Molecular Interactions of Rnf13, a Ubiquitin Ligase in Endosome Membranes

溶酶体蛋白水解酶在细胞内介导囊泡内的切割事件，但它们也可以被分泌，从而介导细胞外的蛋白分解。溶酶体蛋白酶前体L可以储存在特殊的细胞囊泡中，至少在某些类型的细胞中，这些囊泡可以被诱导将其内容物分泌到细胞外部。该项目试图确定蛋白酶靶向这些小泡是否由Rnf13介导，Rnf13是一种新发现的整合膜蛋白，其结构域结构类似于一种蛋白质，其结构类似于介导酶靶向结构相似的植物蛋白储存体的蛋白质。为了了解RNF13在细胞靶向和溶酶体蛋白酶分泌中的作用，我们将对RNF13的生物合成进行表征，并检测该膜蛋白与该酶的结合情况。这些研究将确定这种膜蛋白所遵循的细胞路线，以及它在合成后所经历的修饰的性质和作用。作为建立RNF13生理功能的一步，将进行结合分析和质谱分析，以确定与这种未知的多结构域完整膜蛋白相互作用的蛋白质。该项目的一个核心优先事项是招募本科生加入研究小组，特别关注代表人数不足的少数群体。这些学生将广泛参与实验工作的方方面面。

Lysosomal proteases mediate cleavage events within cellular vesicles, but they can also be secreted and so mediate proteolysis outside cells. The lysosomal protease precursor procathepsin L can be stored in specialized cellular vesicles that can, at least in certain cell types, be induced to secrete their contents to the cell exterior. This project seeks to determine whether targeting of the proprotease to these vesicles is mediated by Rnf13, a newly discovered integral membrane protein that has a domain structure similar to that of a protein shown to mediate targeting of enzymes to structurally similar plant protein storage bodies. To understand the role of Rnf13 in the cellular targeting and secretion of a lysosomal protease, the biosynthesis of Rnf13 will be characterized and binding of this membrane protein to the protease will be assayed. The studies will establish the cellular route followed by this membrane protein and the nature and role of modifications that it undergoes after its synthesis. As a step toward establishing physiological functions of Rnf13, binding assays and mass spectrometry will be performed to identify proteins that interact with this uncharacterized multi-domain integral membrane protein. A central priority of the project is the recruitment of undergraduates, with a particular focus on underrepresented minorities, to the research group. These students will participate extensively in all aspects of the experimental work.