The role of Sec16p in the organization and function of mammalian ER export sites.

哺乳动物细胞的分泌途径是必不可少的。几乎所有由细胞分泌的蛋白质都要经过这条途径，就像大多数组成细胞膜的蛋白质一样。这一途径的缺陷是脂肪代谢和凝血障碍等疾病的直接原因。我的工作旨在解决这一途径的第一步（所有后续步骤都依赖于此）是如何被控制的。我们从酵母实验中对这一途径的机制了解很多，但我们已经确定了人类的显著差异，特别是基于这一过程的组织方式。在人类和其他高等真核生物中，细胞中有数百个点，在那里，合成后的分泌货物被包装出口。在较简单的生物中（如通常研究的模式生物，如面包酵母），这一事件不是以这种方式组织的。我们的问题涉及到这个过程是如何以及为什么在人类中以一种不是酵母的方式组织起来的。与酵母细胞相比，这与人类细胞的复杂性增加有直接关系吗？据估计，人类基因组编码的蛋白质中约有三分之一通过这条途径，这凸显了它对健康细胞的重要性。我们是世界上第一个确定并公布这一过程中关键参与者之一（一种称为Sec16的蛋白质）数据的实验室，我们现在希望对其在构建人类分泌途径的第一个组织单元中的作用及其在控制和协调人类细胞分泌中的功能进行深入研究。我们还确定了这种关键蛋白质的第二种形式，我们认为它是该过程的关键调节剂。我们认为这是一种调节剂，而不是关键成分，因为它缺失了较长的Sec16蛋白的整个前半部分。此外，我们可以通过数据库搜索来识别多个生物体中的等效蛋白质。这意味着我们可以通过物种之间的身份来排列这些序列并定义关键的功能区域。我们希望解决以下问题：Sec16的功能域是什么？这两种形式的Sec16是如何相互关联的——它们是相互作用还是对这条通路的其他组分起不同的作用？Sec16是否作为单元内定义导出站点的构建块？第二种Sec16形式在这些位点的形成或功能中是否调节了第一种Sec16形式的作用？这些问题将通过经典的生化实验、高分辨率活细胞成像、电子显微镜超微结构成像和生物物理学方法来解决，以确定这些蛋白质在精确分子细节中的作用。我们有非常好的条件来开展这项工作，我们有关键的技术，从我们以前的工作中获得的背景知识，以及已经到位的试剂。我们还将与两个拥有该项目所需技术专长的主要实验室合作。

The secretory pathway in mammalian cells is essential. Nearly all proteins that are secreted by cells pass through this pathway, as do the majority of proteins that go to make up cell membranes. Defects in this pathway are a direct cause of diseases ranging from those of fat metabolism to blood clotting disorders. My work proposes to address the way in which the first step of this pathway (on which all subsequent steps rely) is controlled. We know much about the machinery of this pathway from experiments in yeast but we have identified significant differences in humans, notably based around the way in which this process is organized. In humans and other higher eukaryotes, there are hundreds of points in the cell at which secretory cargo is packaged for export after synthesis. In simpler organisms (such as commonly studied model organisms like baker's yeast) this event is not organized in this way. Our questions relate to both HOW and WHY this process is organized in humans in a way that it is not in yeast. Does this relate directly to the increased complexity of a human cell in comparison to a yeast cell? It is estimated that around one third of proteins encoded by the human genome traverse this pathway, underlining its importance to the healthy cell. We are the first lab in the world to have identified and published data on one of the key players in this process (a protein called Sec16) and we now wish to undertake an in-depth investigation into its role in building the first organizational unit of the human secretory pathway and its function in controlling and coordinating secretion in human cells. We have also identified a second form of this key protein which we believe acts as a key modulator of the process. We believe that this is a modulator rather than key component since it is missing the entire first half of the longer Sec16 protein. Furthermore, we can idenitify the equivalent proteins in multiple organisms through database searching. This means that we can align these sequences and define key functional regions by identity between species. We wish to address the following questions: What are the functional domains of Sec16? How do the two forms of Sec16 relate to one another - do they interact or perform distinct functions with regard to other components of this pathway? Does Sec16 act as a building block defining the export sites within cells? Does the second Sec16 form modulate the role of the first in the formation or function of these sites? The questions will be addressed using classical biochemical experiments coupled with high resolution imaging of living cells, ultrastructural imaging by electron microscopy and biophysical approaches to define the role of these proteins in precise molecular detail. We are exceptionally well placed to undertake this work with key techniques, background knowledge from our prvious work, and reagents in place already. We will also collaborate with two major labs who have technical expertise necessary for this project.