One aspect of this project relates to determining the role of the ubiquitin system in mitochondrial membrane dynamics and how this in turn relates to cellular energetics in cancer. This project was begun in yeast and is now being expanded to mammalian systems. We have determined that in yeast that the E3 SCF-Mdm30 plays a critical role in targeting the mitofusin, Fzo1, for degradation. We also have preliminary evidence for the involvement of other ubiquitin ligases in this process. The importance of understanding the regulation of mitochondrial fusion and fission is of great relevance to intermediary metabolism and potentially to understanding how cancers perform anabolic functions under hypoxic conditions. A major aspect of this project relates to degradation from the endoplasmic reticulum. Together with collaborators we are studying E2s and E3s critical to this process. gp78, also known as the tumor autocrine motility factor receptor (AMFR), was discovered by our laboratory to be a ubiquitin ligase resident to the endoplasmic reticulum. We have recently determined that gp78 plays essential roles in the degradation of multiple substrates functioning together with an E2 known as MmUBC7 (Ube2g2). We have now determined that multiple domains within gp78 function together to mediate its ubiquitin ligase activity. These include its RING finger, a ubiquitin-binding Cue domain and a novel region that specifically recruits Ube2g2 independent of the RING finger, referred to as the G2BR. Moreover, we now know that expressing the G2BR in isolation can block ERAD and induce ER stress. We are exploring the potential for such expression or other means of blocking the interaction between the gp78 and Ube2g2 as a means to target cells that are predisposed to ER stress, such as multiple myeloma cells, to undergo apoptosis. gp78 levels are correlated with the metastatic potential of tumors including melanomas and lung cancers. We have now determined that knocking down gp78 levels results in a decrease in migration of cells in response to AMF as well as to other stimuli and to an inhibition of migration in in vitro wound healing type experiments. In vivo studies in mice using knockdowns of gp78 and reexpression have now determined that gp78 does in fact play an important role in the metastatic potential of multiple different sarcomas and that this potential for metastasis requires intact ubiquitin ligase function of this protein. WE have also determined that gp78 targets the metastasis suppressor, KAI1 (CD82) for degradation in sarcomas. This provides at least a partial explanation for our findings. Further studies are underway to characterized additional substrates for gp78 and to understand the degree to which alleviating ER stress through gp78 might be playing a role in facilitating metastasis.