Mechanisms of Golgi Apparatus Assembly

高尔基体是真核细胞中分泌蛋白和膜蛋白生物合成过程的核心。用于分泌或插入质膜的蛋白质，以及用于溶酶体等各种细胞内区室的蛋白质，通常通过共翻译被迅速隔离到细胞内的区室系统中，从而促进这些蛋白质的翻译后生化处理及其在细胞内功能位置的易位。这些蛋白质被隔离的第一个隔间是内质网（ER）；从那里，蛋白质通过高尔基体运输，并从那里分布到它们的最终位置。高尔基体本身是复杂的，它的各种组成部分（堆叠池）专门用于各种功能，例如蛋白质的翻译后修饰。蛋白质通过高尔基体的各种贮池的运输机制，以及高尔基体在细胞中的生物发生和维持，是对这些过程的理解（并最终有意的生物技术操作）具有相当重要意义的主题。这也是一个有争议的话题，因为最近的发现迫使细胞生物学家重新考虑高尔基体生物发生的工作模型，该模型在过去20年里主导了该领域的细胞生物学思维。这个建议解决了这个问题的一个关键方面，高尔基体与内质网的功能和动态关系。高尔基体从内质网（ER）接收新合成的蛋白质和脂质，并在分泌途径中将它们分配给其他下游细胞器。高尔基体功能的关键是“常驻”蛋白质的不均匀分布，这些蛋白质集中在一个池中，而在细胞器的其他部分则不存在，从而形成“极化”的高尔基膜堆栈。提议的研究集中在极化高尔基堆栈如何形成的问题上；换句话说，关于细胞器的生物发生。Storrie博士之前的工作（Storrie等人，1998；Storrie和Yang， 1998）提出了高尔基体可能在有丝分裂期间“融化”到内质网，随后从内质网重新形成细胞器的可能性。在这个项目中，将进行探索性研究，以测试两个分子家族在假设的极化高尔基堆叠从头形成中的作用。这些分子是COPI类外壳蛋白和rab6和rab33b小gtp酶家族的两个成员。从其他人的工作中，每个人都知道参与了膜贩运。Storrie博士将评估具体阻断高尔基体堆积内囊泡运输的条件，并将使用brefeldin a （BFA）冲洗后ER的高尔基体重组模型系统对重新形成高尔基体的影响进行评分。bfa处理导致高尔基蛋白和脂质向内质网可逆扩散。实验探讨了一个假设，即泡状运输过程可能是将高尔基体内稳定的分子结合塑造成极化高尔基堆所必需的。将采用反向遗传方法，通过显微注射将显性阴性突变蛋白引入细胞。

The Golgi apparatus is central to the biosynthetic processing of secretory and membrane proteins in eukaryotic cells. Proteins destined for secretion or for insertion into the plasma membrane, as well as proteins destined for various intracellular compartments such as the lysosomes, are rapidly sequestered, usually co-translationally, into an intracellular system of compartments that facilitate the post-translational biochemical processing of these proteins and their translocation to their functional locations in the cell. The first compartment into which such proteins are sequestered is the endoplasmic reticulum (ER); from there, the proteins are transported through the Golgi apparatus and from there distributed to their ultimate locations. The Golgi apparatus itself is complex, and its various components (stacked cisternae) are specialized for various functions, e.g., in terms of postranslational modifications of proteins. The mechanism of transport of proteins through the various cisternae of the Golgi apparatus, and the biogenesis and maintenance of the Golgi apparatus in the cell, is a topic of considerable significance to the understanding (and, ultimately, deliberate biotechnological manipulation) of these processes. It is also a controversial topic, since recent discoveries have forced cell biologists to reconsider the working model of Golgi biogenesis that has dominated cell biological thinking in this area for the past twenty years. This proposal addresses a key aspect of this issue, the functional and dynamic relationship of the Golgi apparatus to the endoplasmic reticulum.The Golgi apparatus receives newly synthesized proteins and lipids from the endoplasmic reticulum (ER) and distributes them to other downstream organelles within the secretory pathway. Key to Golgi function is an unequal distribution of 'resident' proteins, that are concentrated in one cisterna and are absent from other parts of the organelle, to give a 'polarized' stack of Golgi membranes. The proposed research centers on the question of how polarized Golgi stacks form; in other words, on the biogenesis of the organelle. Prior work by Dr. Storrie (Storrie et al., 1998; Storrie and Yang, 1998) raises the possibility that the Golgi apparatus may 'melt' into the ER during mitosis followed by subsequent de novo formation of the organelle from the ER. In this project, exploratory research will be performed to test the role of two families of molecules in the postulated de novo formation of polarized Golgi stacks. These molecules are the COPI class of coat proteins and two members of the rab family of small GTPases, rab6 and rab33b. Each is known from the work of others to be involved in membrane trafficking. Dr. Storrie will assess conditions that specifically block vesicular transport within the Golgi stack and will score for effects on de novo Golgi formation using as a model system Golgi reassembly from the ER following brefeldin A (BFA) washout. BFA-treatment results in a reversible dispersal of Golgi proteins and lipids to the ER. The experiments probe the hypothesis that vesicular transport processes may be necessary to mold stabilized molecular associations within the Golgi apparatus into polarized Golgi stacks. A reverse genetic approach will be taken, in which dominant negative mutant proteins will be introduced into cells by microinjection.