| Ubiquitin (Ub) mediated proteolysis is the primary strategy employed by cells to rapidly and selectively modulate their proteomes in response to stimuli. It involves an E1-E2-E3 enzymatic cascade to create a poly-Ub chain on the target protein. The ubiquitinated protein is then recognized by Ub-receptors, which facilitate its delivery to the 26S proteasome for degradation. With close to 700 dedicated proteins involved in the conjugation of Ub, challenges exist to determine the specific degradative route(s) for individual proteins. Of particular importance is the identification of the E3 Ub-ligases because they confer substrate specificity to the pathways by directly binding to the target protein.;Our work has uncovered specific proteolytic pathways that regulate neuronal function and depend on the SCFbeta-TRCP E3 Ub-ligase complex. By identifying RapGAP SPAR and transcriptional repressor REST as substrates of the SCFbeta-TRCP complex, we discovered novel roles for this E3 Ub-ligase in neural development and in the maintenance of postmitotic neurons. The SCFbeta-TRCP complex directly recognizes a characteristic phosphodegron motif in its substrates that depends on antecedent kinase activity. We characterized the beta-TRCP phosphodegrons in both SPAR and REST, and further explored characteristics of other beta-TRCP phosphodegrons found in substrates Cdc25A and Per1.;How do these substrates arrive at the proteasome? Previous studies indicated that this required a diverse group of proteins termed Ub-receptors. Our data in mammalian cells suggested both specificity and partial redundancy among candidate Ub-receptors with regard to their selection of substrates. However, the mechanisms by which individual receptors achieve substrate specificity and the identities of substrates for individual receptors are currently unclear. We have laid the groundwork for a GPS profiling screen to monitor the stability of 12000 proteins upon depletion of Ub-receptor S5a (Rpn10). We identified protein phosphatase Cdc25A as a target of Ub-receptor S5a. Interestingly, simultaneous depletion of Ub-receptors 55a and PLIC-1 resulted in additive stabilization of this substrate. Together, these results suggest a complex interplay among mammalian Ub-receptors in regulating substrate proteolysis.;Overall, we have identified and characterized novel Ub-mediated proteolytic pathways, and framed a study to elucidate the unexplored mechanisms of substrate delivery to the proteasome. |
