The Ubiquitin Proteasome Pathway (UPP) controls the activities of signaling systems through reversible conjugation of ubiquitin and ubiquitin like proteins (Ubls). The UPP system contains more than 900 genes and functions in virtually all aspects of biology, including aging, neurodegeneration and cancer, yet the functions of only a subset of these genes are known. This proposal seeks to continue our development a systems-based approach for identifying functions and targets of UPP components in the cell division cycle and checkpoint pathways in mammalian cells. The platform we are developing employs global analysis of the UPP in these processes using loss of function genetics, proteomics, and newly established genetic approaches for the identification of substrates for E3 ubiquitin ligases. The integration of these systems with a focus on cell cycle and checkpoint controls provides a powerful approach to gene and pathway discovery. Using these systems, we have identified several new genes that control the spindle checkpoint controlling chromosome segregation. One of these, the deubiquitinating enzyme Usp44, controls the ubiquitination status of the APC regulatory protein Cdc20, a recently identified step in APC activation that promotes dissociation of Mad2. Usp44 therefore functions to maintain Cdc20 in its Mad2-inhibited state. Usp44 itself is also regulated during the cell cycle and in response to checkpoint activation. In Aim 1, we will continue to elucidate how Usp44 is regulated and how its activity controls the checkpoint. In addition, we will perform a systematic mechanistic analysis of 4 additional deubiquitinating enzymes we have already validated as being required for the checkpoint. In Aim 2, we propose to validate and analyze candidate genes from a recently performed RNAi screen to identify UPP genes involved in the response of cells to ionizing radiation. In Aim 3, we will systematically analyze the role of the UPP in cell proliferation using new barcode-based shRNA screening capabilities coupled with high throughput flow cytometry. In Aim 4, we continue to develop a general solutions to the problem of finding substrates for E3s. We have devised a novel flow-cytometry based system that allows degradation substrates for particular E3s to be identified using microarray hybridization and can also be used to identify proteins whose abundance is altered in response to a particular stimulus.