Substrate degradation by the proteasome: A single-molecule kinetic analysis

Single-molecule assay of ubiquitylation Many biological processes in cells are regulated by ubiquitin peptides that are attached to proteins. Measurement of single fluorescent molecules in cell extracts can be used to trace the kinetics of such reactions. Lu et al. refined assay conditions to follow ubiquitination by an E3 ubiquitin ligase (see the Perspective by Komander). They visualized the activity of the anaphase-promoting complex (APC), a ubiquitin ligase critical for control of the cell division cycle. The processive initial reaction catalyzed by the APC was replaced by slower reactions. The results show how small, commonly occurring recognition motifs can guide specific and highly controlled enzymatic events. In a companion paper, Lu et al. explored how the number and arrangement of added ubiquitin chains affected the interaction of ubiquitylated proteins with the proteasome (a protein complex that recognizes ubiquitylated proteins and degrades them). The extent of ubiquitylation determined the strength of interaction of a substrate protein with the proteasome, and the arrangement of the ubiquitin chains determined the movement of the protein into the proteasome and thus the rate of degradation. Science, this issue 10.1126/science.1248737, 10.1126/science.1250834; see also p. 183 How added ubiquitin chains regulate protein degradation at the proteasome is shown. [Also see Perspective by Komander] INTRODUCTION Protein degradation, mediated by the ubiquitin-proteasome system (UPS), plays a critical and complementary role to transcription, splicing, and translation in the control of gene expression. Effective regulation of the UPS relies on the specificity of substrate recognition, which is conferred largely by the upstream ubiquitylation process. As a specific example, the anaphase-promoting complex (APC) acting on a series of substrates promotes waves of ubiquitylation and degradation, leading to key transitions in the cell cycle. However, in this cascade the proteasome itself also plays a role in the specificity of degradation. There have been various efforts to characterize this role, leading to a commonly held view that a substrate protein must be conjugated with a chain of at least four ubiquitins to be recognized by the proteasome. Yet mass spectrometry studies show that ubiquitin chains on APC substrates (such as cyclin B) contain, on average, only two ubiquitins. But these can have complex ubiquitin configurations created by the multiplicity of ubiquitylated lysine residues. Therefore, the tetraubiquitin chain selection rule may not be generally applicable, and the mechanism by which the proteasome recognizes substrates is still shrouded in mystery. RATIONALE The multiple lysines on a substrate and on ubiquitin itself generate a large number of possible ubiquitin configurations. To understand the “ubiquitin code” that must be read by the proteasome and converted into a rate of substrate degradation, we examined the kinetics of degradation with defined ubiquitin configurations by conjugating preformed ubiquitin chains on substrate molecules. Further, to reveal the molecular basis through which some ubiquitin configurations promote more efficient degradation than others, we investigated the degradation process using single-molecule (SM) methods that are capable of identifying transient intermediates and measuring their kinetic parameters and sensitivity to ubiquitin configurations. RESULTS Contrary to the tetraubiquitin chain selection rule, we find that for APC substrates with multiple ubiquitylated lysine residues, diubiquitin chains are more efficient than tetraubiquitin chains in promoting degradation, given the same number of conjugated ubiquitins. Ubiquitin chains are essential for degradation of most substrates. Nevertheless, a multiple monoubiquitylated form of securin, a regulator of chromatid separation, interacts with the proteasome as strongly as securin containing the same number of ubiquitins grouped in chains. By dissecting the degradation process using SM methods, we find that ubiquitin chain structures on substrates promote the passage of a bound substrate into the translocation channel on the proteasome. CONCLUSION This systematic study of synthetically constructed ubiquitylated substrates with defined configurations revealed no simple length threshold for ubiquitin chains for degradation by the proteasome. A distributed array of short ubiquitin chains, as appears naturally on APC substrates, is a superior and perhaps optimal signal for degradation; this conclusion will most likely extend to substrates of other E3 ligases. The rate of degradation is an aggregate of two sequential processes: substrate binding and kinetic postbinding events. In the past, it was widely assumed that ubiquitin chains mostly promoted binding to the proteasome. Our SM studies demonstrate that the strength of interaction with the proteasome, for substrates with multiple ubiquitylated lysines, is largely determined by the total number of ubiquitins and is less sensitive to ubiquitin chain configurations. For most substrates, binding alone is not sufficient for degradation. Rather, degradation depends strongly on a process that initiates passage into the substrate translocation channel; this transition, in contrast to binding, is determined by the particular configuration of ubiquitin chains. Key transitions in the degradation of ubiquitylated substrates by the proteasome. A polyubiquitylated substrate molecule explores multiple configurations on the proteasome through stochastic binding. Rearrangements of proteasomal subunits, driven by the binding and hydrolysis of adenosine triphosphate, allow a deeper engagement of the substrate with the proteasome, when there are appropriate ubiquitin chain structures on the substrate. As a result of this engagement, the substrate or its terminus is moved closer to the substrate entry port, promoting expeditious initiation of translocation and ensuing degradation. To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation.