Proteasome activators cap the proteasome core particle (CP or 20S proteasome) and enhance proteasome activity. To date three activators are known: the regulatory particle (RP or 19S proteasome), the PA28 proteins in higher eukaryotes and the universally conserved Blm10 proteins. Proteasome activators compete for CP binding. The modular nature of proteasome complexes most likely serves regulatory functions, potentially by targeting different substrate subsets. The objective of this project is to investigate the impact of Blm10 activators on proteasome activity and function in S. cerevisiae. Aim 1: While the RP and PA28 are oligomeric ring structures, Blm10 is a monomeric 240 kDa protein. The reduced complexity of Blm10-proteasomes facilitates the investigation of proteasome activation at the molecular level. Proteasome activation involves a gating mechanism, which controls substrate access to the central proteolytic chamber of the CP. CP activation by PA28 regulators is achieved by internal loop structures. CP activation by the RP ATPases appears to involve the docking of their C-termini into surface pockets of the CP. Our data suggest that both mechanisms apply to Blm10 CP activation. Using combined mutagenesis, biochemical, enzymatic and genetic approaches, we will elucidate the molecular details of proteasome activation by Blm10 proteins. Aim 2: Our data show elevated levels of the protein Dnm1 in BLM10 deleted cells, suggesting that Dnm1 is a proteolytic target for Blm10-proteasomes. Dnm1 is an essential factor for mitochondrial fission. A potential function for Blm10-proteasomes in the regulation of mitochondrial dynamics is supported by our observations that Blm10 mutants exhibit dysfunctional mitochondria and altered mitochondrial morphology. We found Blm10-proteasomes to interact with the kinase Yak1. Similar to BLM10 deletion, YAK1 deletion stabilizes Dnm1, suggesting a functional link between Yak1 kinase activity and Blm10-proteasomes. In Aim 2 we will investigate the functional relevance and molecular details of this interaction by establishing an in vitro degradation system involving purified Dnm1, Yak1 and Blm10-proteasomes or mutant versions of the proteins. We furthermore propose to investigate the function of Blm10-proteasomes and Yak1 in mitochondrial dynamics using fluorescence microscopy and colocalization studies. We will also investigate, whether additional factors required for mitochondrial dynamics are targets for Blm10-proteasomes. Aim 3: To fully understand this proteolytic system, it is necessary to identify its targets. Many substrates of the 26S proteasome (RP+CP) have been detected, two example are known for PA28- proteasomes and none for Blm10-proteasomes. Through utilizing advances in yeast genetics we will screen for Blm10-proteasome substrates on a genome wide scale using SGA technology. PUBLIC HEALTH RELEVANCE Malfunction of the ubiquitin/proteasome system is the cause for many human diseases, such as cancer, neurodegenerative, and immune-related diseases and proteasome inhibition has developed into a promising strategy for chemotherapy. Understanding the mechanistic details of proteasome activity and regulation is crucial for the development of novel proteasome-related drugs and is the main objective of this project.