Molecular Mechanisms Relating to Protein Sorting within the Yeast TGN and Early Endosomal System

诺斯韦尔博士的实验室研究了一种名为Ste13p的蛋白质，这种蛋白质在细胞内一个膜封闭的隔室中发挥作用，即反式高尔基网络（TGN）。Ste13p的一个重要作用是，当交配信息素从细胞分泌时，它可以改变交配信息素；没有这种修饰，分泌的信息素就没有功能。Ste13p不只是停留在TGN内，而是不断地在TGN和其他两个区室（早期核内体（EE）和晚期核内体（LE））之间运输。PI的兴趣在于破译Ste13p在这些隔间之间有序移动并避免被运送到其他目的地的机制。膜运输通常通过囊泡的形成发生，小的隔室从大的隔室（如EE）中挤压出来。外壳蛋白组件催化囊泡的形成，并决定哪些货物蛋白进入囊泡。PI的实验室已经证明了一种叫做网格蛋白/AP-1的外壳与Ste13p相关，数据表明这种相互作用是Ste13p被招募到ee衍生的囊泡中并运输回TGN的机制的一部分。该项目将在原子水平上研究这些蛋白质之间的相互作用，以精确定义相互作用是如何发生的。PI的实验室还确定了Ste13p的特定磷酸化，该磷酸化决定了Ste13p是从EE到LE还是从EE到TGN。该项目将确定使Ste13p磷酸化的酶，然后评估决定磷酸化的条件。最后，该项目将确定磷酸化如何影响Ste13p的贩运。PI的假设是，除了网格蛋白/AP-1外，Ste13p上磷酸基团的存在影响了它与其他蛋白质的相互作用。了解蛋白质如何在膜封闭的隔室之间运输和分类与细胞中的许多重要过程有关，包括细胞如何响应来自其他细胞的指令。这个项目的更广泛影响将是培训研究生和本科生，特别是为代表人数不足的群体的个人参与提供支助。该项目将利用密苏里-哥伦比亚大学（University of Missouri-Columbia）现有的两个项目，即路易斯·斯托克斯少数族裔参与联盟和研究生教育与教授联盟，提高少数族裔在研究生阶段的参与度。此外，学生将从科学学生研究项目中招募，该项目旨在促进少数民族和第一代大学生在本科阶段参与研究。

Dr. Nothwehr's laboratory studies a protein called Ste13p that carries out its function in a membrane-enclosed compartment within the cell, the trans-Golgi network (TGN). An important role of Ste13p is that it modifies mating pheromone as the pheromone is secreted from the cell; without this modification the secreted pheromone is not functional. Ste13p does not simply stay in the TGN, but is constantly being transported between the TGN and two other compartments, the early endosome (EE) and late endosome (LE). The PI's interests lie in deciphering the mechanism by which Ste13p moves between these compartments in an orderly fashion and avoids being transported to other destinations. Membrane transport typically occurs by the formation of vesicles, small compartments that pinch off from larger compartments such as the EE. Coat protein assemblies catalyze the formation of vesicles and determine which cargo proteins enter the vesicle. The PI's lab has shown that a coat called clathrin/AP-1 associates with Ste13p and the data suggest that this interaction is part of the mechanism by which Ste13p is recruited into EE-derived vesicles for transport back to the TGN. The project will study the interaction between these proteins at the atomic level to precisely define how the interaction takes place. The PI's lab also identified a specific phosphorylation of Ste13p that dictates whether Ste13p moves from the EE to the LE or from the EE to the TGN. The project will identify the enzyme that phosphorylates Ste13p and then assess the conditions that determine phosphorylation. Finally, the project will determine how phosphorylation affects the trafficking of Ste13p. The PI's hypothesis is that the presence of the phosphate group on Ste13p affects its interaction with other proteins in addition to clathrin/AP-1. Understanding how proteins are transported between membrane-enclosed compartments and are sorted has relevance to many important processes in cells, including how cells respond to instructions from other cells. The broader impact of this project will be in the training of graduate and undergraduate students, particularly in providing support for participation of individuals from under-represented groups. The PI will take advantage of two programs in place at the University of Missouri-Columbia for increasing minroity participation at the graduate level, the Louis Stokes Alliance for Minority Participation and Alliances for Graduate Education and the Professoriate. In addition, students will be recruited from the Exposure to Research for Science Students program which is designed to foster participation in research at the undergraduate level for minority and first-generation college students.