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

Nothwehr博士的实验室研究了一种名为Ste 13 p的蛋白质，该蛋白质在细胞内的膜封闭区室（trans-Golgi network，TGN）中发挥作用。 Ste 13 p的一个重要作用是，当交配信息素从细胞分泌时，它修饰交配信息素;没有这种修饰，分泌的信息素就没有功能。 Ste 13 p并不简单地停留在TGN中，而是不断地在TGN和另外两个隔室（早期内体（EE）和晚期内体（LE））之间运输。 PI的兴趣在于破译的机制，其中步骤13 p之间的移动，这些车厢在有序的方式，并避免被运送到其他目的地。 膜运输通常通过囊泡的形成而发生，囊泡是从较大的隔室（如EE）夹断的小隔室。外壳蛋白组装体催化囊泡的形成并决定哪些货物蛋白进入囊泡。 PI的实验室已经表明，一种名为网格蛋白/AP-1的涂层与Ste 13 p相关，数据表明这种相互作用是Ste 13 p被招募到EE衍生囊泡中以转运回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如何发生。 PI的实验室还确定了Ste 13 p的特异性磷酸化，这决定了Ste 13 p是从EE移动到LE还是从EE移动到TGN。 该项目将确定磷酸化Ste 13 p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Ste 13 p的运输。 PI的假设是，除了网格蛋白/AP-1之外，Ste 13 p上磷酸基团的存在影响其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并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。 这一项目的更广泛影响将是对研究生和本科生的培训，特别是为代表性不足群体的个人参与提供支助。 PI将利用密苏里-哥伦比亚大学的两个项目，增加研究生阶段的少数民族参与，即路易斯·斯托克斯少数民族参与联盟和研究生教育联盟以及教授。 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.