Project Summary The eukaryotic nucleus is enclosed by a double-membrane structure, the nuclear envelope, which separates the nucleoplasm from the cytoplasm. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER), whereas the inner nuclear membrane (INM) is a specialized cellular compartment with a unique proteome. In order to ensure compartmental homeostasis, the INM-associated degradation (INMAD) pathway is required for both protein quality control and regulated proteolysis of INM proteins. We have recently discovered a novel INMAD branch functioning through the E3 ubiquitin ligase: the anaphase promoting complex/cyclosome (APC/C), which degrades the integral INM protein Mps3 in budding yeast. Furthermore, APC/C-dependent INMAD regulates nuclear envelope reorganization, a key factor controlling replicative lifespan and cell aging. Our hypothesis is that APC/C directly ubiquitinates INM substrates, targeting them for proteasomal degradation, which process is critical for maintaining nuclear envelope homeostasis. Three specific aims will be fulfilled to test this hypothesis: (1) determine the modes of APC/C- dependent INMAD substrate recognition, (2) determine the modes of APC/C-dependent INMAD substrate extraction and translocation, and (3) determine the pathological consequences of impaired APC/C-mediated INMAD. Under the first aim, the critical cis and trans factors responsible for APC/C-mediated INM protein ubiquitination and degradation will be characterized. Under the second aim, the mechanism by which the AAA-ATPase Cdc48/p97 and its cofactors extract and translocate APC/C-dependent INMAD substrates will be characterized. Under the third aim, the biological function of nuclear envelope remodeling and replicative lifespan regulated by APC/C-mediated INMAD will be determined. The proposed research is expected to provide key insights into the mechanism controlling APC/C-dependent INMAD substrate ubiquitination, translocation and degradation, and the pathological consequences of INMAD impairment. Aggregation of SUN1, the Mps3 homolog, has been implicated in premature cell aging and dystrophic laminopathies in mammals. Unraveling how APC/C-mediated INMAD regulates its substrate turnover in yeast is a crucial step to understanding the molecular mechanisms governing INM protein turnover in human cells and may provide a direct link between nuclear envelope-associated diseases and cell-cycle regulation.