The ubiquitin-proteasome system (UPS) is the major cytosolic and nuclear mediator of protein turnover in eukaryotes. It controls many cellular processes through targeted degradation of transcription factors and other regulatory proteins, and degrades misfolded proteins as part of the cell's stress response (1
A two-part degron targets proteins to the proteasome for efficient degradation (4
). The first part is a proteasome-binding tag, typically a poly-ubiquitin chain that is added to one or more lysines within the substrate through the action of E1, E2 and E3 enzymes. The second part is an unstructured initiation region, which functions best when it is close in space to a poly-ubiquitin chain, as displacing it by ~40 Å largely inhibits degradation of model substrates (7
). The ubiquitin tag is recognized by receptors in the proteasome's 19S regulatory cap and the initiation region is likely engaged by ATPase motors at the base of the cap (1
). The motors pull at the initiation region, which leads to the sequential unfolding and translocation of the substrate to the protease active sites buried within the 20S proteasome core particle. Thus, degradation typically begins at the initiation region and proceeds linearly along the polypeptide chain (6
). The proteasome denatures any folded domains as it encounters them so that the end product is the complete degradation of the substrate into small peptides (9
). This processivity prevents the formation of fragments that could have undesired biological activities.
The proteasome can also initiate degradation at internal unstructured loops within larger proteins (6
) and this appears to be common in cells. However, we do not know how the location of the initiation region affects the rest of the degradation reaction and its end products. The proteasome can degrade circular and disulfide-linked proteins, indicating that the channel that leads to the degradation chamber can accommodate more than one polypeptide chain at once (10
) but crystal structures of the proteasome core particles show that the channel will be a tight fit for two chains (17
). This tight fit or the load put on the proteasome by the simultaneous presence of two polypeptide chains and their folded domains could reduce the effectiveness of the unfolding and degradation machinery, and thus the proteasome's processivity.
In a few cases, proteins are degraded incompletely by the proteasome in a process referred to as proteasomal processing. The known physiological examples of processing are the p105 and p100 precursors of the p50 and p52 subunits of mammalian transcription factor NFκB, which functions in immune and inflammatory responses, the yeast Spt23 and Mga2 transcription factors, which are distantly related to NFκB and regulate unsaturated fatty acid biosynthesis, and the Drosophila
transcription factor Cubitus interruptus (Ci) and its vertebrate homologs Gli2 and Gli3, which function in Hedgehog signaling (18
). Partial degradation of these proteins releases fragments with new biological activities. In the case of p105, p100, Spt23 and Mga2, an inert precursor protein is converted into an active transcription factor, whereas in the case of Ci and the Gli proteins, a transcriptional activator is converted into a competitive repressor of transcription. Thus, proteasomal processing represents an additional layer of post-translational regulation that can be used to control biological activity and cellular fate. However, the biochemical mechanism of this processing reaction is only poorly understood and this is a major stumbling block to the discovery of other examples.
Internal initiation may play an important role in processing. For Spt23 and Mga2, degradation must begin internally because both ends of the polypeptide chain are blocked: the N-terminus by the tightly folded IPT domain that is released by the processing reaction and the C-terminus by a membrane anchor (11
). The ubiquitination sites responsible for processing of p105, Ci and Gli3 are in the middle of the full-length polypeptide chain (26
) and thus the proteasome likely initiates their degradation internally as well. Ci also contains degrons near its termini but these do not lead to processing (29
Here we determine whether internal initiation is directly related to processing by following how the structure of model substrate proteins affects proteasome processivity in a purified degradation system. We discover that domains flanking the initiation region stabilize each other without interacting directly. This remote stabilization decreases processivity and can tune proteasomal degradation from complete proteolysis to almost quantitative fragment formation.