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

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.

This work sets out to define the localization and establish the role of Sec16 in mammalian cells. Sec16 is a key molecule involved in assembly of the COPII coat in Saccharomyces cerevisiae. The COPII coat is highly conserved across species and mediates the export of secretory cargo from the endoplasmic reticulum. One key difference between COPII in Saccharomyces cerevisiae and humans is that in humans, coat assembly is organized in to discrete locations continuous with the endoplasmic reticulum membrane known as exit sites. We are the first lab in the world to have published a characterization of the human homologue of Sec16. We have now also identified a second, much shorter form of Sec16 which we believe might act as a modulator of the COPII assembly process. We propose to evaluate the roles of these two Sec16 proteins (which we have now termed Sec16A and Sec16B) in defining the organization of exit sites in human cells, and their roles in relation to other COPII coat components. We will address the mechanism of localization of these proteins and their roles in coordinating assembly and controlling the localization of other COPII components. We will address specific questions relating to the potential for Sec16 proteins to self-assemble in to higher ordered structures and their potential role in binding to curved membranes, and/or modulating membrane curvature during vesicle budding. These experiments will also reveal key information about how the generation and maintenance of this intracellular architecture relates to secretory pathway function in human cells. From our previous work, we now have the technical expertise, reagents and knowledge base to undertake this work in a timely and dynamic manner. We have also established collaborations with two major labs with which we will undertake specific technical assays within the proposal to expedite the generation of data and progress of the project.