Eukaryotic cells have a highly conserved enzymatic system for the ligation of ubiquitin (Ub) to proteins. Moreover, polypeptides distinct from but related to Ub, called Ub-like proteins or Ubls, can also be conjugated to proteins. Ligation to each Ubl has unique mechanistic and functional consequences. SUMO is a highly divergent Ubl. Both SUMO and Ub have crucial roles in many organisms, including important contributions to human biology. These modifications are reversed by specialized proteases. Under the auspices of this grant, our group has been analyzing both deubiquitylating enzymes (DUBs), and a set of Ubl-specific proteases (ULPs) specific for SUMO. The proteins we have been studying are conserved from yeast to humans. The long-term objective of the project is to gain a molecular understanding of the physiological and mechanistic roles played by DUBs and ULPs. In this renewal application, the proposed experiments concentrate on two distinct topics. The first is an exciting new area recently entered by the lab through the demonstration that a specific DUB from an obligate intracellular bacterium, Wolbachia pipientis, is responsible for a long mysterious type of reproductive parasitism in insects called cytoplasmic incompatibility (CI). The DUB functions as a toxin encoded as part of a two- gene Wolbachia operon. This novel connection has potentially important implications for controlling insect pests and disease vectors. The second topic focuses on how an enigmatic yeast SUMO protease, Ulp2, contributes to the function of the SUMO system in transcription and how its loss, unexpectedly, leads to a specific multi-chromosome aneuploidy. The latter could lead us to an understanding of the essential function(s) of Ulp2 in cell division and insights into aneuploidy in human cells, which is a hallmark of cancer. The following Specific Aims are proposed: (1) Examine the mechanism of CI induced by CidB, a Wolbachia Ulp1/C48-family DUB, using both Drosophila and yeast models. (2) Determine if the Wolbachia CinB protein, a putative nuclease, also contributes to CI and if so, how its mechanism of cell division disruption differs from CI induced by CidB. (3) Investigate how the Ulp2 SUMO protease regulates transcription in yeast by gene-specific and genomic approaches, and determine the basis of the specific aneuploidy induced by loss of Ulp2.