Molecular Interactions of Rnf13, a Ubiquitin Ligase in Endosome Membranes

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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.