Cellular Control of Endoplasmic Reticulum Biogenesis

真核细胞由一组基本的细胞器“积木”组成，其中包括细胞核、内质网（ER）、高尔基体、溶酶体、线粒体、过氧化物酶体，以及光合生物中的叶绿体。这种相对较少的独立细胞成分掩盖了真核生物中存在的细胞结构和功能的惊人多样性。在每种情况下，细胞结构和功能的特化都反映在细胞器组成的变化上。因此，细胞生物学的一个关键特征是对亚细胞组织的调节，即对存在哪些细胞器及其大小、组成、位置、数量和寿命的调节。令人惊讶的是，考虑到这种调节的重要性，还没有一个例子表明这种调节的分子性质被很好地理解。实验将通过关注酵母内质网结构和功能的调节来探索这个问题。正如在所有其他被检测的细胞类型中一样，酵母菌内质网的组织对内质网蛋白子集的水平很敏感。其中一种蛋白质，HMG-CoA还原酶，催化了甾醇和异戊二烯生物合成的第一步。在酵母中，HMG-CoA还原酶表达水平的增加诱导内质网特化区域的组装，称为胼胝体，由成对的光滑膜组成，与细胞核密切相关。仅通过改变单个蛋白质的水平来控制卡梅尔菌组装的能力为探索细胞在生理需求下增加特定内质网结构域生物发生的分子机制提供了独特的机会。为了揭示这些机制，赖特博士将使用一种遗传方法，利用酵母基因组计划完成后可用的新资源。具体地说，她将使用群体遗传方法来识别缺失突变体，这些突变体在组装胼胝体时表现出生长速度的缺陷。结合双杂交方法鉴定与HMG-CoA还原酶相互作用的基因产物的信息，该方法将揭示在卡尔默菌组装中起重要作用的基因。卡米拉组装突变体的分析将以体内延时光学显微镜和卡米拉组装的电镜分析为指导。这些实验的完成将揭示随着生理需求的变化而协调报告和调节内质网功能的通讯网络的基本特征。这些知识将对理解内质网结构和功能的细胞调节有特定的应用，但也应该提供关于细胞器生物发生的细胞调节的一般见解。

Eukaryotic cells are composed of a basic set of organelle "building blocks" that include (among others) the nucleus, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, mitochondria, peroxisomes and, in photosynthetic organisms, chloroplasts. This relatively small number of separate cellular components belies the amazing diversity of cell structure and function that exists in eukaryotic organisms. In each case, the specialization of cell structure and function is mirrored by changes in organelle composition. Thus, a key feature of cell biology is the regulation of subcellular organization, that is, the regulation of which organelles are present and their size, composition, location, number, and life spans. Surprisingly, given the importance of this regulation, there is not yet a single example in which the molecular nature of this regulation is well understood.The experiments that will be performed explore this question by focusing on the regulation of endoplasmic reticulum structure and function in the yeast, Saccharomyces cerevisiae. As in all other cell-types examined, the organization of the ER in yeast is sensitive to the levels of a subset of ER proteins. One of these proteins, HMG-CoA reductase, catalyzes the first committed step in sterol and isoprene biosynthesis. In yeast, expression of increased levels of HMG-CoA reductase induces assembly of specialized regions of ER termed karmellae, consisting of stacks of paired smooth membranes that are closely associated with the nucleus. The ability to control karmella assembly by merely changing the levels of a single protein provides a unique opportunity to explore the molecular mechanisms by which cells increase biogenesis of a particular ER domain when dictated by physiological demands. To uncover these mechanisms, Dr. Wright will use a genetic approach that takes advantage of new resources available as a result of completion of the Yeast Genome Project. Specifically, she will use a population genetic approach to identify deletion mutants that display defects in growth rate when they assemble karmellae. Coupled with information from a Two-Hybrid approach to identify gene products that interact with HMG-CoA reductase, this approach should reveal genes that have important roles in assembly of karmellae. The analysis of karmella assembly mutants will be guided by in vivo time-lapse light microscopy and electron microscopic analysis of karmella assembly. Completion of these experiments should uncover basic features of the communication network that reports and regulates ER function coordinately with changing physiological demands. Such knowledge will have specific application to understanding the cellular regulation of ER structure and function, but should also provide general insights concerning the cellular regulation of organelle biogenesis.