A High Density Interactome for the Ubiquitin-like Protein Systems

摘要

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Like ubiquitin, most Ubls are covalently attached to a lysine residue on target proteins. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, such as proliferation and regulation of the cell cycle, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we are developing and utilizing a robust affinity purification-mass spectrometry (AP-MS) technique to create high-density protein-protein interaction maps for the Ubl systems. An inducible plasmid-based expression system was used to systematically express C-terminal HA-tagged fusion proteins in S. cerevisiae. A standardized method in which affinity purification against the HA epitope followed by mass spectrometry analysis was performed. Data were transferred to a novel LIMS system, searched by multiple search engines, and bona fide interactors identified using a novel statistical analysis algorithm. The data generated were used to build high-density interaction maps. Interacting partners identified new roles for each modifier in previously unknown pathways.