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中重要的持续目标。我们的长期目标是识别高尔基体结构所需的蛋白质，并阐明其机制。这项工作始于以siRNA为基础的筛选，针对候选者，特别是高尔基蛋白，以及基于细胞的高尔基体组装分析，然后结合生化、渗透细胞、结构和计算分析，在击倒后拯救。我们的筛查确定Golgin-160(G160)是高尔基体膜向内运动的关键要求，并表明它的缺失阻止了分散的部分阶段的高尔基体组装，损害了创伤愈合过程中的定向分泌和细胞迁移。这一观察提出了我们可以回答有关未知的身份和调节的运动受体复合体的基本问题的可能性，该复合体将分泌货物和高尔基膜沿着微管向内移动。为了验证G160招募或激活动力蛋白运动复合体的假设，我们将1)鉴定并从功能上表征其与运动细胞质动力蛋白的相互作用，2)鉴定并功能表征其与高尔基体膜依赖Arf1的相互作用，3)通过开发一种在几秒钟内用光灭活G160的技术来测试高尔基体对基于G160的运动性的严重依赖，以及4)阐明允许高尔基体在细胞分裂过程中扩散和遗传的控制机制。这些目标得到了关键的初步调查结果的支持。高尔基膜的微管+尖端捕获需要G160。G160C项直接与动力蛋白复合体的中间链结合。G160是动力蛋白高尔基体结合所必需的，也是功能性运动招募所必需的，因为靶向线粒体的G160招募动力蛋白并诱导线粒体内向运动。G160N-Term介导其依赖Arf1的膜结合，其结合受到调控，使G160循环到细胞外围，并在微管上向内返回。对G160的光灭活表明高尔基体强烈地依赖于基于G160的内向运动，因为早期的高尔基体酶不断地循环到细胞外围。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，并聚集在纺锤体极。因此，我们的实验准备揭示动力蛋白马达的长期寻找的高尔基体受体的成分，并阐明将高尔基体膜与基于微管的运动解偶联以允许高尔基体分裂为子细胞的调节。 公共卫生相关性：这项提案的目标是阐明高尔基体组装和维护的分子机制。高尔基体处理新合成的蛋白质和脂类，这些反应在预防和治疗人类疾病方面非常重要。膜转运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，在针对缺陷蛋白产品引起的多种疾病的治疗开发中，了解运输及其分泌室的建立是一个至关重要的问题，在这些缺陷蛋白产品中，这些蛋白依赖于诸如蛋白质折叠、质量控制、糖基化、蛋白分解激活和定位等分泌过程。这些疾病包括囊性纤维化、普里恩相关疾病、糖尿病和阿尔茨海默氏症，仅举几例。人类疾病也是由脑室功能本身的缺陷引起的。例如，葡聚糖合成障碍是一个重要且迅速增长的群体，越来越明显的是，主要缺陷可能是由内质网和高尔基体组成的膜运输系统中的葡聚糖转移者的运输和定位。

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.