BIOGENESIS OF THE GOLGI APPARATUS

描述（由申请人提供）：了解在发育、细胞分裂和细胞应激过程中建立和维持高尔基体的分子机制是生物医学研究的重要目标。我们的长期目标是确定高尔基体结构所需的蛋白质，并阐明其机制。这项工作是使用基于siRNA的筛选靶向候选人，特别是golgins，和基于细胞的高尔基体组装测定，然后结合生物化学，透化细胞，结构和计算测定敲低后救援。我们的筛选确定高尔基体蛋白-160（G160）作为高尔基体膜向内运动的关键要求，并表明它的缺乏逮捕高尔基体组装在分散的ministack阶段损害伤口愈合过程中的定向分泌和细胞迁移。这一观察结果提出的可能性，我们可以回答有关未知的身份和运动受体复合物的调节，内沿着微管的分泌货物和高尔基体膜的基本问题。为了检验G160募集或激活动力蛋白运动复合体的假设，我们将1）鉴定并功能性表征其与运动细胞质动力蛋白的相互作用，2）鉴定并功能性表征其与高尔基体膜的Arf 1依赖性相互作用，3）通过开发一种在秒的时间尺度内使用光抑制G160的技术来测试高尔基体对基于G160的运动性的急性依赖性，（4）阐明了高尔基体在细胞分裂过程中扩散和遗传的控制机制。这些目标得到了主要初步调查结果的支持。G160是微管+尖端捕获高尔基体膜所必需的。G160 C-端直接结合到动力蛋白复合物的中间链。G160是动力蛋白与高尔基体结合所必需的，它也足以进行功能性运动募集，因为G160靶向线粒体募集动力蛋白并诱导线粒体向内运动。G160 N端介导其Arf 1依赖性膜结合，其结合受到调节，使得G160循环至细胞外周并向内返回微管。G160的光失活表明，高尔基体严重依赖于G160为基础的向内运动，因为早期的高尔基体酶不断循环到细胞周边。最后，G160在有丝分裂时从高尔基体解离，但仍与动力蛋白结合并聚集在纺锤体两极。因此，我们的实验准备揭示长期寻求高尔基体受体的动力蛋白马达的组件，并阐明调控，使高尔基体膜从微管为基础的运动，让高尔基体分区到子细胞。 公共卫生相关性：该提案的目标是阐明高尔基体组装和维持的分子机制。高尔基体处理新合成的蛋白质和脂质，这些反应在预防和治疗人类疾病方面很重要。膜运输缺陷是许多人类疾病的原因，我们对这些缺陷的分子基础的理解为未来有效的治疗铺平了道路。此外，理解运输及其分泌区室的建立是靶向由缺陷蛋白质产物引起的多种疾病的治疗剂开发中的重要关注点，其中这些蛋白质依赖于分泌过程，如蛋白质折叠、质量控制、糖基化、蛋白水解活化和定位。这些疾病包括囊性纤维化、朊病毒相关疾病、糖尿病和阿尔茨海默病，仅举几例。人类疾病也是由隔室功能本身的缺陷引起的。例如，聚糖合成障碍是一个重要且快速增长的群体，并且越来越明显的是，主要缺陷可能是在由内质网和高尔基体组成的膜运输系统内聚糖被转移物的运输和定位中。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms that establish and maintain the Golgi during development, cell division, and cellular stress are significant ongoing goals in biomedical research. Our long-term goal is to identify proteins required for Golgi structure and elucidate their mechanism. This work was initiated using an siRNA-based screen targeting candidates, especially golgins, and cell-based Golgi assembly assays followed by rescue after knockdown in conjunction with biochemical, permeabilized-cell, structural, and computational assays. Our screen identified golgin-160 (G160) as a critical requirement for inward motility of Golgi membranes and showed that its absence arrests Golgi assembly at the dispersed ministack stage impairing directed secretion and cell migration during wound healing. This observation raised the possibility that we could answer fundamental questions concerning the unknown identity and regulation of the motor receptor complex that moves secretory cargo and Golgi membranes inward along microtubules. To test the hypothesis that G160 recruits or activates the dynein motor complex we will 1) identify and functionally characterize its interaction with the motor cytoplasmic dynein, 2) identify and functionally characterize its Arf1-dependent interaction with the Golgi membrane, 3) test the acute dependence of the Golgi on G160-based motility by developing a technique to inactivate G160 using light in a time scale of seconds, and 4) elucidate the control mechanism that allows Golgi dispersal and inheritance during cell division. These aims are supported by key preliminary findings. G160 was required for microtubule +tip capture of Golgi membranes. The G160 C-term bound directly to the intermediate chain of the dynein complex. G160 was required for dynein Golgi association and it was also sufficient for functional motor recruitment as G160 targeted to mitochondria recruited dynein and induced mitochondrial inward motility. The G160 N-term mediated its Arf1-dependent membrane association and its binding was regulated such that G160 cycled to the cell periphery and returned inward on microtubules. Photo-inactivation of G160 showed that the Golgi acutely depends on G160-based inward motility because early Golgi enzymes constantly cycle to the cell periphery. Finally, G160 dissociated from the Golgi at mitosis but remained bound to dynein and collected at spindle poles. Thus, our experiments are poised to uncover components in the long-sought Golgi receptor for the dynein motor and elucidate the regulation that uncouples Golgi membranes from microtubule-based motility to allow Golgi partitioning into daughter cells. PUBLIC HEALTH RELEVANCE: The goal of this proposal is elucidating the molecular mechanisms in assembly and maintenance of the Golgi apparatus. The Golgi processes newly synthesized proteins and lipids and these reactions are important in preventing and treating human disease. Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. Further, understanding trafficking and its establishment of secretory compartments is a vital concern in the development of therapeutics targeting the multitude of diseases that arise from defective protein products in which these proteins depend on secretory processes such as protein folding, quality control, glycosylation, proteolytic activation, and localization. Such diseases include cystic fibrosis, prion-related diseases, diabetes, and Alzheimer's disease, to name just a few. Human disease also arises from defects in compartment function itself. For example, disorders of glycan synthesis are a substantial and rapidly growing group and it is becoming increasingly evident that the primary defect can be in the transport and localization of the glycan transferees within the membrane trafficking system comprised by the endoplasmic reticulum and the Golgi apparatus.