Kinetic Silver Staining of Proteins

Abstract

INTRODUCTION: Protein degradation, me-diated by the ubiquitin-proteasome system (UPS), plays a critical and complementary role to transcription, splicing, and translation in the control of gene expression. Effective reg-ulation of the UPS relies on the specificity of substrate recognition, which is conferred largely by the upstream ubiquitylation pro-cess. As a specific example, the anaphase-promoting complex (APC) acting on a series of substrates promotes waves of ubiquitylation and degrada-tion, 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, lead-ing 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 protea-some. 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 ap-plicable, and the mechanism by which the proteasome recog-nizes 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 degrada-tion, we examined the kinetics of degrada-tion 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 degra-dation than others, we investigated the degra-dation process using single-molecule (SM) methods that are capable of identifying tran-sient intermediates and measuring their ki-netic 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 es-sential for degradation of most substrates. Never-theless, a multiple monoubiquitylated form of securin, a regulator of chromatid separa-tion, 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 syn-thetically constructed ubiquitylated substrates with defined configurations revealed no sim-ple 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: sub-strate binding and kinetic postbinding events. In the past, it was widely assumed that ubiquitin chains mostly promoted binding to the protea-some. Our SM studies demonstrate that the strength of interaction with the proteasome, for substrates with multiple ubiquitylated ly-sines, is largely determined by the total num-ber of ubiquitins and is less sensitive to ubiquitin chain configurations. For most substrates, bind-ing alone is not sufficient for degradation. Rather, degradation depends strongly on a pro-cess that initiates passage into the substrate translocation channel; this transition, in con-trast to binding, is determined by the particular configuration of ubiquitin chains.