Research Article

Specificity of the anaphase-promoting complex: A single-molecule study

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Science  10 Apr 2015:
Vol. 348, Issue 6231, 1248737
DOI: 10.1126/science.1248737

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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

Structured Abstract

INTRODUCTION

Regulation of biological processes is generally thought to be conveyed by structurally complementary interactions between molecules, resulting in a bound state that is the most energetically stable. However, the limited size of most recognition motifs poses a very general question as to how specificity is actually achieved. In the cell cycle, timing of events from exit of mitosis through passage into G1 is regulated by the anaphase-promoting complex (APC), an enzyme that catalyzes ubiquitin conjugation to multiple lysines on substrate molecules, for proteasome-mediated degradation. The difference in how APC ubiquitylates its substrates leads to the ordering of events. APC-substrate interactions are also mediated through very short and redundant sequences, such as the well-studied D and KEN boxes found in 69% of human proteins. It is unclear how the APC can distinguish a relatively small number of substrates from nonsubstrates having the same interaction motifs. At a total concentration of approximately 1 mM, many of these nonsubstrates should compete for APC binding, thus reducing the rate of ubiquitylation and degradation of the real targets.

RATIONALE

Simple binding based on structural complementarity seems inadequate to explain the specificity in APC-mediated ubiquitylation. We therefore looked beyond a single binding step to the extensive network of reactions that take place at multiple sites on a substrate molecule and in a cellular environment with competing reactions. To describe these, we developed single-molecule fluorescence assays, capable of identifying multiple reaction intermediates, and applied these methods to the study of the kinetics of APC-mediated ubiquitylation in both cytosolic extracts and in purified reaction systems. Our goal was to understand how the APC selects the right substrates among competing molecules and generates effective ubiquitin configurations that can be recognized for degradation.

RESULTS

In this assay, the rates of ubiquitylation and APC-substrate interaction can be measured simultaneously and traced to individual substrate molecules. We find that the APC-mediated ubiquitylation initially involves a highly processive reaction conjugating several ubiquitins on a substrate molecule, followed by multiple encounters and reactions at a slower rate. The initial ubiquitylation greatly enhances substrate-binding affinity with the APC in subsequent reactions, by both increasing the on-rate and decreasing the off-rate. Results of kinetic studies in bulk assays further substantiate this positive feedback mechanism.

CONCLUSION

The process of converting the reactivity of ubiquitin on a substrate to binding affinity can complement weak recognition by the D/KEN-box motifs. Cycles of positive feedback achieve high specificity for substrates with short recognition motifs in a cellular environment of competing molecules. This process, called processive affinity amplification (PAA), iteratively increases conjugated ubiquitins on substrates with both the recognition motifs and receptor lysine residues. Reaction schemes similar to PAA may be responsible for maximizing specificity, while maintaining efficiency in other biological processes.

Positive feedback enhances specificity and efficiency in APC-mediated ubiquitylation.

The intrinsic affinity between APC and D-box–containing decoy substrates is low, which prevents APC from inhibition by a high concentration of those substrates in the cytosol (A). The initial ubiquitylation on real substrates is highly processive, likely due to their preponderance in the availability and reactivity of receptor lysines. Conjugated ubiquitins enhance substrate’s affinity for the APC and promote further ubiquitylation, enabling proteasomal degradation (B).

Abstract

Biological processes require specific enzymatic reactions, paradoxically involving short recognition sequences. As an example, cell-cycle timing depends on a sequence of ubiquitylation events mediated by the anaphase-promoting complex (APC) based on short redundant motifs. To understand the origin of specificity, we designed single-molecule fluorescence assays that capture transient ubiquitylation reactions. We find that the APC-mediated ubiquitylation involves a highly processive initial reaction on the substrate, followed by multiple encounters and reactions at a slower rate. The initial ubiquitylation greatly enhances the substrate’s binding affinity in subsequent reactions, by both increasing the on-rate and decreasing the off-rate. We postulate that these cycles of positive feedback enable high specificity for substrates with short recognition motifs in a complex cellular environment.

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