原毒素基因的cDNA克隆进行的融合实验，我们 确定毒素和免疫决定因素都存在于这个单一的 分子。该毒素由两个9.0-9.5kd组分组成，Alpha和Beta 来源于43kd的糖基化原毒素，其序列如下 Delta-Alpha-Gamma-Beta。Delta是一个44个氨基酸的N末端先导，并且 伽马大约有103个氨基酸，是糖基化的部位。成熟 遵循SEC基因定义的标准酵母分泌途径，以及 涉及染色体KEX(杀手表达)的附加功能和 REX(抗性表达)基因座。 我们计划通过解决几个具体问题来继续我们的研究。我们 将评估原毒素蛋白结构域的结构和功能 通过定点突变。这将包括标识 负责免疫的细胞成分。我们将定义组件 相互作用并参与毒素和免疫的产生和功能 通过分离和鉴定不同类型的第二位点抑制子 原毒素突变体。最后，我们将分析N端的作用 细胞内转运中的Delta序列和中心Gamma序列 前原毒素的加工。

We are attempting to elucidate basic molecular mechanisms underlying the production and specific immunity to a mycoviral toxin in the type 1 killer dsRNA system in S. cerevisiae. Our approach is a coordinate one utilizing both biochemical and genetic techniques. We anticipate that results from these studies will provide new insights into a variety of interrelated aspects of protein secretion and endoproteolysis in yeast relevant to protein processing in higher eucaryotes, and will further develop our understanding of the molecular biology of this mycoviral system, particularly with respect to mechanisms of toxin action and immunity function. The K1 system consists of a segmented dsRNA genome packaged cytoplasmically in virus-like particles (VLPs). The two predominant constituents are a 4.7 kilobase pair (kb) L-dsRNA, which encodes the major VLP capsid protein, and a 1.9 kb M-dsRNA. In previous studies we identified a 34.8 kilodalton (kd) preprotoxin as the primary translation product of M-dsRNA. Through gene fusion experiments employing a cDNA clone of the preprotoxin gene, we determined that both toxin and immunity determinants reside in this single molecule. The toxin consists of two 9.0-9.5 kd components, Alpha and Beta derived from a 43 kd glycosylated protoxin which has the sequence Delta-Alpha-Gamma-Beta. Delta is a 44 amino acid N-terminal leader, and Gamma, about 103 amino acids, is the site of glycosylation. Maturation follows the standard yeast secretory pathway defined by the SEC genes, and involves the additional function of chromosomal KEX (killer expression) and REX (resistance expression) loci. We plan to continue our studies by addressing several specific issues. We will evaluate the structure and function of the protein domains of protoxin by site-specific mutagenesis. This will include identification of the cellular component responsible for immunity. We will define components that interact and participate in toxin and immunity production and function by isolation and characterization of second-site suppressors of various protoxin mutants. Finally, we will analyze the role of the N-terminal Delta sequence and the central Gamma sequence in intracellular transit and processing of the preprotoxin.