SecYEG activates GTPases to drive the completion of cotranslational protein targeting

Abstract

Signal recognition particle (SRP) and its receptor (SR) comprise a highly conserved cellular machine that cotranslationally targets proteins to a protein-conducting channel, the bacterial SecYEG or eukaryotic Sec61p complex, at the target membrane. Whether SecYEG is a passive recipient of the translating ribosome or actively regulates this targeting machinery remains unclear. Here we show that SecYEG drives conformational changes in the cargo-loaded SRP-SR targeting complex that activate it for GTP hydrolysis and for handover of the translating ribosome. These results provide the first evidence that SecYEG actively drives the efficient delivery and unloading of translating ribosomes at the target membrane.

Figure 1.

SecYEG destabilizes the early intermediate in the RNC–SRP–FtsY complex. (A) Conformational states of the SRP–FtsY complex. Green and blue shapes denote FtsY and Ffh, respectively. T denotes GTP and D denotes GDP. (B) Equilibrium titration of the early complex in the absence (green) and presence of 0.05% DDM (black) or 24 µM SecYEG (red). The data were fit to Eq. 1 and gave the K_d values and FRET end points shown in F. (C) Effect of SecYEG on the stability of the early complex. The K_d values of the early complex were determined as in A at the indicated concentrations of SecYEG. (D) Equilibrium titration of the closed/activated complex was performed in the presence of 0.02% DDM (black) or 12 µM SecYEG (red), using 100 nM acrylodan-labeled SRP, 230 nM RNC, and 200 µM GppNHp. The data were fit to Eq. 1 and gave the K_d values shown in F. (E) Equilibrium titration of the activated complex was performed in the presence of 0.01% DDM (black) or 10 µM SecYEG (red), using 50 nM acrylodan-labeled FtsY C356, 300 nM RNC, and 200 nM GppNHp. The data points of the two titrations overlapped at 0.2 µM SRP, concealing the data point for the –SecYEG titration. The data were fit to Eq. 1 and gave the K_d values shown in F. (F) Summary of the K_d values and FRET end points from the experiments in B–E. The values are averages of two to four experiments ± SD. B–E show representative data from two to four experiments.

Figure 2.

SecYEG reactivates GTP hydrolysis from the RNC–SRP–FtsY complex. (A) Stimulated GTP hydrolysis by SRP and FtsY in the absence (circles) and presence (squares) of RNC, and in the presence of both RNC and SecYEG (triangles). The data were fit to Eq. 3 and gave k_cat values of 80, 22, and 66 min⁻¹ for the SRP–FtsY, RNC–SRP–FtsY, and RNC–SRP–FtsY–SecYEG complexes, respectively. (B and C) Effect of SecYEG (B) or DDM (C) on GTP hydrolysis from the RNC–SRP–FtsY complex. The data in B were fit to Eq. 3 and gave a K_d value of 2 µM and a k_max value of 57 min⁻¹. (D) DDM reduces the GTP hydrolysis rate from the SRP–FtsY complex. k_cat values were determined as in A in the absence and presence of 0.02% DDM. (E) SecYEG does not affect the basal GTPase activity of FtsY. Reactions were performed in the presence of 4 µM FtsY, 100 µM GTP, and the indicated concentrations of SecYEG. (F) In the absence of RNC, SecYEG does not significantly affect GTP hydrolysis from the SRP–FtsY complex. The data were fit to Eq. 3 and gave k_cat values of 60 and 51 min⁻¹ in the absence (closed) and presence (open) of 12 µM SecYEG, respectively. The data in A, B, D, E, and F are the average of two experiments ± SD (error bars).

Figure 3.

SecYEG forms a quaternary complex with RNC, SRP, and FtsY. (A) Kinetics of the closed complex assembly were measured in the absence (red) and presence of 100 nM RNC (green), and in the presence of 100 nM RNC and 7 µM SecYEG (blue). Reactions contained 40 nM DACM-labeled SRP, 100 nM BODIPY-FL–labeled FtsY, and 200 µM GppNHp. (B) Kinetics of the early → closed rearrangement of the RNC–SRP–FtsY complex in the absence (black) and presence (red) of 12 µM SecYEG. The data were fit to Eq. 2. Single exponential fits to the data gave rearrangement rate constants of 0.403 ± 0.027 and 0.489 ± 0.008 s⁻¹ with and without SecYEG, respectively. (C) Summary of the early → closed rearrangement rate constants. The value of 1.5 s⁻¹ was obtained in the absence of SecYEG and RNC. A and B show representative data from three replicates. Error bars indicate average ± SD.

Figure 4.

Mutations in basic cytosolic loops of SecYEG abolish its stimulatory effects. (A) Stimulated GTP hydrolysis by the RNC-bound SRP and FtsY in the absence (open circles) and presence (closed circles) of 12 µM mtSecYEG. The data were fit to Eq. 3. The broken line is the data for wild-type SecYEG from Fig. 2 A. (B) Effect of SecYEG charge reversal mutants on GTP hydrolysis from the RNC–SRP–FtsY complex. Solid black, R255E/R256E/R357 (mtSecYEG); red, R357E; blue, R255E/R256E. The broken line is the data for wild-type SecYEG from Fig. 2 B. (C) Equilibrium titration of the early complex in the presence of 24 µM mtSecYEG (closed circles). The data were fit to Eq. 1 and gave a K_d value of 162 ± 4 nM. Titration in the presence of DDM (solid line) and wild type SecYEG (broken line) are from Fig. 1. (D) Effect of mtSecYEG on the stability of the early complex. K_d values were determined as described in C. The data with wild-type SecYEG (broken line) are from Fig. 1 C. (E) Equilibrium titration of the early targeting complex formed by FtsY-NG in the presence of 0.05% DDM (black) or 24 µM SecYEG (red). The data were fit to Eq. 1 in the Materials and methods, and K_d values are given in F. (F) Summary of the effects of SecYEG on the stability of the early targeting complex formed with full-length FtsY and FtsY-NG. The data with full-length FtsY are from Fig. 1. A, C, D, and E show representative data from two to three replicates. Error bars in B and F are standard deviations from two to three experiments.

Figure 5.

SecYEG drives conformational changes in the RNC–SRP–FtsY complex. (A) Free energy profile for the FtsY–SRP interaction in the absence (black) and presence (red) of SecYEG. The red arrows denote the effect of SecYEG. Activation energies were calculated from the rate constants using ΔG^‡ = –RTln[kh/(k_BT)], where R = 1.987 cal·K⁻¹·mol⁻¹, h = 1.58 × 10⁻³⁷ kcal−s, k_B = 3.3 × 10⁻²⁷ kcal·K⁻¹, and T = 298 K. The relative free energies were calculated from the equilibrium stability of the complexes using ΔG = –RTlnK, where K is the equilibrium constant. A standard state of 1 µM was used. T denotes GTP and D denotes GDP. (B) Model for the role of SecYEG in driving GTPase rearrangements in the targeting complex and completing cotranslational protein targeting. The M domain of Ffh is also shown. The question mark denotes questions regarding the fate of the signal sequence and the interaction of SecYEG with FtsY in the quaternary complex.