Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

Abstract

The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP-FtsY complex. This leads to accelerated SRP-FtsY complex assembly, and allows the SRP-FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP-FtsY GTPase complex exposes FtsY's lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting.

Figure 1.

Phospholipids stimulate FtsY’s basal GTPase activity and its complex assembly with SRP. (A) Effect of liposomes on the basal GTPase reaction of FtsY (red), FtsY-NG+1 (green), and FtsY-NG (black and inset). The data were fit to Eq. 2, and gave Hill coefficients of 2.9 and 3.4 for FtsY and FtsY-NG+1, respectively, and an apparent K_d value of 2.0 and 2.2 mM for lipid binding to FtsY and FtsY-NG+1, respectively. (B) Effect of liposomes on the reaction: ^GTP•SRP + FtsY^•GTP → products for FtsY (red), FtsY-NG+1 (green), and FtsY-NG (black). (C) A domain–specific lipid stimulation of complex assembly with FtsY (red) and FtsY-NG+1 (green), after subtraction of the rate constants from FtsY-NG. The data were fit to Eq. 2, and gave Hill coefficients of 4.4 and 3.8 for FtsY and FtsY-NG+1, respectively. Error bars indicate SD.

Figure 2.

Role of the FtsY A domain in preprotein targeting and translocation. (A) SDS-PAGE analysis of the translocation efficiency of pPL mediated by FtsY, FtsY-NG+1, and FtsY-NG. (B) Quantitation of the results in A. Error bars indicate SD.

Figure 3.

Phospholipids accelerate formation of the activated SRP–FtsY complex. (A) Fluorescence emission spectra of acrylodan-labeled FtsY C356 in the presence (closed circles) or absence (open circles) of 5 µM SRP, with (red) or without (black) 2 mM of liposomes present. The scattering from buffer, SRP, and liposomes has been subtracted from the respective spectra. (B and C) Time courses for complex assembly were measured in the presence of 200 nM of acrylodan-labeled FtsY C356 and 200 µM GMPPNP without (B) or with (C) 2 mM of liposomes present. The data were fit to single exponential functions to yield observed rate constants at individual SRP concentrations. (D) Liposomes accelerate formation of the activated SRP–FtsY complex. Observed rate constants for complex formation are from B and C. GNP denotes GppNHp. The inset shows the data in the absence of liposomes on an expanded scale. Linear fits of the data to Eq. 4 gave association rate constants of k_on = 3.0 × 10⁶ and 1.8 × 10⁴ M⁻¹s⁻¹ in the presence and absence of liposomes, respectively. Error bars indicate SD from three or more measurements.

Figure 4.

FtsY preferentially stabilizes the SRP–FtsY complex in the closed and activated states. (A) Equilibrium titration of the early intermediate in the presence (red) and absence (black) of 2 mM of liposomes. Titrations used 100 nM of coumarin-labeled SRP C235, 200 nM RNC, and 200 µM GDP. (B) Equilibrium titration of the closed/activated complex in the absence (left) and presence (right) of 2 mM of liposomes. Titrations used 50 nM of coumarin-labeled SRP C235 and 200 µM GppNHp. (C) Equilibrium titration of the activated SRP–FtsY complex in the absence (left) and presence (right) of 2 mM of liposomes. Titrations used 100 and 40 nM of acrylodan-labeled FtsY C356 in the absence and presence of liposomes, respectively, and 200 µM GppNHp. The data were fit to Eq. 3, and the values of K_d are summarized in D. Representative fluorescence measurements are shown in A–C, and the K_d values reported in D are averaged from three or more measurements.

Figure 5.

The stable SRP–FtsY complex binds more strongly to lipids than free FtsY. (A and B) Density gradient flotation analysis of the binding of FtsY (left) and the SRP–FtsY complex (right) to E. coli liposomes for full-length FtsY (A) and FtsY-NG (B). (C) The effect of liposomes on the reaction ^GTP•SRP–FtsY^•GTP → products with FtsY (red) and FtsY-NG (black). The data with FtsY was fit to Eq. 2, and gave a Hill coefficient of 4.8 and an apparent K_d value of 39 µM for FtsY–lipid binding in the complex. Error bars indicate SDs from two measurements.

Figure 6.

Formation of the GTP-dependent SRP–FtsY complex exposes FtsY’s lipid binding helix. (A) Crystal structure of E. coli FtsY-NG+1 (PDB accession no. 2QY9). The amphiphilic lipid-binding helix at the A–N domain junction is highlighted in orange, and residue E229, which served as a negative control, is shown in blue. (B) EPR spectra of the nitroxide spin probe at residue T206 of FtsY-NG+1 for apo-FtsY (black), the early intermediate formed in GDP (red), and the closed/activated complex formed in GppNHp (green). ΔH indicates the central linewidth, and im and m denote the population of immobile and mobile molecules, respectively. (C and D) Summary of the central linewidth (C) and fraction of mobile molecules (D) for nitroxide probes placed at different positions along FtsY’s lipid-binding helix. Color coding is the same as in B. Error bars indicate SDs from two or more measurements.

Figure 7.

FtsY is specifically stimulated by anionic phospholipids. (A and B) Effects of E. coli liposomes on FtsY’s basal GTPase rate (A) and on SRP–FtsY complex assembly (B). (C and D) The effect of 7:3 PE/PC liposomes on FtsY’s basal GTPase reaction (C) and on SRP–FtsY complex assembly (D). (E and F) Effects of PG (circles) liposomes on FtsY’s basal GTPase rate (E) and on SRP–FtsY complex assembly (F). (G and H) Effects of cardiolipin (closed circles) on FtsY’s basal GTPase rate (G) and on SRP–FtsY complex assembly (H). The dashed lines depict the data for PG/PE liposomes and are shown for comparison. Error bars indicate SDs from two or more measurements.

Figure 8.

FtsY binds specifically to anionic phospholipids. (A) SPR traces depicting resonance changes on FtsY-immobilized biosensor chips due to liposome binding. The numbers above each line denote the corresponding liposome concentration. (B–D) Equilibrium binding curves of FtsY (red) or FtsY-NG (black) to liposomes composed of 7:3 PE/PC (B), 7:3 PG/PE (C), and E. coli (D) lipids. The data were fit to Eq. 5 to obtain the apparent K_d values and hill coefficients (h) for FtsY-lipid binding.

Figure 9.

Phospholipids drive conformational changes of FtsY to regulate protein targeting. (A) Free energy profile depicting the effect of phospholipids in shifting the conformational equilibrium of FtsY from the open to the closed/activated states by ∼100-fold (∼2.8 kcal/mol). A standard state of 1 µM FtsY was used to calculate the free energy differences and activation energy for GTP-dependent SRP–FtsY complex formation. (B) Model for how lipid binding of FtsY is coupled to the SRP–FtsY interaction and protein targeting. Step 1, dynamic association of free FtsY with the phospholipid membrane. Step 2, membrane-bound FtsY is more efficient at forming a stable closed/activated SRP–FtsY complex. Step 3, cytosolic FtsY forms an early complex with cargo-loaded SRP. Step 4, the cargo–SRP–FtsY complex shifts between the early and closed conformations with an equilibrium of ∼1. Step 5, the closed complex binds more strongly to the membrane than free FtsY. Step 6, the closed complex rearranges to the activated state, during which it completes the transfer of cargo to the translocon. Step 7, GTP hydrolysis drives complex disassembly, returning a fraction of FtsY molecules to the cytosol. (C) Movement of the αN1 helix (red) accompanies the open → closed rearrangement and membrane binding of FtsY.