Research Article

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

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Science  10 Apr 2015:
Vol. 348, Issue 6231, 1250834
DOI: 10.1126/science.1250834
  • 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.

  • Fig. 1 Quantitative degradation assay.

    (A) The assay strategy. Preformed, methylated ubiquitin chains were conjugated to PKA-labeled securin using purified APC and E2 UbcH10. After reaction, the product was subject to degradation by purified 26S human proteasome. The decay constant for each ubiquitylated species, separated on a gel, was measured from a time (T) series. Signal from unmodified substrates (Ub0) was used as a control for loading and nonspecific dephosphorylation, which is the main reason for the decrease of Ub0 signals (Materials and methods section). Ub, ubiquitin; K, lysine residue; M-DiUb, methylated diubiquitin chain; M-TetraUb, methylated tetraubiquitin chain. (B to D) 160 nM geminin, securin, and cyclin B–NT (Xenopus) were ubiquitylated using indicated constructs of ubiquitin. Their rates of degradation by 3 nM of purified human 26S proteasome were measured and shown as a function of total ubiquitins per substrate molecule. Error bars represent the SD of three experimental replicates. The asterisk in (C) indicates that the rate for this species is 1.4. The lack of data for certain species is due either to these species not having been tested or to their signals being too weak to quantify. (E and F) Human cyclin B–NT mutants carrying lysines only at indicated positions were ubiquitylated with either methylated ubiquitin (M-Ub) or WT Ub and tested in a quantitative degradation assay. Original autoradiography for retrieving the rate information is compiled in fig. S8. In (E), the inset shows the location of the D-box (DB) on cyclin B–NT and relevant ubiquitylatable lysine residues identified by mass spectrometry.

  • Fig. 2 Proteasome-substrate interaction kinetics by the SM method.

    (A) Schematics showing the experimental design, where purified 26S proteasome was immobilized on passivated coverslip using anti-20S antibody. Ubiquitin was fluorescently labeled and conjugated to substrates in solution. Sample pictures capture ubiquitin signals on the surface with either 26S proteasome (+26S) or antibody only (no 26S). (B) Average dwell time on the proteasome for different substrates (or free Ub chain) with varying numbers of conjugated ubiquitins, measured by the SM method. The distributions of individual dwell times are shown in fig. S15. Error bars represent SD of the mean. The asterisk indicates that data for chains of 6 to 9 ubiquitins are not shown due to insufficient events. (C) Cooperative and stochastic mechanisms affect binding enthalpy and entropy, respectively. By each mechanism, the expected relationship between dwell time tb and the number of ubiquitins N is shown below. ΔG, change in Gibbs free energy; ΔH, change in enthalpy; T, temperature; ΔS, change in entropy. (D) Dwell time on the proteasome (right), or its logarithm (left), for securin and cyclin B versus the number of conjugated ubiquitins. Red lines show linear fitting. The ratio of P values by fitting the red segment (in linear scale) with either a linear model (pl) or an exponential model (pe) is shown for each plot. ∆gub is the binding free energy per ubiquitin on the proteasome, calculated from the slope of the binding curve.

  • Fig. 3 Interaction with the proteasome is mainly determined by the total number of ubiquitins on a substrate, insensitive to Ub chain structures.

    (A and B) Securin or cyclin B–NT was ubiquitylated by APC with either WT Ub or Ub0K and was tested for interaction with the proteasome, as in Fig. 2B. (C and D) Interaction of WT Ub– or Ub0K-conjugated cyclin B with the proteasome in the presence of ATP or ATP-γ-S. rep1 and rep2 are two experimental replicates. The data for the ATP-proteasome experiment are plotted for comparison; these data are identical to those shown in (B). Cyclin B–ATP data in (C) are identical to those in (B). In (A) to (D), error bars denote SD of the mean. (E) Degradation rate and proteasomal dwell-time relationship for WT Ub– and Ub0K-conjugated securin. Degradation rates were measured in the quantitative degradation assay (Fig. 1C), and dwell time on the proteasome was measured using the SM method. The inset shows the Ub0K result on a smaller y axis. (F) Ratio of “commitment efficiency” for securin–WT Ub over securin-Ub0K, as a function of total conjugated ubiquitins. Commitment efficiency is defined as the degradation rate divided by the dwell time.

  • Fig. 4 Ubiquitin chains on substrates promote translocation initiation.

    (A) Examples of structureless and stepped traces from a SM experiment on WT Ub–conjugated securin. Stepped traces are due to progressive deubiquitylation by Rpn11, as illustrated in (B). (C) The fraction of stepped traces, as a function of the number of ubiquitins, for WT Ub (N = 663) or Ub0K-conjugated (N = 133) securin. Error bars represent SD of the mean.

  • Fig. 5 An integrated model for the degradation of ubiquitylated substrates by the 26S proteasome.

    Polyubiquitylated substrates could simultaneously interact with multiple ubiquitin receptors on the proteasome. While remaining bound, a substrate molecule explores multiple configurations on the proteasome through intramolecular diffusion (1). Rearrangements of proteasomal subunits, fueled by the ATPases in response to binding and hydrolysis of ATP, expose or activate a deeper ubiquitin chain receptor and facilitate the transfer of ubiquitylated substrates from initial binding to a deeper engagement (2 and 3). In this engagement, the substrate or its terminus is closer to the substrate entry port, which expedites translocation initiation (3) and ensuing degradation (4).

Supplementary Materials

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

    Ying Lu, Byung-hoon Lee, Randall W. King, Daniel Finley, Marc W. Kirschner

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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