Sequential checkpoints govern substrate selection during cotranslational protein targeting

Abstract

Proper protein localization is essential for all cells. However, the precise mechanism by which high fidelity is achieved is not well understood for any protein-targeting pathway. To address this fundamental question, we investigated the signal recognition particle (SRP) pathway in Escherichia coli, which delivers proteins to the bacterial inner membrane through recognition of signal sequences on cargo proteins. Fidelity was thought to arise from the inability of SRP to bind strongly to incorrect cargos. Using biophysical assays, we found that incorrect cargos were also rejected through a series of checkpoints during subsequent steps of targeting. Thus, high fidelity of substrate selection is achieved through the cumulative effect of multiple checkpoints; this principle may be generally applicable to other pathways involving selective signal recognition.

Figure 1. Potential fidelity checkpoints in the SRP pathway

(A) Model for potential checkpoints during co-translational protein targeting. A cargo (RNC) with a signal sequence (magenta) enters the pathway upon binding SRP, and is either retained (black arrows) or rejected (red arrows) at each step (numbered 1–5). T and D denote GTP and GDP, respectively. (B) Signal sequence variants used in this study. Bold highlights the hydrophobic core. Blue highlights the N-terminal signal sequence extension of EspP. (C, D) Equilibrium titrations of SRP-RNC binding. Nonlinear fits of data gave K_d values of 0.55±0.20, 8.4±2.0, 13.6±3.0, 108±11 and 130±12 nM for RNC_1A9L (C, ●), RNC_3A7L (C, ■), RNC_EspP (C, ◆), RNC_phoA (D, ■) and RNC_luciferase (D, ●), respectively. Error bars are SDs from three independent experiments. (E) Summary of the binding affinities of SRP for different cargos. The dashed line denotes the cellular SRP concentration of 400±58 nM. Error bars are SEs of the fits.

Figure 2. Correct cargos stabilize the early intermediate and mediate faster rearrangement to the closed complex

(A, B) Equilibrium titrations of the early intermediate. Nonlinear fits of data gave K_d values of 78±5, 110±8, 311±21 and 2060±201 nM and FRET endpoints of 0.68±0.02, 0.64±0.02, 0.41±0.03, and 0.34±0.02 for RNC_1A9L (A, ●), RNC_2A8L (A, ■), RNC_EspP (B, ■), and RNC_luciferase (B, ●), respectively. Error bars are SDs from three independent experiments. (C, D) Summary of the K_d values (C) and FRET end points (D) of the early intermediates formed by different cargos. Error bars are SEs of the fits in C and SDs from three independent experiments in D. (E, F) Measurements of the early to closed rearrangement. Nonlinear fits of data gave rate constants of 0.31±0.02 s⁻¹ with RNC_1A9L (E) and 0.039±0.003 s⁻¹ with RNC_luciferase (F). Error bars are SDs from three independent experiments. (G) Summary of the rate constants for the early to closed rearrangement with different cargos. Error bars are SEs of the fits.

Figure 3. Correct cargos accelerate GTP-dependent complex formation but delay GTP hydrolysis

(A, B) Rate constants of SRP-SR complex assembly in GMPPNP measured by FRET. Linear fits of data gave k_on values of 9.9±1.3×10⁶, 8.8±1.6×10⁶, 2.0±0.2×10⁵, 6.3±0.4×10⁴, 1.1±0.2×10⁴ and 1.8±0.3×10³ M⁻¹s⁻¹ for RNC_1A9L (A, ●), RNC_2A8L (A, ■), RNC_3A7L (B, ●), RNC_phoA (B, ■), RNC_5A5L (B, ◆) and RNC_luciferase (B, ▲), respectively. Error bars are SDs from three independent experiments. (C) Summary of GTP-dependent complex assembly rate constants with different cargos. Error bars are SEs of the fits. (D, E) Effects of cargo on GTP hydrolysis from the SRP•SR complex. Nonlinear fits of the data gave maximal GTPase rate constants (k_cat) of 0.72±0.03 s⁻¹ without cargo (●), and 0.11±0.01, 0.38±0.02, 0.51±0.08, and 0.65±0.22 s⁻¹ with RNC_1A9L (D, ■), RNC_5A5L (D, ◆), RNC_EspP (E, ■) and RNC_luciferase (E, ◆), respectively. Error bars are SDs from three independent experiments. (F) Summary of GTPase rate constants in the presence of different cargos. Error bars are SEs of the fits.

Figure 4. Stepwise rejection of incorrect cargos from the SRP pathway

(A) Top panel, cargos are either retained (black arrow) or rejected (red arrow) during each checkpoint. Lower panel, predicted fraction of cargos retained in the SRP pathway during each checkpoint (Supplementary text). (B) SRP-dependent protein targeting and translocation of the model substrates. pPL and PL denote the precursor and processed forms of the substrate, respectively. (C) Predicted protein targeting efficiencies (● and ○) agree well with the experimentally determined values (■), quantified from the data in (B). Translation elongation rates of 20 (●) and 10 amino acids/s (○) were used for the E. coli and eukaryotic ribosomes, respectively, to calculate the targeting efficiencies.