The Transitional ER-Golgi System in Budding Yeasts

描述（由申请人提供）：我们的目标是阐明分泌途径的结构如何反映特定分子的作用。本项目重点研究过渡性内质网（tER）和高尔基体。tER位点是产生COPII转运囊泡的膜结构域。在许多真核生物中，高尔基体与tER位点相邻。这种排列可以根据池成熟模型来理解，该模型假设tER位点产生新的高尔基池，这些池随后成熟。然而，对于tER位点是如何产生的，高尔基池是如何组织成堆栈的，或者高尔基堆栈是如何定位在tER位点旁边的，人们知之甚少。我们假设早期分泌室的结构是通过自组织建立的，并受到膜交通动力学的影响。这些想法将在毕赤酵母和酿酒酵母的帮助下进行探索。P. pastoris含有大量稳定的tER位点，与高尔基体相邻，而S. cerevisiae则含有破碎的tER和高尔基体元素。具体目标#1：测试Sec16是否定义tER位点，或调节tER动态，或两者兼而有之。我们的研究表明外周膜蛋白Sec16是tER组织的关键决定因素。但是Sec16的功能是未知的。分析Sec16将为我们对COPII囊泡生物发生和tER组织的了解增加一个新的维度。我们已经确定了Sec16和五个COPII组件之间的相互作用。为了阐明Sec16反应周期，我们将使用生化和体内方法来解剖COPII-Sec16相互作用，并确定这些相互作用如何调节COPII囊泡形成和tER动力学。Sec16的一个基本n端区域对tER定位很重要。我们将研究这种定位机制，它可能对确定tER位点至关重要。具体目标#2：鉴定出芽酵母中ter -高尔基基质的关键成分。在大多数真核生物中，排除核糖体的“基质”将高尔基池连接成一堆。在像帕斯德酵母这样的生物体中，这种基质也将tER位点与顺式高尔基体连接起来，我们现在已经在酿酒酵母中看到了类似的tER位点与顺式高尔基体并置。我们计划确定ter -高尔基矩阵的核心成分。一种方法将涉及基于显微镜的巴斯德酵母筛选。第二种方法将涉及测试酿酒葡萄球菌的候选基质蛋白，以确定那些负责连接tER和顺式高尔基体的蛋白。公共卫生相关性：分泌通路功能异常是癌症和发育障碍等疾病的致病因子。充分的治疗需要对定义分泌区室的过程有细胞生物学的理解。这项研究旨在揭示细胞组织的这些基本原理。

DESCRIPTION (provided by applicant): Our goal is to elucidate how the architecture of the secretory pathway reflects the actions of specific molecules. This project focuses on the transitional ER (tER) and the Golgi apparatus. tER sites are membrane domains that produce COPII transport vesicles. In many eukaryotes, Golgi stacks are adjacent to tER sites. This arrangement can be understood in light of the cisternal maturation model, which postulates that tER sites give rise to new Golgi cisternae that subsequently mature. Yet little is known about how tER sites are generated, how Golgi cisternae are organized into stacks, or how Golgi stacks are positioned next to tER sites. We hypothesize that the structure of early secretory compartments is established by self-organization and influenced by the dynamics of membrane traffic. These ideas will be explored with the aid of the budding yeasts Pichia pastoris and Saccharomyces cerevisiae. P. pastoris contains large, stable tER sites that are adjacent to Golgi stacks, whereas S. cerevisiae contains fragmented tER and Golgi elements. Specific Aim #1: To test whether Sec16 defines tER sites, or regulates tER dynamics, or both. Our work has implicated the peripheral membrane protein Sec16 as a key determinant of tER organization. However, the function of Sec16 is unknown. Analyzing Sec16 will add a new dimension to our knowledge of both COPII vesicle biogenesis and tER organization. We have identified interactions between Sec16 and five COPII components. To clarify the Sec16 reaction cycle, we will use biochemical and in vivo approaches to dissect the COPII-Sec16 interactions, and to determine how these interactions regulate COPII vesicle formation and tER dynamics. An essential N-terminal region of Sec16 is important for tER localization. We will examine this localization mechanism, which may be crucial for defining tER sites. Specific Aim #2: To identify key components of the tER-Golgi matrix in budding yeasts. In most eukaryotes, a ribosome-excluding "matrix" links Golgi cisternae into stacks. In organisms such as P. pastoris, this matrix also links tER sites to the cis-Golgi, and we have now seen a similar juxtaposition of tER sites with the cis-Golgi in S. cerevisiae. We plan to identify the core components of the tER-Golgi matrix. One approach will involve microscopy- based screens in P. pastoris. A second approach will involve testing candidate matrix proteins in S. cerevisiae to identify those responsible for linking the tER to the cis-Golgi. PUBLIC HEALTH RELEVANCE: Abnormal secretory pathway function is a causative agent in diseases such as cancer and developmental disorders. Adequate treatments will require a cell biological understanding of the processes that define secretory compartments. The proposed study aims to reveal these basic principles of cellular organization.