Signaling efficient degradation by the proteasome and related ATP-dependent proteases

ATP-dependent proteases degrade many regulatory proteins in the cytosol and nucleus and thereby control processes such as the cell cycle, gene expression and signal transduction. The oligomeric structure of ATP-dependent proteases resembles a hollow cylinder formed by distinct subunits that are assembled as a ring and sequesters the active sites of proteolysis within a central chamber. The small diameter of the entrances leading to the active sites permits access to unfolded polypeptides but not to globular folded proteins. The proteases, therefore, use ATP-hydrolysis to denature their native substrate proteins and translocate them into the active site for degradation. I have analyzed this activity for the proteasome, the mitochondrial Lon and the bacterial FtsH ATP-dependent proteases and found that they vary in their protein unfolding ability with the proteasome being the most robust of these enzymes. I have further characterized the signals in proteasome substrates that lead to their efficient destruction. It is known that proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. I have found that the ubiquitin modification serves as the proteasome recognition element, but by itself it is not sufficient for efficient removal of folded proteins. I show that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to them. This disordered region serves as the initiation site for degradation and is hydrolyzed first, followed by the sequential digestion of the rest of the target protein. Thus, the initiation site is a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery fully. By analyzing the proteolysis of two naturally long-lived proteasome interacting proteins Cdc34 and Rad23, I have been able to show that the initiation site must also have a complex amino acid composition. Interestingly, the proteasome initiation site and the ubiquitin modification can also work in trans, i.e. when separated onto different polypeptide chains in a complex, and the subunit with the initiation site is selectively degraded. Presence of an effective proteasome initiation site in proteins and the susceptibility of their folded domains to unfolding together determine selectivity in proteasome-mediated degradation.