Fig 1.
SCF-mediated substrate degradation and Cand1 cycle.
A: Scheme of SCF-mediated substrate degradation: (1) Substrate (S) binding to substrate receptors (Skp1/SR) and UBC12-mediated neddylation (N8) of Cul1, (2) E2 recruitment, ubiquitin (Ub) transfer by E2 to the substrate and Ub chain elongation, (3) substrate degradation by the 26S proteasome and (4) deneddylation of Cul1 by the COP9 signalosome. Relative sizes of protein subunits are not to scale. B: Model of the Cand1-mediated exchange cycle for two substrate receptors (Skp1/SR1 and Skp1/SR2). Ksr, Kca, and
denote dissociation constants whereas ksr, kca,
and
are dissociation rate constants (cf. Table 1). The parameter η, defined in Eq (2), measures the preference of Cand1 and SR for binding to Cul1. Similarly, α and β account for relative differences in the dissociation rate constants for the binary complexes (α) and the ternary complex (β).
Table 1.
Default parameter values.
Fig 2.
Trade-off between high SCF occupancy and fast SR exchange rate.
A: Left axis shows SCF activity as measured by the steady state concentration of Cul1.SR1. Right axis shows the exchange rate as measured by the leading eigenvalue of the Jacobian matrix (ρl). As the total Cand1 concentration increases the SCF activity (solid lines) decreases while the exchange rate (dashed lines) concomitantly increases. As the relative binding affinity η (Eq 2) decreases both the SCF response curve as well as the curve characterizing the exchange rate develop a sharp threshold near Cand1T = Cul1T (marked by arrow head). The horizontal dotted line indicates the maximal exchange rate (cf. Eq 9). B: Exchange rate (|ρl|) vs. SCF occupancy ([Cul1.SR1]) drawn from the curves in panel A. Note that the curves are overlapping. We have also indicated the positions along the curve where, depending on the value of η, the concentration of Cand1 equals that observed in cells (cf. Table 1). C: Left panel shows the time scale for the exchange of substrate receptors (τs = 1/|ρl|) as a function of the total Cand1 concentration for the parameters listed in Table 1. Right panel shows the corresponding time course for the assembly of Cul1.SR1 after adding 100nM SR1 to a steady-state mixture containing 300nM Cul1, 560nM SR2 and Cand1 as indicated by the dashed lines in the left panel. The dashed lines in the right panel indicate the value of τs obtained from the intersection of the dashed lines with the black solid line in the left panel.
Fig 3.
A: Extension of the model depicted in Fig 1B. Substrate (S1) reversibly binds to Cul1.SR1. The substrate in the resulting Cul1.SR1.S1 complex is degraded with effective rate constant kdeg. B: Time courses showing the degradation of total substrate ST = [S1] + [Cul1.SR1.S1]. At t = 0 substrate (300nM) was added to a steady state mixture containing SR1T = 30nM, SR2T = 630nM and Cand1T as indicated. Note that t1/2 changes non-monotonically as a function of Cand1T. C: Half-life for substrate degradation (t1/2) as a function of Cand1T for decreasing binding affinity. As the association rate constant kon for substrate binding decreases t1/2 increases and the dependence of t1/2 on Cand1T becomes monotonic. Parameters for substrate binding: kon = 108M−1s−1, koff = 1s−1, kdeg = 0.004s−1. Parameters other than those mentioned are listed in Table 1.
Fig 4.
Effect of Cand1 dose on the stability of the SCF substrate Rum1p in S. pombe.
A: S. pombe cells stably overexpressing the indicated versions of Cand1 were treated with 100 μg/ml cycloheximide (CHX) for the indicated periods of time and analyzed for the expression of Rum1p, Cul1p, and Cand1 by immunoblotting. Cdc2p signals are shown for reference. Representative results of two independent experiments. B: Immunoblotting signals for Rum1p and Cdc2p were quantified using Image Studio Lite, and Rum1p intensities normalized to Cdc2p were plotted on a log scale. C:Exponential decay lines fitted through the data points in (B) were used to calculate protein half-lives in strains overexpressing the indicated Cand1 proteins. Results are averages of two independent experiments. Error bars represent standard deviations.
Fig 5.
Dynamic readjustment of the SCF repertoire.
A: Transient response of the SCF complexes Cul1.SR1, Cul2.SR2 and Cul1.SR1.S1 upon substrate addition (300nM at t = 0) to a steady state mixture containing SR1T = 30nM, SR2T = 630nM and Cand1T = 100nM. Between 1-100min the drop in [Cul1.SR2] is accompanied by a peak in [Cul1.SR1.S1] indicating that Cul1 is redistributed from Cul1.SR2 into Cul1.SR1 and Cul1.SR1.S1. B, C: Redistribution of Cul1 from Cul1.SR2, Cul1.SR1(2).Cand1 and Cul1.Cand1 into Cul1.SR1 and Cul1.SR1.S1 for Cand1T = 100nM (B) and Cand1T = 1000nM (C). In both panels the solid violet line shows the transient increase of the concentration of “engaged” ligases ([Cul1.SR1] + [Cul1.SR1.S1]) upon substrate addition as described in (A). The remaining curves indicate the contribution to the transient response by any of the other complexes. For example, δCul1.SR2(t) = [Cul1.SR2](0) − [Cul1.SR2](t) denotes the amount of Cul1 that is redistributed into Cul1.SR1 and Cul1.SR1.S1 upon disassembly of Cul1.SR2. Note that in panels B and C the solid violet curve is the sum of the other curves. Parameters for substrate binding: kon = 108M−1s−1, koff = 1s−1, kdeg = 0.004s−1. Parameters other than those mentioned are listed in Table 1.
Fig 6.
Temporal hierarchy of substrate degradation.
A, B, C: Transient response upon substrate addition. At t = 0 two substrates, S1 and S2 (each 300nM), are added to a steady state mixture containing Cul1, Cand1 and SR1-SR3. The resulting decline of the total amount of substrates is displayed together with the t1/2 (dotted lines). Substrates with a higher SR affinity (A), substrates for SRs with a higher affinity for Cul1 (B) and substrates for more abundant SRs (C) are preferentially degraded.D, E, F: Assembly and disassembly of SCF ligases upon substrate addition. Depicted are changes in the fraction of SRs that are bound in a SCF complex. The blue and violet curves correspond to ([Cul1.SR1] + [Cul1.SR1.S1])/SR1T and ([Cul1.SR2] + [Cul1.SR2.S2])/SR2T, respectively, whereas the light red curve denotes [Cul1.SR3]/SR3T. In each case Cul1 is redistributed from Cul1.SR3 into Cul1.SR1(.S1) and Cul1.SR2(.S2). In (A-F) if not indicated otherwise reference parameters are: KS1 = KS2 = 10nM (koff = 1s−1), Ksr,1 = Ksr,2 = Ksr,3 = 0.225pM, SR1T = SR2T = 60nM. To preserve detailed balance has been increased by a factor of 5 in (B) and (E). SR3T = 660nM − (SR1T + SR2T), Cand1T = 400nM, kdeg = 0.004s−1. Parameters other than those mentioned are listed in Table 1.
Fig 7.
Alternative network architecture.
A: Extension of the Cand1 cycle model (black solid lines) to analyze different modes of substrate binding to SR1. Sequential mechanism: Substrate (S1) only binds to SR1 if the latter is already bound to Cul1 or Cul1.Cand1 (blue lines). Random order mechanism: S1 binds to free SR1, Cul1.SR1 and Cul1.Cand1.SR1. In addition, SR1.S1 binds to Cul1 or Cul1.Cand1 (red lines). By increasing the factor γ the binding affinity between Cul1 and SR1 can be lowered while still satisfying the detailed balance condition in Eq 1. For SR2 we used the scheme depicted in Fig 1B (without substrate), but with ksr and kca multiplied by γ. Ksr, Kca, and
denote dissociation constants whereas ksr, kca,
and
are dissociation rate constants (cf. Table 1). B and C: Comparison of the half-life (t1/2) of S1 for two network designs: one with Cand1 (+Cand1) and tight binding of SRs to Cul1 (Cand1T = 390nM, γ = 1) and another one without Cand1 (-Cand1) and weak binding of SRs to Cul1 (Cand1T = 390nM, γ = 1.67 ⋅ 107). In the latter case γ is chosen such that the pre-stimulus steady state for Cul1.SR1 is the same in both cases (note that dashed and solid lines in lower panels partially overlap). If substrate binds sequentially the system without Cand1 (B, dashed line) outperforms the system with Cand1 (B, solid line) as the t1/2 is 3.4-fold larger in the presence of Cand1. In both cases Cul1 is redistributed from Cul1.SR2 to Cul1.SR1 and Cul1.SR1.S1 (B, lower panel). In contrast, when substrate binds in a random order (cf. panel A) its degradation is substantially delayed (4.1-fold) in the absence of Cand1 (C, dashed line) and redistribution of Cul1 only occurs in the presence of Cand1 (C, lower panel). Total substrate is defined as S1T = [S1] + [SR1.S1] + [Cul1.SR1.S1] + [Cul1.Cand1.SR1.S1]. Parameters: At t = 0 substrate S1 (300nM) was added to a steady state mixture containing Cul1T = 300nM, SR1T = 30nM and SR2T = 630nM. The values of Cand1T and γ are indicated in the upper panels. kon = 107M−1s−1, koff = 0.01s−1, kdeg = 0.004s−1. Parameters other than those mentioned are listed in Table 1.
Fig 8.
A: Scheme showing the reactions as used in the experimental setup of Pierce et al. [10]. States and reactions have the same meaning as in Fig 1B. Note that Cul1 and Fbxw7 are bound to fluorescent dyes.B: Fit of the model simulations to a single exponential function. Data points for the first 5 seconds were discarded to obtain a better fit. Kinetic parameters were taken from Table 1.