Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) play a pivotal role in vertebrate intracellular signaling. They are a family of three very similar proteins that form tetrameric ion channels in endoplasmic reticulum (ER) membranes. For the last 15 years, I have been studying the molecular biology of IP3Rs and for the last 10 years or so, have been examining how they are degraded by the ubiquitin-proteasome pathway (UPP). During these studies, it became obvious that IP3Rs are degraded by the ERAD pathway (the facet of the UPP that accounts for the disposal of aberrant ER proteins) and that given their unique properties, IP3Rs might be particularly useful tools for identifying the proteins and mechanisms that mediate ERAD in mammalian cells. Thus, in the last grant cycle we sought to identify and characterize the proteins and mechanisms that mediate IP3R ERAD, and discovered that the ubiquitin conjugates that become attached to activated IP3Rs are unexpectedly complex, and that a completely novel, and very large (>1MDa) complex composed of two uncharacterized ER membrane proteins, termed SPFH1 and 2, mediates the ERAD of activated IP3Rs and participates in the degradation of model ERAD substrates. Interestingly, we have also very recently found that RNF170, an uncharacterized, putative ubiquitin ligase, interacts strongly with the SPFH1/2 complex. The current proposal is designed to extend this work. As in the past, the bulk of the work will be done with endogenous proteins in mammalian cells, ensuring that the results obtained closely mirror the physiological situation. The Specific Aims of the current proposal are to (i) identify and characterize the proteins that govern the levels of ubiquitin conjugates on activated IP3Rs, and (ii) investigate the structure and function of the SPFH1/2 complex. These goals are significant for three main reasons. First, the studies in Aim 1 on the fine details of IP3R ubiquitination, should help solve the recently revealed mystery of how and why UPP substrates are modified with diverse ubiquitin conjugates. Second, Aim 2 should generate a clearer understanding of the structure and function of the recently discovered, but poorly understood, SPFH1/2 complex. Third, our focus on endogenous proteins in mammalian cells means that the results obtained will accurately model the in vivo situation, and will provide information on the ERAD pathway under near- physiological conditions. Results from this work should have a significant impact on our understanding of both ERAD and the UPP in general, and will hopefully facilitate efforts aimed at developing drugs targeting these pathways. PUBLIC HEALTH RELEVANCE: The degradation of proteins via the proteasome, which is governed by another protein called ubiquitin, is a fundamental cellular process that controls the levels of many key proteins, and has enormous potential as a therapeutic target in diseases such as cystic fibrosis, neuropathies, diabetes and cancer. The studies I propose will investigate in mammalian cells some of the mechanisms that underpin this process with the aim of obtaining a clearer understanding of this area.