Analysis of the Inheritance and Positioning of the Golgi Apparatus

9604342高尔基体具有独特的结构和蛋白质组成，但赋予该细胞器独特特性的过程才刚刚开始阐明。例如，在细胞分裂过程中高尔基体结构是如何遗传的，或者高尔基体如何定位于细胞质的特定区域，目前还不清楚。理解这些现象的一个关键是发现高尔基体堆叠通常位于内质网(ER)泡化的“过渡”区域附近。该项目的工作假设是高尔基体堆叠位于过渡性内质网部位附近，因为高尔基体组装发生在这些部位，新的高尔基体池是由内质网衍生的囊泡合并形成的。在这种观点下，高尔基可以被认为是ER的动态副产品。这项工作的最终目标是确定细胞如何创建和传播导致高尔基体堆叠的过渡性ER亚域。实验方法是分析高尔基体在酿酒酵母和巴斯德毕赤酵母这两个萌芽酵母中的遗传和定位。在酿酒酵母中，高尔基体具有碎裂结构，很少显示堆叠的池状形态。然而，通过监测高尔基体元素进入芽中的运动来研究高尔基体在酿酒酵母中的遗传是可能的。对于高尔基体定位的研究，巴氏假单胞菌是首选的有机体，因为它含有少量离散的高尔基体堆叠，类似于高等真核生物中发现的细胞器。巴斯德毕赤酵母中的高尔基体位于囊泡膜的旁边，明显对应于过渡性内质网。由于巴氏假单胞菌与酿酒酵母有密切的亲缘关系，而且可以以大致相同的方式进行遗传操作，因此对这两种酵母中的高尔基体进行比较研究将是有价值的。该项目有两个具体目标。一是分析蛋白质定位于过渡内质网的机制。牧羊人。针对定义过渡性内质网的COPII包被的囊泡系统中的巴氏杆菌成分，正在产生抗体。将进行免疫定位实验，以确认这些蛋白质仅限于内质网的离散亚域。根据对酿酒酵母的功能研究，COPII组分在过渡性内质网中的特异性募集似乎依赖于启动COPII组装途径的蛋白质Sec12p的限制性定位。这一预测将通过修改巴斯德毕赤酵母的SEC12基因来验证，试图改变其定位。此外，将使用亲和方法来分离可能在过渡性ER区锚定Sec12p的伙伴蛋白。第二个目标是进行遗传筛选，以确定在酿酒酵母中介导高尔基体遗传的蛋白质。一种用绿色荧光蛋白(GFP)标记高尔基体的酵母菌株已经被创造出来。这些细胞将被诱变并进行目视筛选，以确定显示高尔基体遗传缺陷的突变。将在突变的细胞中检查ER和高尔基体之间的关系，并将克隆和鉴定负责突变表型的基因。高尔基体是真核细胞的复杂细胞器，它由高度专业化的膜封闭隔室组成，在这些隔室中，新合成的蛋白质和某些复杂的糖结合物被共价修饰，其他复杂的糖结合物被合成。高尔基复合体中的大多数蛋白质和复杂的糖偶联物，无论是溶解在隔室内还是与膜结合，都只是暂时驻留在那里；一旦合成和修饰完成，这些大分子就被分类并运输到细胞内外的其他位置，在那里它们执行它们的特定功能。高尔基复合体可能具有不同的形态和亚细胞位置(甚至特定的代谢功能)，这取决于特定的细胞类型和细胞生命周期的阶段。尽管高尔基复合体已经被深入研究了几十年，关于特定的高尔基酶和通过该复合体的大分子的命运也知道了很多，但关于它的许多方面仍然知之甚少。该项目利用尖端技术和方法，解决有关高尔基复合体生物发生的基本问题。如果研究结果支持格利克博士的假说，他们将迫使研究人员改变对高尔基复合体的看法，从其功能和细胞内起源的角度来看。***

9604342 Glick The Golgi apparatus has a distinctive structure and protein composition, but the processes that give this organelle its unique character are only beginning to be elucidated. For example, it is still unclear how Golgi structures are inherited during cell division or how the Golgi is localized to a particular region of the cytoplasm. A key to understanding these phenomena comes from the finding that Golgi stacks are often situated near vesiculating "transitional" regions of the endoplasmic reticulum (ER). The working hypothesis for this project is that Golgi stacks are located near transitional ER sites because Golgi assembly occurs at these sites, with new Golgi cisternae forming by the coalescence of ER-derived vesicles. In this view, the Golgi can be considered a dynamic outgrowth of the ER. The ultimate goal of this work is to determine how a cell creates and propagates the transitional ER subdomains that give rise to Golgi stacks. The experimental approach is to analyze Golgi inheritance and positioning in two budding yeasts, Saccharomyces cerevisiae and Pichia pastoris. In S. cerevisiae, the Golgi has a fragmented structure and rarely displays a stacked cisternal morphology. Nevertheless, it is possible to study Golgi inheritance in S. cerevisiae by monitoring the movement of Golgi elements into the bud. For studies of Golgi positioning, P. pastoris is the organism of choice because it contains a small number of discrete Golgi stacks that resemble the organelles found in higher eukaryotes. Golgi stacks in P. pastoris are located next to vesiculating membranes that evidently correspond to transitional ER. Because P. pastoris is closely related to S. cerevisiae and can be genetically manipulated in much the same way, it will be valuable to perform a comparative study of the Golgi in these two yeasts. The project has two Specific Aims. The first is to analyze the mechanism by which proteins are localized to the transitional ER in P . pastoris. Antibodies are being raised against P. pastoris components of the COPII- coated vesicle system, which defines the transitional ER. Immunolocalization experiments will be performed to confirm that these proteins are restricted to discrete subdomains of the ER. Based on functional studies in S. cerevisiae, it seems likely that the specific recruitment of COPII components to the transitional ER depends upon a restricted localization of Sec12p, the protein that initiates the COPII assembly pathway. This prediction will be tested by modifying the P. pastoris SEC12 gene in an attempt to alter its localization. In addition, affinity methods will be used to isolate partner proteins that may anchor Sec12p in transitional ER zones. The second aim is to carry out genetic screens for identifying proteins that mediate Golgi inheritance in S. cerevisiae. A strain of yeast has been created in which Golgi organelles are labeled with Green Fluorescent Protein (GFP). These cells will be mutagenized and screened visually to identify mutants that show defects in Golgi inheritance. The relationship between ER and Golgi elements will be examined in the mutant cells, and the genes responsible for the mutant phenotypes will be cloned and characterized. The Golgi apparatus is a complex organelle of eukaryotic cells which consists of a highly specialized collection of membrane-enclosed compartments in which newly synthesized proteins and certain complex glycoconjugates are covalently modified and other complex glycoconjugates are synthesized. Most of the proteins and complex glycoconjugates within the Golgi complex, whether soluble inside the compartments or membrane-bound, are only transiently resident there; once the syntheses and modifications are completed, these macromolecules are sorted and transported to other sites inside or outside the cell, where they carry out their specific functions. The Golgi complex may assume different morphologies and subcellular locations (and even specific metabolic functions) depending on the specific cell type and the stage of the life cycle of the cell. Although the Golgi complex has been studied intensively for decades, and much is known about specific Golgi enzymes and the fate of macromolecules that pass through the complex, there is still much about it that remains poorly understood. This project addresses fundamental questions about the biogenesis of the Golgi complex, utilizing cutting-edge technologies and approaches. If the results support Dr. Glick's hypothesis, they will force a change in the way investigators think about the Golgi complex, in terms of its function and its origin within the cell. ***