Structure and Function of Fission Yeast Signal Recognition Particle

信号识别颗粒（SRP）是一种由6个蛋白质亚基和1个7SL RNA分子组成的核糖核蛋白，是识别真核细胞通过分泌途径输出的蛋白质的专门机制的第一个元件。根据体外实验，假设在核糖体出现信号序列后，SRP结合并阻止翻译。当复合物到达内质网（ER）膜时，SRP与特定受体相互作用，解离，蛋白质的翻译恢复并伴随着载体转运进入内质网腔。通过利用分裂酵母Schizosaccharomyces pombe的独特优势，我们将从遗传和生化角度剖析分泌蛋白靶向，首先关注SRP的基本7SL成分。克隆基因的随机突变，随后转化和筛选条件或部分分泌缺陷，将确定对体内功能至关重要的核苷酸。保守的序列和结构将通过比较来自不同生物的7SL同源物来确定，从而允许生成其作用模式的特定模型，这些模型将通过S. pombe基因的定点诱变进行测试。抑制因子选择将识别与7SL RNA相互作用的其他成分，最小程度上是裂变酵母SRP蛋白；其他可能的靶标包括SRP受体和核糖体蛋白。在基因实验的同时，将建立一个同源的体外系统来研究SRP的功能；这也将有助于对突变体的缺陷进行生化表征。这些研究将为细胞分类蛋白质的机制提供洞见，这些蛋白质的显著特征在裂变酵母和人类之间是保守的。

Signal recognition particle (SRP), a ribonucleoprotein composed of six protein subunits and one molecule of 7SL RNA, is the first element of the specialized machinery which recognizes proteins destined for export from eukaryotic cells via the secretory pathway. Based on in vitro experiments, it has been postulated that, upon emergence of the signal sequence from the ribosome, SRP binds and arrests translation. When the complex arrives at the endoplasmic reticulum (ER) membrane, SRP interacts with a specific receptor, dissociates, and translation of the protein resumes accompanied by vectorial translocation into the ER lumen. By exploiting the unique advantages of the fission yeast Schizosaccharomyces pombe, we will genetically and biochemically dissect secretory protein targeting, focusing first on the essential 7SL component of SRP. Random mutagenesis of the cloned gene followed by transformation and screening for conditional or partial secretory defects will identify nucleotides which are critical for function in vivo. Conserved sequences and structures will be determined by comparison of 7SL homologs from divergent organisms, allowing generation of specific models for its mode of action which will be tested by site- directed mutagenesis of the S. pombe gene. Suppressor selection will identify other components which interact with 7SL RNA, minimally the fission yeast SRP proteins; other likely targets include the SRP receptor and ribosomal proteins. Concurrent with the genetic experiments, a homologous in vitro system will be developed to study SRP function; it will also be useful for biochemically characterizing defects in the mutants. These studies will provide insight into the mechanism by which cell sort proteins, the salient features of which are conserved between fission yeast and humans.